Abstract
Abstracts
CONTENTS
Plenary Lecture
Overcoming translational challenges for the new era of regenerative medicine
1Wake Forest Institute for Regenerative Medicine, Winston Salem, North Carolina, U.S.A.
Advances in tissue engineering and regenerative medicine have provided new therapeutic opportunities for repairing damaged tissues and organs. Aligned with the goals of tissue engineering, we have followed a strategy that involves the use of biocompatible matrices, either with or without cells. The matrices are either used as a cell delivery vehicle or scaffolds to promote and enhance tissue regeneration. When cells are used, target cells obtained from donor tissue are isolated and expanded in culture, attached to a support matrix, and re‐implanted into a recipient to recover tissue function. This strategy has been successfully applied to many tissue and organ systems in the clinic. However, developing and translating viable solutions for complex tissue systems has been delayed. This is primarily due to various scientific and technological challenges inherent to the tissue‐building process and implant integration, such as establishing vascularization and innervation of volumetric tissue mass. Furthermore, processes involving manufacturing, regulation, and commercialization logistics present an additional layer of complexity.
This presentation intends to provide a better understanding of the translational processes and discusses approaches to overcome some of the challenges that impede the development and delivery of therapeutic solutions.
An integrated smart electroactive dressing that promotes wound healing and noninvasively monitors healing progress
1Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan (ROC)
Traditional wound dressings do not promote cellular activities that heal wounds or assist in monitoring the progress of that healing. This work develops an engineered electroactive dressing that comprises a backing film of collagen, a layer of polydopamine‐crosslinked carboxymethyl chitosan conductive hydrogel, and an interdigitated array (IDA) electrode. The dressing is evaluated in a mouse model with a full‐thickness skin defect. The incorporated conductive hydrogel provides a channel for transmitting the endogenous bioelectrical signals to the wound; stimulating electrical stimuli‐responsive cells, and accelerating the restoration of the wounded tissue. The IDA electrode, which is connected in series with a conventional power source or a self‐powered triboelectric nanogenerator, is able to detect the electrical resistance or output current across the wounded tissue to monitor its entire healing process noninvasively. This wound monitoring system is integrated with a WIFI‐based system for wireless data collection and transmission, using a personal smartphone on which is installed an app. Such a real‐time wound monitoring system can be worn by patients in daily life; it issues early warnings to them of potential infections and wirelessly sends wound progression data to remote medical staff for dynamic intervention and the provision of telemedicine as required.
Matrix mechanics to regulate regeneration
1School of Engineering and Applied Sciences and Wyss Institute, Harvard University
Mechanical cues regulate many aspects of biology, and we are developing materials capable of providing either defined resistance to cell intrinsic mechanical forces or application of specific extrinsic forces to tissues. These biomaterials can enhance the effectiveness of stem cell therapies, directly support and regenerate damaged tissues, and have led to bio‐inspired new medical devices with unprecedented properties.
Endogenous stem cells in the bone marrow for tissue repair and disease development
1Department of Genetic Engineering, Graduate School of Biotechnology, Kyung Hee University Director of Kyung Hee Institute of Regenerative Medicine (KIRM), Kyung Hee University Hospital, Seoul, Korea
Tissue injury may create a specific microenvironment, which brings up the systemic participation of reparative stem cells in the repair process. We identified a new role of substance‐P (SP, 11 aa peptide) as an injury‐inducible messenger to mobilize bone marrow MSC and EPC to the blood, home to the injured tissue, and be engaged in the tissue repair in a variety of acute and chronic tissue injury models such as alkali‐burn corneal injury, spinal cord injury, acute myocardiac infarction, stroke, limb ischemia, aortic injury, diabetics, radiation‐induced BM injury and gastrointestinal injury, elucidating endogenous healing mechanism recalling BM stem cells. In addition, SP can play a role as a novel anti‐inflammatory cytokine to directly induce M2 type monocyte/macrophages and stimulate specific trafficking of CD163+/CD206+ subset of monocytes from the bone marrow to the blood, which suppresses the injury‐evoked tissue inflammation and clean up dead cells for tissue repair. This unique role of SP seems to orchestrate tissue repair by reducing the inflammation‐provoked tissue damages at early stage and creating favorable microenvironment for the engraftment of incoming stem cells and then recruiting endogenous stem cells from bone marrow to the injured tissue as reparative stem cells for the tissue repair. Collectively, SP may be proposed as a potential stem cell stimulating agent to cure a variety of acute and chronic diseases requiring the reduction of inflammation load and engagement of endogenous reparative stem cells. This work was supported by Projects NRF‐2016M3A9B4917320, HI13C1479, and HI18C1492.
Symposia
Engineering scaffold‐free tissue constructs via modular assembly, cell‐ only bioprinting and 4D strategies
1University of Illinois at Chicago
Many tissues form and heal through the initial formation of a condensation of cells. Scaffold‐free tissue engineering aims to partially mimic these processes. Traditional scaffold‐free tissue engineering has focused primarily on cell condensations in the form of simple spheres or sheets. In this talk, three technologies enabling the biofabrication of more complex cell condensation tissue architectures and organizations will be presented. In the 1st technology, hydrogel molds are used to form modular cell condensation tissue building blocks that may then be assembled to form multi‐tissue organs. The 2nd technology involves a platform system for bioprinting cell‐only bioinks in complicated structures. Finally, the 3rd technology permits the formation of 4D high cell density and cell condensation constructs that can change their shape over time.
Micro/nano engineered biomaterials for manufacturing biomimetic tissues and biomedical applications
1Harvard Medical School/Brigham and Women's Hospital
Native tissues and organs exhibit highly organized 3D complex architectures composed of several cell types and an extracellular matrix (ECM), which are difficult to replicate in vitro using conventional microfabrication methods. Recent bioprinting technologies offer significant potential for advances in rapid prototyping methods that have enabled the building of functional and complex 3D tissue constructs. We have developed an advanced multi‐material bioprinting platform that employs ECM‐based colloidal gels as biodegradable self‐healing supporting baths, and a programmable microfluidic device, which can quickly switch between different biomaterials, reagents, and cells. We addressed the limitations, such as poor construct positioning or bed stability, by using a self‐healing supporting gel. This technique allows us to deposit highly complex patterns using human induced pluripotent stem cells (iPSCs)‐laden bioinks within the biodegradable self‐healing supporting matrix to maintain their architectural integrity in a facile yet highly rapid manner for creating personalized and physiologically relevant human tissue constructs. These obtained biomimetic tissue constructs can be used for regenerative medicine and organs‐on‐a‐chip platforms to discover and evaluate drug toxicity. Such human cell‐based organs‐on‐a‐chip platforms have become increasingly prominent in drug discovery since they allow for evaluating the cytotoxic effects of pharmaceutical compounds or nanomaterials on human tissue models, thereby averting the premature need for expensive animal tests or clinical trials. The outcomes of this study may be extended to academic and pharmaceutical research applications appertaining to personalized medicine, in that drug pharmacokinetics, efficacy, and toxicity can be potentially monitored on a patient‐by‐patient basis.
Creating advanced bio‐inks using microfluidics to engineer multiscale tissue
1University Twente
The modular design of tissues is indispensable for proper organ function. For example, matrix is naturally produced in a modular way. Cells are coated in a thin layer of pericellular matrix, which is located within a bulk of extracellular matrix. However, recreating pericellular matrix has remained a grand challenge as it requires the coating of individual single cells in a micrometer thin layer of biomaterial. To overcome this challenge, we developed microfluidic droplet generation platforms to produce enzymatically crosslinked ultra‐thin shelled single‐cell‐microgels. Using this platform, we engineered 3D single stem cell microniches with on‐demand tunable biophysical and biochemical properties to controllably program stem cell differentiation along chosen lineages. The microgels' Young's modulus can be accurately tuned from 2 to 50 kPa. Single cell analysis revealed that softer microgels stimulated adipogenesis, while stiffer microgels induced osteogenesis. Temporally stiffening the microgels revealed that the first three days of differentiation where of key importance for stiffness‐induced stem cell fate decisions. We then combined our microgels with distinct biomaterials to create advanced bioinks. This modular approach effectively uncoupled the engineered tissues pericellular and extracellular environments. This allowed for an unprecedented control over the design and behavior of the living implant. Lastly, to endow our tissues with macroscale properties such as tissue shape, we used our bioinks in combination with various biofabrication techniques including injection molding, photolithography, and 3D bioprinting to engineer multiscale hierarchical living implants. In short, we here present several microfluidic microgel‐based concepts that focus on advancing the engineering of functional living matter.
A 3D culture platform promotes maturation of midbrain‐fated iPSCs into functional, physiologically relevant synaptic networks
1University of Texas at Austin, 2Cedars Sinai Los Angeles, 3University of California Los Angeles
There is increasing recognition of the importance of cell‐cell and cell‐matrix interactions during development, regeneration, and disease progression in the central nervous system. We developed hyaluronic acid‐based (HA‐based), matrices which enhance these microenvironmental matrix in 3D culture of human iPSC‐derived, midbrain‐fated neural cells. After maturation, dopaminergic and GABAergic neurons were present in both 3D matrices and conventional 2D cultures. However, single‐cell RNAseq results indicate 3D cultures yielded more cells with neurons (e.g., β‐III tubulin and synaptophysin transcripts) and, in particular, dopaminergic neurons (e.g., tyrosine hydroxylase transcripts) than 2D cultures. In 3D culture, dopamine release was consistently detected in response to repeated potassium exposure. As evidence that neuronal circuits mimic the nigrostriatal pathway, central to substance abuse disorders, morphine stimulation increased dopamine release, which was reversal by naloxone. As further evidence of GABAergic synapses evoking post‐synaptic cell dopamine release, GABAergic antagonists and agonist decreased or increased dopamine release, respectively. With a mechanically and molecularly defined environment, our 3D culture platform enhanced iPSC maturation in functional synaptic networks that are physiologically relevant in substance abuse, and perhaps neurodegenerative conditions including Parkinson's disease.
Biofabrication of engineered tissues by 3D bioprinting of tissue specific, high cell density bioinks
1University of Illinois Chicago
Scaffold‐free engineering of three‐dimensional (3D) tissue has focused on building sophisticated structures to achieve functional constructs. Although the development of advanced manufacturing techniques such as 3D printing has brought remarkable capabilities to the field of tissue engineering, it is still difficult to create precisely controlled complex structures and organization of tissue specific condensations with high cell density bioink‐based bioprinting. Recently, we introduced a cell printing platform using a biodegradable and photocrosslinkable microgel supporting bath which addresses the aforementioned challenge and permits 3D printing and long‐term culture of a living cell‐only bioink lacking a biomaterial carrier for functional tissue formation. The biodegradable and photocrosslinkable microgel bath serves initially as a fluid‐like support, allowing free movement of the printing nozzle for high‐resolution cell extrusion, while also presenting solid‐like properties to sustain the structure of the printed constructs after extrusion. In this study, we present newly biofabricated tissues from directly assembled tissue specific high cell density (THCD) bioinks. THCD bioinks have been prepared with individual stem cells or stem cell aggregates by incorporation of growth factor‐loaded gelatin microparticles. The bioprinted THCD bioinks in the photocrosslinked microgel supporting bath condense together and differentiate down tissue‐specific lineages to form osteochondral and cartilage tissues. By changing the growth factors presented and/or cell types, these THCD bioinks enable engineering various functional tissues with controlled architecture and organization of cells.
Are there hopeful new strategies to rejuvenate myocardium?
1The Catholic University of Korea
Ischemic heart disease is the leading cause of morbidity and mortality in the world. While pharmacological and surgical interventions developed in the late twentieth century drastically improved patient outcomes, mortality rates over the last two decades have begun to plateau. Following ischemic injury, pathological remodeling leads to cardiomyocyte loss and fibrosis leading to impaired heart function. Cardiomyocyte turnover rate in the adult heart is limited, and no clinical therapies currently exist to regenerate cardiomyocytes lost following ischemic injury. In this summary, I introduce new therapeutic strategies including cell‐based interventions and non‐cellular therapeutics to regenerate the heart: (1) 3D engineered cardiac patches, (2) stem cell engineering, and 3) exosome‐mimetic extracellular nanovesicles. Moreover, I highlight recent mechanistic insights governing these strategies to promote heart regeneration and identify current challenges in translating these approaches to human patients.
Effect and application of cryopreserved three‐dimensional microcardiac spheroids inmyocardial infarction therapy
1T&R Biofab, 2The Catholic University of Korea
Worldwide, attention is focused on developing therapeutics using human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs), and some clinical trials have demonstrated therapeutic efficacy in heart failure. However, cardiomyocytes do not have the proliferation capacity, strategies to increase the survival efficiency of transplanted cardiomyocytes in vivo are required in order to maximize the efficacy of cardiomyocyte therapeutics. Recently, we have developed the 3D microcardiac spheroid and cell patch system for improving the survival rate of transplanted cardiomyocytes. Animal experiments confirmed that 3D microcardiac spheroid was effective for survival efficiency in hypoxic environment in the early stage of transplantation, and cell patch was effective for long‐term survival efficiency of transplanted cardiomyocytes by promoting angiogenesis. Our results are expected to be developed as a system to improve the effectiveness of hPSC‐CMs therapy.
Novel photobiomodulation for enhancing angiogenic efficacy of adult stem cells toward future biomedical application
1Sungkyunkwan University
In this presentation, the contents of improving the angiogenic efficacy of stem cells using photobiomodulation will be introduced. The reproducible and optimized photobiological techniques that can enhance the angiogenic efficacy of stem cells by irradiating the stem cells with red, blue, and green light will be discussed in detail. With the development of various medical and cosmetic‐cosmetic devices, photobiological regulation has become widely known. However, the conditions for the optimized methodology that meet the criteria and purpose are not clearly set. In this presentation, we aimed to introduce a commercialized light source that can break away the use of customized in‐house device and unify the conditions for enhancing the therapeutic efficacy of stem cells. We will also introduce how to select light sources and control physical properties required for the optimization. In addition to the previously reported molecular mechanisms photobiomodulation, newly discovered molecular mechanisms and following cellular behavior changes will be discussed. Our new photobiomodulation technique introduced in this lecture might open a novel platform that can be used in the fields of tissue regeneration and disease treatment in future
Three‐dimensional microenvironments boost direct cardiac reprogramming
1Yonsei University Medical College
Direct cardiac reprogramming technology represents great potential to generate autologous target cells for tissue repair and restoration of tissue and organ function. Chemical reprogramming driven by small molecules has gained considerable interest to generate cardiomyocytes as it avoids safety questions associated with genetic manipulations. While this technology is promising, there are a number of critical limitations must overcome to translate from the laboratory to clinical applications. These include low conversion efficiency and incomplete cardiomyocyte maturation. Here, we demonstrate that reprogramming of fibroblasts in three‐dimensional microenvironment boosts reprogramming efficiency and promotes cardiac maturation, displaying electrophysiological features and drug responses comparable to primary cardiomyocytes. We also show the therapeutic potential of the induced cardiomyocytes in a rat model of myocardial infarction. This research was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) (2020M3A9I403845513, and 2022M3E5E8081187).
Vascular patches comprising polycaprolactone/decellularized extracellular matrix (PCL/dECM)‐based core/shell nanofibers for arterial healing and vascular reconstruction
1Department of Chemical Engineering, Faculty of Engineering, Graduate School, Kyushu University, 744 Motooka, Nishi‐ku, Fukuoka, Japan, 2Department of Chemical Engineering, Graduate School of Engineering, Kyushu University, Kyushu, Japan
Vascular malformations pose huge morbidity and mortality worldwide, which becomes further severe with bleeding complications. While autografts and allografts are gold‐standard treatment options for vascular reconstruction, host‐site associated infection risks as well as limited availability of these grafts constrict their widespread applicability. Alternatively, non‐degradable and biodegradable polymers, while perform satisfactorily, face shortcomings due to thrombotic occlusion and poor cell recognition. Since extracellular matrix (ECM) is endowed with a myriad of biological cues, it provides an invaluable platform for tissue engineering (TE) and biomanufacturing. Herein, we attempted to harness decellularized ECM (dECM) from porcine aorta to develop core/shell type polycaprolactone/dECM based patches. The dECM was characterized by microscopic, histological, and physico‐chemical assays to ascertain the success of decellularization and the preservation of ECM. Electrospun patches were further characterized physico‐chemically and morphologically. Hematoxylin and eosin (H&E) staining and deoxyribonucleic acid (DNA) assay revealed successful decellularization. Besides, biochemical assays, including collagen and glycosaminoglycan (GAG) content showed the preservation of the key ECM components in dECM. Scanning electron microscopy and transmission electron microscopy revealed core/shell type morphology of nanofibers. While human umbilical vein endothelial cells (HUVECs) grew well on patches, platelets were only scarcely adhered, establishing their good cytocompatibility. The vascular patches will be next evaluated as arterial substitutes and systematically evaluated histologically and immunohistochemically for endothelialization, vascular remodeling, and immunomodulation. Conclusively, these dECM‐based arterial patches may have broad implications for cardiovascular reconstruction as well as for other bio‐related research disciplines.
3D bioprinted biomimetic bone scaffolds for mandibular reconstruction
1Wake Forest University School of Medicine
Medical imaging and 3D bioprinting strategy converged to allow the fabrication of a structure with complex shape and inner architecture based on patient anatomy and biomimicry. The objective of this study is to demonstrate the effect of biomimetic strategy by a geometric control in the scaffold design for in situ bone tissue regeneration. We tested a biomimetic design based on the architectural arrangement of bone, which provides a porous core for bone ingrowth and a dense external layer resisting fibrous tissue ingrowth. Biomimetic bone constructs were made of a composition of poly(ɛ‐caprolactone) and β‐ tricalcium phosphate (PCL/TCP). We examined this biomimetic bone construct in a critically sized mandibular bony defect of rabbits. After implantation, the new bone has been detected by CT analysis and shows the increasing occupation of the defected region with new bone. The biomimetic scaffolds show improved bone regeneration that new bone was filled in the entire defected region, while the control scaffolds only present new bone formation at the boundary. Histological examination of the bioprinted biomimetic bone constructs showed more matured bone regeneration in the biomimetic scaffolds, while fibrotic tissue ingrowth was observed in the control scaffolds. Our finding suggests that bone regeneration could be enhanced by the biomimetic bone construct designed to minimize competition for fibrotic tissue forming in the bony defect. We demonstrated the concept that patient‐specific anatomy could be translated into 3D bioprinting strategy through medical imaging and image processing software with strong clinical relevance.
Vessel‐derived decellularized extracellular matrices (VdECM): Novel bio‐engineered materials for the wound healing
1The Catholic University of Korea
Introduction
Decellularized ECM is a processed form of ECM that provides the non‐cellular ECM scaffold for various purposes in tissue engineering. One of the components is elastin, a chain of tropoelastin molecules that plays an important role in providing tissue elasticity. It is known to be closely related to scarless and fetal wound healing. Abundantly found especially in lung, blood vessel, and skin tissues, it has been studied to build scaffolds that reduce wound contraction and improve skin elasticity. In this study, we studied the characteristics of blood vessel‐derived decellularized extracellular matrix (VdECM) and its effect in wound healing via in vitro and in vivo studies.
Materials and Methods
VdECM composition, cell compatibility, cell proliferation and migration tests were performed through in vitro and in vivo study.
Results
Compared to 3% atelocollagen, VdECM showed superior results in fibroblast migration in scratch test. VdECM hydrogel showed better cell proliferation, with 0.35% VdECM promoting highest cell count at day 14. Fluorescent microscopic findings of L/D assay showed highest percentage of cell survival in 1% VdECM compared to 0% or 0.35% VdECM.
In the animal study model, the epithelialization rate was highest in the VdECM group compared to that of control, oxytetracycline, and epidermal growth factor ointments. Cross‐sections showed an enriched layer of collagen fibers.
Conclusion
From this study, VdECM promotes fibroblast migration, proliferation, and survival, which leads to a more effective epithelialization and collagen sedimentation compared to other methods of widely used topical agents.
ASC/chondrocyte‐laden alginate hydrogel/PCL hybrid scaffold fabricated using 3D printing for auricle regeneration
1Chonnam National University, 2Department of Biomechatronic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan University (SKKU)
Tissue engineering using adipose derived stem cells (ASCs) has become one of the most promising treatments for defective articular cartilage owing to the stability and dynamic differentiation of ASCs. In this study, we fabricated a 3D hybrid scaffold using poly(ɛ‐caprolactone) (PCL) to support the mechanical properties of the regenerating auricle cartilage, and injected a cell‐laden alginate hydrogel, containing a mixture of ASCs and chondrocytes, into the PCL scaffold. Using the cell‐laden 3D auricle structure, the in vitro chondrogenesis of the ASCs with and without the presence of chondrocytes was examined. Additionally, the feasibility of utilizing the 3D cell‐laden auricle structure for cartilage tissue engineering was evaluated in a rat model. In our in vitro and in vivo experiments, we observed that as the ASCs were co‐cultured with the chondrocytes, chondrogenic differentiation was encouraged, and the regeneration of cartilage was significantly increased.
Development of the in‐situ monitoring system for molecular response to mechanical stimuli in three‐dimensional tissue‐engineered cartilage
1Department of Biomedical Engineering, Doshisha University, 2Graduate School of Life and Medical Sciences, Doshisha University
Gene expression levels involved in anabolism and catabolism of chondrocytes change in response to changes in the mechanical field. Therefore, many previous studies have searched for appropriate mechanical stimulation conditions for the efficient production of cultured cartilage. However, since heterogeneous strain distribution is generated in the inside of cultured cartilage under mechanical stimulation and changes with culture time, it is not known whether the mechanical stimulation is optimally applied to the tissue during entire culture period. Therefore, it is necessary to obtain information on the molecular response of chondrocytes in the tissue during the culture process over time to evaluate how the applied mechanical stimuli affected the synthesis and degradation of the cultured cartilage.
In this study, we developed a system for in‐situ monitoring on molecular response of chondrocytes related to extracellular matrix (ECM) production under mechanical stimulation. The system was designed to visualize gene expression on ECM composition in different strain fields generated in the tissue using fluorescent reporter genes connected to the promoter of Col2a1 or Mmp13. In addition, we evaluated the local strain which changes with culture period owing to the anabolic and catabolic effects of chondrocytes, by measuring the distance between cells before and after deformation. This system enabled the spatial and temporal evaluation of the in‐situ molecular response to the production and degradation of cartilage ECM during the culture process.
Novel chitosan dermal filler with enhanced moldability and elasticity
1Department of Plastic and Reconstructive Surgery, SMG‐SNU Boramae Medical Center, 2R&D Center, Medifab Co. Ltd
Dermal fillers are injectable biomaterials that are used for tissue recovery and supplementation. Currently, dermal fillers are largely based on commercialized cross‐linked hyaluronic acid (HA) injections, which require a large injection force. Additionally, HA can be easily decomposed by enzymes, and HA‐treated tissues present a risk of developing granuloma.
In this study, we present a chitosan‐based dermal filler consisted of two fomulations: Chitosan dissolved in HCl with sodium phosphate dibasic and anhydrous glycerol. This filler operates on a liquid‐to‐gel transition and allows the injection force to be kept ∼4.7 times lower than that required for HA injections. Evaluation of the physical properties of the chitosan filler indicates high viscoelasticity and recovery rate after gelation at 37 °C.
Furthermore, in an in‐vivo evaluation, the liquid injection‐type chitosan filler transitions to a gel state within 5 min after injection into the body, and exhibits a compressive strength that is ∼2.4 times higher than that of cross‐linked HA. The filler also exhibits higher moldability and maintains a constant volume in the skin of mouse photoaging model for a longer time than the commercial HA filler, Restylane.
Therefore, a novel chitosan filler based on liquid‐to‐gel transition was prepared, which exhibited superior moldability and elasticity because of its thermosensitivity. The filler could maintain a constant volume in the skin for a longer compared to commercial HA filler.
3D printed bi‐layer biomimetic artificial periosteum for boosting bone regeneration
1Tianjin Hospital, 2Tianjin University, 3Tianjin Hospital; Tianjin medical University
Periosteum, a membrane covering on the surface of bone, plays an essential role in maintaining the whole function of bone tissue, especially in providing nourishment and vascularization during the bone regeneration process. Currently, most artificial periosteum has a relatively weak mechanical strength and fast degradation rate, and lacks integrated angiogenesis and osteogenesis function. In this study, a bi‐layer biomimetic artificial periosteum composed of methylacryloylated gelatin‐nano hydroxyapatite (GelMA‐ nHA) cambium layer and poly (N‐acryloyl 2‐lycine) (PACG)‐GelMA‐Mg2+ fibrous layer is fabricated via 3D printing technique. The GelMA‐nHA layer is shown to undertake the function of improving osteogenic differentiation of rMSCs with the sustainable release of Ca2+ from nHA nanoparticles. The hydrogen bonding strengthened P(ACG‐GelMA‐L)‐Mg2+ hydrogel layer serves to protect the inner defect site and prolong degradation time (60 days) to match the new bone regeneration. Furthermore, the released magnesium ions exhibit a prominent effect in regulating the polarization phenotype of macrophage cells into M2 phenotype and thus promoting angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. This bilayer artificial periosteum was implanted into the critical‐ sized cranial bone defect of rats, and 12‐week post‐operative outcomes demonstrate optimal new bone regeneration.
Extracellular vesicles derived from mesenchymal stem cells for the treatment of intractable neonatal diseases
1Samsung Medical Center, Sungkyunkwan University School of Medicine
Recent data have shown that extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are mainly responsible for the paracrine efficacy of parent MSCs through the transfer of mRNA, miRNA, and proteins. Previously, we have demonstrated that the MSCs could be a therapeutic option through their paracrine action for the various neonatal intractable disorders such as bronchopulmonary dysplasia, severe intraventricular hemorrhage, and severe hypoxic‐ischemic encephalopathy, which are major causes of mortality and significant acute and life‐long morbidities of the newborn infants. Therefore, EVs derived from MSCs can be a promising new therapeutic modality since this therapy is cell‐free and thus may bypass concerns associated with viable MSC treatment through direct transport of extracellular messages and mediate cell‐to‐cell communication. We have investigated and compared the amount and cargo content of EVs secreted from MSCs according to various preconditioning methods of MSCs and tested the preclinical efficacy and the mechanisms of EVs derived from MSCs in various intractable neonatal disorders of the neonatal rat pup models.
Magnet‐assisted systemic delivery of artificial extracellular vesicles to injured central nervous system
1Catholic University of Korea
Central nervous system (CNS) injuries such as ischemic stroke, hemorrhage, traumatic brain injury, and spinal cord injury (SCI) are leading cause of long‐term disability or death. As a novel tissue regenerative strategy, application of mesenchymal stem cell‐derived extracellular vesicles (MSC‐EVs) for treatment of CNS injuries has drawn much attention. However, it still remains challenging to improve the therapeutic outcomes of using EVs to treat CNS injuries. MSC‐EVs poorly accumulate in target tissue after systemic administration. In this study, iron oxide nanoparticles were introduced to MSC‐derived artificial EVs and served as navigating tool toward external magnetic field. In presence of external magnetic field, fabricated artificial EVs were intravenously injected to CNS injury models; mouse SCI model and rat ischemic stroke model. Magnetic guidance markedly enhanced the targeting efficacy of intravenously injected artificial EVs toward injured spinal cord or brain, alleviated tissue damage, and improved motor functions. Our results collectively demonstrate that the artificial EVs with magnetic properties can be served as nano‐sized therapeutic agent capable of precise targeting the injured neuronal tissues such as spinal cord and cerebral lesion with help of external magnet. Their application may be a potential new therapy for CNS injuries, and can be expanded to treat various diseases including myocardial infarction that involve the sequence of tissue damage, inflammation, and tissue repair.
Engineered small extracellular vesicles displaying sACE2 to protect against SARS‐CoV‐2 infection
1Sungkyunkwan University
Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) entry is mediated by the interaction of the viral spike (S) protein with angiotensin‐converting enzyme 2 (ACE2) on the host cell surface. Although a clinical trial testing soluble ACE2 (sACE2) for COVID‐19 is currently ongoing, our understanding of the delivery of sACE2 via small extracellular vesicles (sEVs) is still rudimentary. With excellent bio‐compatibility allowing for the effective delivery of molecular cargos, sEVs are broadly studied as nanoscale protein carriers. In order to exploit the potential of sEVs, we design truncated CD9 scaffolds to display sACE2 on the sEV surface as a decoy receptor for the S protein of SARS‐CoV‐2. Moreover, to enhance the sACE2‐S bind‐ ing interaction, we employ sACE2 variants. sACE2‐loaded sEVs exhibit typical sEVs characteristics and bind to the S protein. Furthermore, engineered sEVs inhibit the entry ofwild‐type (WT), the globally dominant D614G variant, Beta (K417N‐E484K‐ N501Y) variant, and Delta (L452R‐T478K‐D614G) variant SARS‐CoV‐2 pseudovirus, and protect against authentic SARS‐CoV‐2 and Delta variant infection. Of note, sACE2 variants harbouring sEVs show superior antiviral efficacy than WT sACE2 loaded sEVs. Therapeutic efficacy of the engineered sEVs against SARS‐CoV‐2 chal‐lenge was confirmed using K18‐hACE2 mice. The current findings provide opportu‐ nities for the development of new sEVs‐based antiviral therapeutics.
Use of sheep intervertebral disc cells to evaluate extracellular vesicle (EVs) therapies in vitro
1Inserm, Nantes Universite, 2CNRS, Universite de Paris, 3Oniris College of Veterinary Medicine
Intervertebral disc (IVD) degeneration is a leading cause of low back pain. Aiming to reduce our reliance on animal models, we set up an in vitro platform to evaluate EVs.
NP and AF
Sheep AF and NP cells exhibited differential RNA expression, notably a higher expression of COL1A1 in AF cells while NP cells favored COL2A1. Cells from older sheep also displayed a higher expression of metalloproteinases and inflammatory cytokines (IL6, CXCL8). Prolonged culture of young cells or treatments with IL‐1β or H2O2 led to a similar profile as for older cells. While EVs consistently increased basal metabolic activity at early and late passages, they had little effect on gene expression. On the other hand, direct cocultures with human ASCs profoundly affected the transcriptional profile of disc cells. Notably, both types of cocultures led to a drastic downregulation of CXCL8 in disc cells, reduced by over 60% in indirect coculture and even undetectable in direct coculture.
Development of stem cell EV therapeutics from bench to bedside
1Hanyang University ERICA
Loss or damage of tissues that result from traumatic injury and tumor resection need reconstructive approaches, such as cell/tissue transplantation or tissue engineering. Although stem cell‐based therapies have clear beneficial effects on tissue regeneration, there are still a number of concerns, such as limited survival and the reduced regenerative capacity of engrafted stem cells, as well as immune‐mediated rejection. Stem cells secrete extracellular vesicles (EV) containing various proteins and genetic materials, which could act as critical signals of cell‐to‐cell communication for tissue regeneration. Stem cell EVs provide a cell‐free therapeutic approach for the regeneration of various tissues. Small EVs, commonly called exosomes, were isolated from conditioned media of stem cells. The tissue regeneration potency of exosomes was analyzed in different animal models. Exosomes contained various cytokines and microRNAs related to each tissue development. New tissue formations were observed in the exosome injection sites of animal models. We are now developing several exosome products for the treatment of various human diseases such as osteoarthritis, liver/lung fibrosis, tendinitis, etc. We plan the future hopefully for entry to clinical trials of some exosome products. In this presentation, we hope to discuss several important issues for entry to clinical trials of exosome therapeutics such as physical/chemical/biological characterization, mass production in GMP, quality control, CMC documentation, pre‐clinical experiments, clinical protocols, etc.
NeuroRegen® scaffold for spinal cord injury repair: From animal models to clinical study
1Center for Regenerative Medicine, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences
Spinal cord injury (SCI) is a devastating injury resulting in changes in the spinal cord's motor, sensory, or autonomic functions. Following SCI, an inhibitory environment develops at the injury site to inhibit neural regeneration. We have developed collagen based NeuroRegen® Scaffold for SCI repair. NeuroRegen® scaffold was able to guide the neural regeneration along its fibers and decrease the formation of glial scars. When combined with collagen binding neurotrophic factors or antagonists to myelin‐associated inhibitors, NeuroRegen® scaffolds promoted neuronal regeneration and functional recovery in SCI animals. NeuroRege®n scaffold has been in clinical study of spinal cord injury repair since 2014. Over 100 SCI patients were enrolled and the nearly 7 year‐observation report show its safety and efficacy.
Engineering vascularized nerve tissue construct
1National Tsing‐Hua University
Timely vascularization and innervation of tissue into a patient plays an important role in the successful translation of transplanted tissues into clinically relevant therapies. Here, to shorten the time needed to vascularize engineered tissue, suitable local microenvironments provided by collagen or gelatin‐based hydrogels to support cell‐based functional vascular‐network formation have been investigated. After mixing blood‐vessel‐forming cells into suitable hydrogels with controlled material properties, these embedded cells first form vascular lumens, and quickly connect and anastomose with the patient's vasculature. After connection occurs, blood flows to every corner inside the implanted hydrogel and quickly form perfused vascularized tissue at subcutaneous site in mice within four days, which can shorten the time of angiogenesis and greatly improve the survival rate of the transplanted tissue. At the same time, host neurons migrated along growing blood vessels, and the density of neurons numbers was proportional to the blood vessels. How hydrogel properties mediated vascular network formation and further guided host neurons were investigated. Furthermore, this vascularized nerve tissue construct was served as the implant to support the repair of abdominal and skeleton muscle defect, which demonstrated that engineered vascular and neuronal network benefits to muscle tissue repair.
Spinal cord progenitor cells encapsulated in hydrogel for spinal cord injury treatment
1School of Chemical and Biomedical Engineering, Nanyang Technological University, 2School of Chemical and Biomedical Engineering, Nanyang Technological University; Lee Kong Chian School of Medicine, Nanyang Technological University; School of Materials Science and Engineering, Nanyang Technological University
Spinal cord injury (SCI) may result in the irreversible impairment of the sensory, motor and autonomic functions. Correspondingly, cell‐based therapies have been used to replace the lost neural cells. However, cell survival in the hostile in vivo microenvironment remains a challenge. Here, we will discuss our efforts in designing suitable scaffolds for the transplantation of human induced pluripotent stem cells‐ derived spinal cord progenitor cells (SCPCs). Specifically, SCPCs were encapsulated in a gelatin‐ hyaluronan hydrogel to assess SCPC behaviour with varying cell and crosslinking densities. Longer neurite extensions and more iPSC‐derived spinal motor neurons are observed in scaffolds with lower stiffness. Additionally, the SCPCs differentiated into ventral interneurons and astrocytes after 18 days of culture in the hydrogels. Hence, soft bioinks are favoured by the SCPCs for better cell viability, attachment and differentiation. Correspondingly, freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting was also implemented to achieve a small construct with microchannels for in vivo SCI treatment. Immunofluorescence staining shows the proliferation and differentiation of SCPCs into neural cells with axonal outgrowth. Therefore, 3D bioprinting may be a promising method for neural construct fabrication with precise architecture and controlled cell distribution for spinal cord treatment.
Engineered myoglobin as an oxygen vector for stem cell transplants
1University of Melbourne
The development of sophisticated biomaterials and engineered tissue scaffolds has greatly improved the efficacy of stem cell transplants by providing not only physical support, but also metabolic and anti‐ inflammatory cues. However, issues relating to poor cell survival, differentiation, and integration with the host tissue remain prevalent. This can be attributed to an insufficient supply of molecular oxygen (O2) prior to angiogenesis, culminating in a hypoxic and cytotoxic environment within the graft. Unfortunately, current methods to improve O2 homeostasis in the absence of a mature vascular system suffer from suboptimal biocompatibility due to the production of reactive oxygen species (ROS).
Here we present an approach to engineer a myoglobin:peptide hydrogel that simultaneously delivers stem cells to the brain and maintains O2 homeostasis until endogenous vascularisation can occur. Myoglobin is a natural protein found in most vertebrates that acts as an O2 reservoir during muscle contraction. It is also believed to possess ROS scavenging properties. Utilising site‐directed mutagenesis, myoglobin was modified to bind and release O2 at a rate that sustains the specific O2 partial pressure required for neural differentiation, prolonged stem cell survival, and functional integration. Additionally, by adjusting the surface charge of myoglobin, it was possible to create favourable non‐covalent interactions with the peptide scaffold to prevent protein leaching without disrupting structure or functionality. The superior biocompatibility and stimulus‐responsiveness of this hybrid biomaterial makes it an ideal candidate for a diverse range of applications, including regenerative medicine and 3D in vitro disease models.
Fabrication of ultrathin and flexible basement membrane for enhanced human iPS‐derived blood‐brain barrier model
1Ulsan National Institute of Science and Technology, 2Pohang University of Science and Technology
Given the importance of the blood‐brain barrier (BBB) for delivery of neuroactive drugs to the brain, and its role in central nervous system (CNS) function, many in vitro models have been developed in an attempt to recapitulate the barrier functions and pathophysiology of the human BBB. The basement membrane (BM) of the BBB, a thin film of extracellular matrix (ECM) underlying the brain microvascular endothelial cells (BMECs), plays a vital role in maintaining brain homeostasis. However, current in vitro BBB models have poor physiological relevance due to the difficulties in mimicking the specific biophysical and chemical properties of BM in in vitro systems. Here, we present a highly ameliorated human BBB model on nEBM, an ultra‐thin ECM hydrogel membrane, supported by a sparse electrospun nanofiber scaffold. Due to physiological relevant stiffness (∼500 kPa) of nEBM, the nEBM provides in vivo BM‐like microenvironment to BMECs. BMEC mechano‐responses of physiological relevant stiffness on the nEBM induces a stronger formation of cell‐cell junctions, which allows the recapitulation of the high physical barrier function of the in vivo human BBB. Also, it exhibits high efflux pump activity which is essential for an accurate drug screening platform. These features of the proposed BBB model enable modelling of ischemic stroke, reproducing the dynamic changes of BBB, immune cell infiltration, and drug response. Therefore, the proposed BBB model represents a powerful tool for studying drug transport and cell transmigration across the BBB, and for developing therapeutic strategies for brain diseases.
The effects of matrix‐bound nanovesicles (MBVs) derived from high‐ hydrostatic pressure decellularized tissues on neural regeneration
1Tokyo Medical and Dental University, 2Kyoto University, 3Kagoshima University, 4KM biologics Co., Ltd., 5Shinshu University
Decellularized tissues are promising materials for tissue engineering and regenerative medicine. Although several research have been studied focusing on the decellularized extracellular matrix (d‐ECM) microstructure and components to improve the current understanding of decellularized tissue functionality, the details are still unclear. Recently, the presence and characterization of matrix‐bound nanovesicles (MBVs) embedded within the d‐ECM have been reported. We have found that MBVs can be derived from high hydrostatic pressure (HHP) decellularized tissues, and they induce endothelial cell proliferation, indicating that MBVs may play a significant role in tissue remodeling. In the present study, since another essential factor for tissue remodeling is neural regeneration, the effects of HHP decellularized derived‐MBVs on neural regeneration and functional recovery were investigated. Using nanoparticle tracking assay (NTA), transmission electron microscopy (TEM), and RNA analysis, nanosized (100–300 nm) and membranous particles that contain small RNA were detected within MBVs derived from HHP‐decellularized mini‐pig brain and placenta. To evaluate the potential functionality of MBVs, the PC12 cells and mouse dorsal root ganglion (DRG) were exposed to isolated MBVs. Neurite outgrowth was observed when both brain and placenta derived‐MBVs were added. Then, MBVs were mixed with fibrin gel and applied to a rat sciatic nerve injury model. By week 6, rats implanted with MBVs mixed gel had statistically higher sciatic functional index (SFI) than those with non‐treated. Taken together, these results indicate that HHP decellularized tissue derived‐MBVs can induce nerve regeneration and may play essential role in tissue regeneration.
Interactions between macrophage and human fibroblast‐derived extracellular matrix leads to advanced wound healing
1Korea Institute of Science and Technology
Wound healing is a complex process that deeply involves the immune system, making immune cells, especially macrophages, a new target in the development of novel wound therapeutics. Herein, we investigated direct interactions between macrophages and extracellular matrix and their effect on wound healing efficacy. We fabricated human fibroblast‐derived matrix hydrogel (FDM‐gel) that was transplanted subcutaneously or into full‐thickness skin wounds in mice, followed by the analysis of immune cells responses. A mechanistic study was also performed using human monocytic cell line, THP‐ 1, where THP‐1 derived macrophage‐FDM interactions were analyzed through q‐PCR, ELISA, and western blot. Subcutaneously implanted FDM‐gel showed active cell infiltration inside the FDM‐gel, with 41.9 ± 9.9 % of the whole population were CD45+ and 7.8 ± 2.3 % among them were macrophages (CD45+/CD11b+/F4/80+). Once FDM‐gel was administered to the wounds, we observed faster and regenerative healing as histological analysis exhibited a robust regeneration of hair follicles, coupled with increased levels of VEGF and bFGF in the wound tissues. Decreased TNF‐α and increased Arg‐1 were also noticed, suggesting FDM‐gel may promote an anti‐inflammatory condition. In particular interest, mechanistic study uncovered that FDM could interact with macrophages through the integrins resulting in significantly elevated VEGF and bFGF secretion, which was suppressed when such integrins were blocked by specific inhibitors. This result was substrate‐dependent, only observed with FDM and was not reproducible with other cell types (hDFB, hMSC). Our study demonstrates that FDM‐gel is an excellent wound healing material that is biocompatible, bioresorbable, as well as immunomodulatory.
Exploring the use of regulatory T cells to promote tissue repair and regeneration
1Australian Regenerative Medicine Institute, Monash University, 2Olivia Newton‐John Cancer Research Institute
The immune system is a master regulator of tissue repair and regeneration. In particular, regulatory T cells (Tregs) have emerged as positive modulators of the tissue healing process. Tregs accumulating at a damaged site have been shown to suppress inflammation and express molecules with tissue healing properties. However, the mechanisms by which Tregs exert their pro‐healing effects across multiple tissues remain elusive.
In this study, we performed RNA sequencing of Tregs infiltrating mouse injured bone, muscle, skin and heart. We found that Tregs share a common “injury‐specific” signature and express multiple pro‐ regenerative factors. Notably, Tregs upregulated factors involved in inducing phenotypic and functional changes in monocytes/macrophages. This was confirmed by RNA sequencing of macrophages from the injured tissues of mice depleted of Tregs.
Given the central role of Tregs during tissue repair/regeneration, we explored whether local delivery of exogenous Tregs via a hydrogel promoted healing of mouse injured bone, muscle and skin. Strikingly, Tregs significantly enhanced tissue healing in all tissues tested. Mechanistically, the therapeutic effect of Tregs was attributed to a reduced accumulation of inflammatory cells and an increase in anti‐ inflammatory macrophages. We also recovered the transplanted Tregs and endogenous macrophages from Treg‐treated tissues at various time points to analyse their phenotypic changes. Our preliminary results indicate that Treg delivery promotes the upregulation of macrophage markers associated with a pro‐ healing phenotype.
Overall, we found that exogenous Treg delivery promoted accelerated healing across multiple tissues, demonstrating the potential of Tregs as a novel cell‐based strategy to improve regenerative outcomes.
A designed spacer for resolving collagen hydrogel contraction
1National Tsing‐Hua University
Collagen has been used to form cell‐mediated vascular soft tissue, but the contraction of collagen hydrogel limits its application in engineering large‐volume vascularized tissue. Many studies had tried to concentrate collagen pre‐polymer solution or synthesize collagen hydrogel crosslinked with covalent bonds to prevent its contraction. These methods indeed reduce the shrinkage of collagen hydrogels to a certain extent, but they also limited the vascular network formation in collagen hydrogels. In this study, we designed a spacer to maintain the volume of collagen hydrogel. After two days of culture, the contraction of cell‐laden collagen hydrogel was proportional to the cell density, and the resulted volume of hydrogel with the designed spacer was two‐fold greater than that without the spacer. The results demonstrated that the designed spacer did not affect the cell spreading and could resist the cell‐mediated hydrogel contraction in vitro. Interestingly, the function of pillars in the spacer not only avoided the hydrogel contraction but also control the topography of collagen hydrogel through controlling the arrangement of pillars. Moreover, to investigate whether the designed spacer could maintain the vascularized tissue volume in vivo, cell‐laden collagen hydrogels with or without a spacer were co‐ implanted into the subcutaneous site of mice for 7 days. Under the same blood vessel density, the volume of explants with the spacer was two‐fold larger than that measured in without the spacer. Taken together, integrating a designed spacer into the collagen hydrogel successfully supports cell‐mediated blood vessel formation and maintains the volume of vascularized tissue.
Trizonal, tissue‐engineered meniscus microtissues for treatment of meniscal defects in a micropig partial meniscectomy model
1Department of Biomedical Sciences, Graduate School of Ajou University, 2Department of Orthopedic Surgery, School of Medicine, Ajou University, 3Department of Molecular Science and Technology, Ajou University, 4Cell Therapy Center, Ajou Medical Center
There is a critical clinical need for regenerative medicines to treat meniscal lesions induced by degenerative meniscus tears and post‐arthroscopic partial meniscectomy. Despite some advances in the recent decade, we are still a long way from meniscus regeneration with mechanical, structural, and biochemical properties comparable to the native meniscus. To address these issues, we developed trizonal meniscus microtissues by self‐aggregating zone‐specific DMECM and autologous synovial mesenchymal stem cells. We verified that DMECM‐augmented self‐aggregation may induce the formation of a meniscus‐like construct that reproduces the features of the region‐specific meniscus in vitro by analyzing histological, biochemical, and biomechanical properties of meniscus microtissues. The construct displayed an adhesive, semiliquid‐type texture that covers meniscus lesions of different sizes and locations in a well‐established micropig partial meniscectomy model. Histological evaluation including SF‐O, collagens, and scores by the Zellner method exhibited mimicking aligned microstructure of native meniscus tissue as well as enhancement of matrix formation at meniscus microtissues implanted groups. Finally, we used tekscan to map graphically the load on the medial tibia. The data indicated that the groups including implanted meniscal microtissues had significantly lower mean and peak contact press than the defect group. These findings demonstrate that the microtissues fabricated by guiding self‐ aggregation of synovial mesenchymal stem cells and DMECM can recapitulate the properties of native meniscus tissue, offering new possibilities for clinical translation.
Biomimetic semi‐flexible hydrogel with reduced inflammation for bone defects
1Department of Dentistry, Graduate School, Kyung Hee University, 26 Kyungheedae‐ro, Dongdaemun‐ gu, Seoul 02447, 2Department of Dental Materials, School of Dentistry, Kyung Hee University, 26 Kyungheedae‐ro, Dongdaemun‐gu, Seoul 02447
Engineered 3D scaffolds must be biomechanically capable to substitute injured tissues, and have typically been made of tissue‐mimicking stiffness. However, this paradigm is shifting towards the understanding that the stiffness of scaffolds can be directly responded to by cells. Herein, we have designed a soft hydrogel to meet two criteria that can shift from soft to progressively hard tissue, similar to tissue development in nature. First, inspired by clotting of blood at the wound site, the goal is to achieve strong bonding with adjacent tissues through physical crosslinking of fibrinogen (FBG). Second, the hydrogel is needed mattress to dissipate energy such as existing collagen (COL) in the body. This scaffold promotes energy dissipation by strengthening the elastic and soft fibers of the FBG through COL to form a dense structure. As a crucial fundamental substrate, soft hydrogels initially induce rapid cell adhesion due to their soft surface. Sequentially, the cells increase to penetration inner part and make rigid themselves, and leading to bone formation. Furthermore, this hydrogel was composed of ECM‐like tissue, which could reduce inflammation in the early stage. Therefore, our dual‐function system provides a promising strategy for natural tissue‐mimicking bone regeneration by reducing inflammation, and concurrently bridging defect area through harmonizing the newly assemble cells and ECMs.
Fabrication of cell bead‐laden 3D structure via oil‐free microfluidic system for using thermoresponsive hydrogel
1Sungkyunkwan University
When microcarriers containing cells are manufactured using microfluidics, oil and aqueous materials are generally used. However, usage of oil has some issues such as large interfacial tension between oil and aqueous materials and difficulty of complete removal oil afterward. One way to overcome the issue is to utilize strong surfactants, which can negatively affect the loaded biological materials or cells. In the present study, therefore, we developed an oil‐free method using the thermoresponsive properties of gelatin methacrylate (GelMA) and microfluidic system to fabricate microfibers containing integrated GelMA‐based cell beads. The sol‐gel transition of GelMA around the physiological temperature enabled the formation of cell‐laden beads in the microfiber. This microfluidic system was then applied to a 3D printer to produce a 3D biomedical scaffold mimicking the motor unit of the muscle. To determine the relative efficiency of the newly developed structure, we used a normal cell‐printed structure, which was directly printed using the same cell density of individual cells suspended in the hydrogel, as a control. We propose that the microfluidic process is a potential solution to the low cell‐to‐cell interactions, a typical shortcoming of the non‐spheroid bioprinting process. In addition, we also suggest that the fabricated structure can be effectively utilized in multiple tissue engineering applications, including organ‐on‐a‐chip models.
Spatiotemporal modulation of skeletal muscle regeneration with varying vasculature patterns in an in‐bath bioprinted skeletal muscle tissue
1POSTECH
For the treatment of volumetric muscle loss (VML), de novo regeneration of damaged sites can be achieved by incorporation of muscle‐resident satellite cells (SC), which possess intrinsic self‐repair capacity upon regenerative stimuli such as tissue injury. However, limited long‐term maintenance (e. g. loss of quiescence) of SCs function hampers their therapeutic efficacy. Engineering an artificial SC niche with tissue‐specific stiffness and ECM composition has been suggested to overcome the addressed limitations. Another promising strategy is to mimic the SC‐endothelial crosstalk that takes place during tissue regeneration by fabrication of three‐dimensional (3D) vasculature in an implantable muscle construct. We suggest a 3D bioprinting‐based direct endothelial cell patterning method which leads to a spontaneous vascular network formation. Human umbilical vein endothelial cells, encapsulated in skeletal muscle decellularized extracellular matrix (mdECM), were directly printed into a supporting bath composed of myogenic cells and mdECM. Immunostaining for CD31 and F‐actin confirmed that printed cells remained viable after printing. With cytokine array and ELISA analysis, we observed the upregulation of myokines upon vascularization that beneficially affects SC self‐repair capacity. Moreover, incorporation of human muscular endothelial cells resulted in skeletal muscle endothelium specificity confirmed by TSPAN7 and FLK1 immunostaining. In addition, angiogenesis‐myogenesis coupling was maintained long‐term (at least 14 days) by optimizing the pattern‐pattern distance and eventually resulted in enhanced muscle maturation, confirmed by qRT‐PCR analysis of late myogenesis genes. We expect that temporal modulation of regeneration in our tissue could be effectively used for treatment of dystrophic muscles such as VML and Duchenne Muscular Dystrophy.
Effect of chondroitin sulfate concentration and matrix stiffness on chondrogenic differentiation of mesenchymal stem cells
1National University of Singapore
Due to the limited capacity of cartilage for self‐repair, a variety of biomaterials to facilitate chondrogenesis emerged as a promising approach to repair cartilage defects. Chondroinduction of mesenchymal stem cells (MCSs) in biomaterials is the key to achieve a satisfactory outcome of cartilage repair. Biomaterial stiffness is well recognized as a determinant to guide stem cell fate. While chondroitin sulfate, a glycosaminoglycan of native cartilage, has shown its potential to promote 3D chondrogenesis of MSCs, how chondroitin sulfate could affect MSCs response to matrix stiffness during chondrogenic differentiation is still unknown. Herein this study aims at 1) assessing the chondrogenic differentiation capacity of MSCs in methacrylated chondroitin sulfate (CSMA) with different matrix stiffness; 2) investigating the influence of CSMA concentration on chondrogenesis. Hydrogels composed of CSMA and gelatin methacryloyl (GelMA) with predefined stiffness were prepared. The mechanical tests showed that CSMA hydrogel was stiffer than GelMA hydrogel of the same concentration and higher CSMA concentration increased the hydrogel modulus significantly. By adjusting the degree of functionalization in CSMA and GelMa and the concentration of photoinitiator, six experimental groups were set in this study, including three groups of high modulus and three groups of low modulus. For each modulus, there were three different compositions of hydrogel with increasing CSMA concentration. MSCs were encapsulated in the hydrogel for 4‐week incubation in chondrogenic differentiation medium. The performance of chondrogenic differentiation in each group will be investigated.
Development of cultured steak by tissue engineering
1The University of Tokyo
Artificially engineered tissues have commonly been used in the fields of regenerative medicine and drug discovery. In recent years, there is a boosting trend to utilize those tissues as alternatives of livestock products such as meat and leather. Cultured meat—the engineered skeletal muscle tissue consisting of culture‐expanded animal cells—is especially attracting attention as a next‐generation food source. There are worries that meat supply by current livestock and poultry production systems will not cover global demand in the near future because the world's population is continuing to grow. In addition, the current industrial large‐scale livestock can cause environmental destruction such as deforestation, water pollution, and greenhouse gas emissions and therefore cannot be expanded any further. On the contrary, cultured meat is expected to be sustainable with lower environmental loads. In 2013, the concept of cultured meat was demonstrated by Prof. Mark Post at Maastricht University with the widely broadcasted live‐cooking and tasting event of beef hamburgers made from cultivated bovine myoblasts. However, their cultured meat at the time was made of separate aggregates of small muscle fibers like minced meat, not steak meat, in which myofibers line up in the same direction in a large length scale to define the chewy texture of whole‐cut steaks. Our group succeeded in constructing millimeter‐thick cultured steak meat with oriented myotubes. In this session, we will introduce the detailed construction method and both histological and food‐science analysis of our cultured steak meat.
Primary culture of bovine muscle cells and fabrication of aligned muscle tissues for cultured meat production
1Tokyo Women's Medical University
Alternative technology for meat production holds the potential to alleviate ethical, environmental, and public health concerns associated with conventional meat production. Cultured meat promises to become a viable alternative to animal‐based meat for the future of the food industry. In this study, we developed a primary culture method to efficiently harvest bovine muscle cells from bovine meat. This method allows the efficient collection of large numbers of primary cells from bovine cheek meat, purifies the muscle cells from the cell mixture, and then continuously grows the muscle cells in vitro. In addition, using immunofluorescence staining methods, we were able to determine the “cell quality”, including the proliferative and differentiation capability. Furthermore, to mimic an aligned structure of native muscle, the bovine muscle cells were cultured on a micropatterned thermoresponsive substrate, which were then transferred onto a fibrin‐based gel. This gel‐based culture environment promoted structural and functional maturation of the muscle cells, resulting in the production of bovine muscle tissues with sarcomere structures and contractile ability. We believe that these biomimetic features of “tissue‐engineered meat” are important for the production of future cultured meat.
Green substrates for sustainable nutrient delivery to enhance seedling development in hydroponics
1School of Materials Science and Engineering, Nanyang Technological University, Singapore, 2Center for Nanotechnology and Nanotoxicology, Harvard T.H. Chan School of Public Health, Harvard University, United States, 3School of Biological Sciences, Nanyang Technological University, Singapore, 4The Connecticut Agricultural Experiment Station, Connecticut, United States, 5Environmental Chemistry and Materials Centre, Nanyang Environment and Water Research Institute, Singapore, 6Singapore‐HUJ Alliance for Research and Enterprise (SHARE), Singapore
The world is facing an unprecedented challenge to produce enough food to feed a growing population. Faced with a continuous loss of arable land, urban farming methods have become the most promising approach to sustain food production to meet demands. However, this must be achieved responsibly with considerations made to reduce negative impacts on the environment. In particular, sustainable agriculture can be achieved by upcycling and repurposing wastes for high value applications. We have identified keratin and cellulose as two natural biopolymers that are plentiful in biowastes and can be upcycled for agriculture applications. Borrowing the concept of a tissue engineering scaffold, waste‐derived keratin and cellulose are converted into three‐dimensional, porous sponges that act as scaffolds to support seed germination and seedling development in hydroponics. Specifically, keratins extracted from human hair and cellulose nanofibers obtained from wood pulp were fabricated into composite sponges by freeze drying. These sponges exhibited microporous structure, superior hydrophilicity and excellent mechanical resilience through disulphide and hydrogen bonding. In addition to amino acid release when keratin degrades, we showed that micronutrient‐doped carbon dots can be incorporated into the sponges to further boost nutrient delivery to the plants. Functional experiments using model plant Arabidopsis and crops including Bok Choy and Arugula indicated that these sponges have the potential to be customized for enhanced crop development. Notably, our approach reduces farm wastes and nutrient loss while providing a sustainable solution for targeted nutrient delivery to crops.
Development of suspension culture method for cultured meat production
1Waseda University, 2Tokyo Women's Medical University
As the demand for meat increases due to the rapid population increase in recent years, the issue of meat production by livestock is expected to become serious. Specifically, it emits greenhouse gases, produces a large amount of breeding materials, uses a large breeding area, uses a huge amount of water, and has a strong risk of infectious diseases such as mad cow disease, cholera, and influenza. In order to solve these global problems, cultured meat produced by cell culture is attracting attention as an alternative to real meat.
Cultured meat is made by collecting muscle satellite cells and myoblasts from muscles collected from animals, proliferating them in large quantities, and then performing three‐dimensional organization. Cost is a major issue in producing cultured meat. As a method for amplifying and culturing a large amount of cells, the usual culture dish method increases the cost of culture work consumables and labor costs, and is extremely expensive. One of the cost reductions is agitated suspension culture in which cells are suspended and amplified. This is because the number of steps such as cell passage work is significantly reduced in the suspension culture, and it is possible to cope with a large scale‐up. In fact, suspension culture is adopted for cell culture for the production of biopharmacy, and it is considered essential to apply it to mass cell proliferation even in cultured meat. Therefore, in this report, we report a method for stirring and floating culture of muscle satellite cells and myoblasts.
Legume‐based scaffolds for the lab‐grown production of cultured meat
1Yeungnam University
Tissue designing methods can be utilized to make meat in vitro by growing cells obtained from little muscle tissue. This process has the potential to reduce greenhouse gas emissions and land requirements while also eliminating the need for large livestock herds. Legume‐based edible scaffold materials suitable for meat growth and subsequent commercialization should ideally be inexpensive and readily available. In this study, we designed and fabricated tofu‐based and Kidney bean (Phaseolus vulgaris)‐based scaffolds that have the potential to mimic a similar structure of the muscle tissue for the culturing of meat along with mechanical properties and nutritional benefits.
Fabricated scaffolds have shown porous microstructure with adequate interpore connectivity. The results of compressive strength, loss, and storage modulus were no significant difference compared to bovine meat. bovine myocyte cells were cultured on the surface of the scaffold for 3,7 and 14 days. There was statistical significance in the proliferation and differentiation of myotubes. These come about proposed that a legume‐based framework may be a cost‐efficient and ecologically neighborly platform, which can quicken the advancement of laboratory‐grown meat by giving a consumable substrate for bovine fawning cells.
Rapid regeneration of a neoartery with elastic lamellae
1University of Sydney
Native arteries contain a distinctive intima‐media comprised of organized elastin and an adventitia containing mature collagen fibrils. In contrast, implanted biodegradable small‐diameter vascular grafts do not present spatially regenerated, organized elastin. The elastin‐containing structures within the intima‐ media region encompass the elastic lamellae (EL) and internal elastic lamina (IEL) and are crucial for normal arterial function. Here, we describe the development of a novel electrospun small‐diameter vascular graft that facilitates de novo formation of a structurally appropriate elastin‐ containing intima‐ media region following implantation. The graft comprise a non‐porous microstructure characterized by tropoelastin fibers that are embedded in a PGS matrix. After implantation in mouse abdominal aorta, the graft develops distinct cell and extracellular matrix profiles that approximate the native adventitia and intima‐media by 8 weeks. Within the newly formed intima‐media region there are circumferentially aligned smooth muscle cell layers that alternate with multiple EL similar to that found in the arterial wall. By 8 months, the developed adventitia region contains mature collagen fibrils and the neoartery presents a distinct IEL with thickness comparable to that in mouse abdominal aorta. We propose that this new class of material can generate the critically required, organized elastin needed for arterial regeneration.
Authors: Ziyu Wang, Suzanne M. Mithieux, Howard Vindin, Yiwei Wang, Miao Zhang, Linyang Liu, Jacob Zbinden, Kevin M. Blum, Tai Yi, Yuichi Matsuzaki, Farshad Oveissi, Reyda Akdemir, Karen M. Lockley, Lingyue Zhang, Ke Ma, Juan Guan, Anna Waterhouse, Nguyen T. H. Pham, Brian S. Hawkett, Toshiharu Shinoka, Christopher K. Breuer, Anthony S. Weiss
Human cardiac tissue engineering for tissue maturation
1Tokyo Women's Medical University
Myocardial tissue engineering is indispensable for regenerative medicine and tissue modeling. We have been developing bioengineered human cardiac tissue and have succeeded to evaluate the physiological property of cardiac tissues using the direct contractile measurement system and flexible electronics. These measurement systems enabled us to clarify the pathological properties such the impaired systolic function of cardiac tissue with the LMNA p.R225X mutation.
Another issue of myocardial tissue engineering is their immaturity. Since the native myocardium is composed of several layers of aligned cardiac tissue, we fabricated aligned human cardiac tissue and the evaluated the functional property. When iPS cell‐derived cardiomyocytes were cultured on a micro‐ processed fibrin gel with inverted V‐shaped ridges, cardiomyocytes were aligned more similarly to those in the native myocardium. The aligned cardiac tissue showed the improvement of contractile properties including contractile force, maximum contractile velocity, and relaxation velocity compared with the control cardia tissue. Gene expression profiles including contractile proteins and Ca2+ handling were not different between aligned and non‐aligned cardiac tissue, suggesting that cardiomyocyte maturation might not be the principal reason for the improved contractile function in the aligned cardiac tissue. On the other hand, motion capture analysis revealed that the cardiomyocytes in the aligned cardiac tissues showed more unidirectional and synchronous contraction than the non‐aligned cardiac tissues, indicating that cardiac tissue maturation involves electrical integration of cardiomyocytes.
Herein, human cardiac tissue engineering along with the development of the functional measurement system, might be the promising platform for fabricating the more functional cardiac tissue.
A human iPSC‐based cardiac tissue model to understand age‐related heart failure
Aswathi Gopalakrishnan1, Ernst Wolvetang4, Joan Li3,
1Australian Institute for Bioengineering and Nanotechnology, School of Chemical Engineering, University of Queensland, 2Sch ool of Chemical Engineering, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, 3School of Clinical Medicine, The University of Queensland, 4Australian Institute for Bioengineering and Nanotechnology, The University of Queensland
Effect of ECM remodeling by cardiac fibroblast on the formation and maturation of 3D cardiac bundle in vitro
1Korea University, 2Professor, 3Ms., 4Ph.D.
Cardiac fibroblasts play a critical role in heart homeostasis, regeneration and disease by producing extracellular matrix (ECM) proteins and remodeling enzymes. In normal condition, fibroblast exists in a quiescent state and maintain homeostasis such as tissue structure and ECM turnover, but become activated upon stimuli such as injury, aging, or mechanical stress, leading to disease through rupture and excessive ECM production. Although it is vaguely known that cytokine and ECM production by fibroblasts influence cardiac maturation, the role of fibroblasts is not fully understood. Here, we established a cardiac bundle development model using human induced pluripotent stem cell derived cardiomyocytes (hiPSC‐CMs) and human cardiac fibroblasts (CFBs) that intrinsically induces functional and structural maturation of cardiac tissue through ECM remodeling. ECM remodeling and fibroblast activity of cardiac bundles were controlled by aprotinin, an antifibrinolytic molecule, and TGF‐β, respectively. Analysis of beating properties, beating force measurement, and mRNA expression showed that absence of CFBs or inhibition of ECM remodeling reduced the functional and structural maturation of cardiac bundles. However, increased ECM remodeling through fibroblast activation by TGF‐β increased the structural maturity, but decreased functional maturity of cardiac bundles, showing an arrhythmia‐like phenomenon. In particular, SHG analysis revealed that the expression of collagen type I in the cardiac bundles was different on fibroblast activity. Our data suggested that fibroblasts are essential for maintaining of myocardial tissue through ECM remodeling, and that differences in collagen deposition following fibroblast activity may lead to cardiac development or disease.
Hyaluronic acid infiltrated collagen type I+elastin scaffolds for myocardial repair
1University of Oxford
Tissue rejection is a common handicap in cardiac tissue engineering due to poor support of cell migration or neovascularisation. An ideal scaffold design to avoid de‐bounding as well as to support ECM formation is a crucial step for achieving this. Therefore, here, collagen type I (100%) or collagen type I and newly characterised‐elastin (90:10; 80:20; 75:25, all percentage, 1% w/v) scaffolds were fabricated by freeze‐casting, and hyaluronic acid (HA) was infiltrated to obtain interconnectivity between pores. Alamar Blue was performed to show IMR90 cell behaviour on HA‐infiltrated scaffolds. Upon proliferation, the cells were induced to differentiate into cardiomyocytes directly on scaffolds. They were then assessed by qPCR and immunocytochemistry to determine cardiac differentiation and ECM formation. Results were evaluated statistically.
The HA‐infiltration and carbodiimide cross‐linking were approved by Fourier transform infrared spectroscopy. Microstructural analysis of scaffolds on scanning electron microscopy showed that area fraction was significantly reduced by both cross‐linking and HA‐infiltration. Tensile moduli of 75:25 cross‐linked (0,11 ± 0,11) and HA‐infiltrated scaffolds (0,13 ± 0,02) were significantly the most ‘close‐fit’ candidates of porcine heart (0,11 ± 0,03). Although cross‐linking increased scaffold stiffness, both elastin content and HA‐infiltration improved bending and swelling properties. The in vitro analysis indicated that cell attachment and proliferation were better on 75:25 HA‐infiltrated scaffolds. Though inverse correlation for cardiac differentiation was obtained following induction, ECM production was significantly higher on the same design, regardless of induction.
In conclusion, both mechanical and in vitro analysis showed 75:25 HA‐infiltrated scaffolds as the more feasible construct for myocardial regeneration.
3D vascularized tissue construct using protein‐based composites
1School of Life and Environmental Sciences, University of Sydney, Sydney, New South Wales 2006, Australia, 2the University of Sydney
Vascularization of tissue constructs by incorporating complex vascular networks into three‐dimensional (3D) bioengineered constructs to achieve proper oxygenation, nutrient delivery, and waste removal after implantation remains a challenge in tissue engineering. To develop a 3D hybrid vascular system, we embedded tubular tropoelastin and silk fibroin composites (TCs) within enzymatically crosslinked gelatin hydrogel to mimic tissue constructs in customized pulsatile flow‐induced bioreactors.
The system utilizes alternative protein‐based systems. Tropoelastin is highly elastic and cell interactive, promotes endothelial cell attachment and angiogenesis, while silk fibroin confers mechanical strength to the vascular system and bestows the ability to withstand sutures and cope with higher blood pressures. The TCs were fabricated by dip coating on sacrificial ice mold then stabilized. They were subjected to analysis and quality control using techniques including scanning election microscopy, immersed in cell‐ encapsulated hydrogel which was then cast around the TC to form a hybrid system. Human umbilical vein and other endothelial cells were seeded into the TC statically and dynamically. Concurrently, human cells including dermal fibroblasts were cultured in the hydrogel. Cells in the surrounding matrix, in the vicinity of vessels, were maintained as the porous TC provided proper nutrient diffusion. The 3D vasculature system was tested in a pulsatile bioreactor, which mimicked the native tissue microenvironment and delivered the further benefit of replicating tissue performance. We conclude that this simple and versatile 3D tissue construct system allows for the growth of complex vascularized tissue models in 3D and has great potential for vascularization, for biological and pharmacological uses.
Engineering nano‐biomaterials for tissue fabrication and regenerative medicine
1Harvard Medical School/Brigham and Women's Hospital
Tissue or organ failure, because of disease, traumatic injury, or tumor ablation, is a severe condition that significantly affects the body functions of patients. In particular, large‐scale injured areas are unable to regenerate, resulting in the repopulation of the area by mainly fibrotic tissues without the recovery of tissue function. Engineering clinically relevant biomimetic tissues has emerged as a potential solution for tissue regeneration. However, a few significant challenges in tissue engineering still exist, such as recapitulating the hierarchical microarchitecture and function of native tissue in vitro, improving survival and integration of cell‐laden implants in the injured area, and consolidating multiple factors in the engineered constructs for improving tissue regeneration. To address these challenges, we are focused on developing nano‐biomaterials to recapitulate biophysical and biological properties of native ECM for controlling stem cell differentiation and maturation, achieving the multiple factors containing functional scaffolds, or improving the viability of engineered tissues after implantation at the injured area. In this presentation, we introduce multi‐functional, nanomaterial‐laden scaffolds which have antibacterial functions and can control the release of growth factors at the specific light stimulation for wound healing applications. We also propose two strategies for improving implants survival at the large‐scale injured area by integrating functional vascular networks within engineered tissue constructs or adding oxygen‐ generating micromaterials to improve the viability and integration of engineered 3D tissue constructs for regenerative medicine. Therefore, incorporating functional vasculature or oxygenating biomaterials could allow the implant to survive its non‐perfused phase and enable living implants' continuous function and maturation.
Nuclear softening expedites interstitial cell migration in fibrous dense connective tissues
1University of Pennsylvania
Dense fibrous matrices impede interstitial cell migration and subsequent repair. We hypothesized that nuclear stiffness is a limiting factor in migration and posited that repair could be expedited by transiently decreasing nuclear stiffness. To test this, we interrogated the interstitial migratory capacity of adult meniscal cells through dense fibrous networks and adult tissue before and after nuclear softening via the application of a histone deacetylase inhibitor, Trichostatin A (TSA). Our results show that transient softening of the nucleus improves migration through microporous membranes, electrospun fibrous matrices, and tissue sections and that nuclear properties and cell function recover after treatment. We also showed that biomaterial delivery of TSA promoted in vivo cellularization of scaffolds by endogenous cells. By addressing the inherent limitations to repair imposed by nuclear stiffness, this work defines a new strategy to promote the repair of damaged dense fibrous connective tissues.
A regenerative dermal template for development of the next generation artificial skin graft
1Bioprocessing Technology Institute, 2Singapore Institute of Manufacturing Technology
Artificial skin graft is an essential medical implant for skin lost emergency such as burn. Existing products aim to mimic the dermis and epidermis structure of the skin, supporting dermis regeneration and re‐epithelialization (one‐stage) or prepare a suitable wound bed for autologous skin grafting (two‐stage). Collagen is the most used material for dermis regeneration. However, current collagen templates lack the regenerative capacity, taking up to 3‐4 weeks for neodermis formation. We have developed a novel dermal template consisting of naturally derived collagen to serve as a base layer of the artificial skin graft. We demonstrated fabrication reproducibility of the template's physical characteristics (pore size, crosslink density and in vitro degradation profile), which is known to influence its regenerative capacity. Ongoing studies include the incorporation of various bio‐additives that enhance angiogenesis to further improve regeneration speed. Following POC fabrication, scale‐up manufacturing techniques will be attempted to demonstrate industrial production potential. This work is leading fabrication efforts under the Additive Manufacturing for Biological Materials (AMBM) Program, Singapore, which aspires to biomanufacture the next generation burn wound healing technology integrating regenerative dermal template, additives, relevant cells, smart sensor, bioprinting and scale‐up fabrication process.
To heal or not to heal: 3D in vitro M2 macrophage model to mimic modulation of tissue repair
1Laboratory for Immuno Bioengineering Research and Applications, Division of Engineering, New York University Abu Dhabi, Abu Dhabi, UAE
Anti‐inflammatory macrophage (M2) subtypes, namely M2a and M2c, are reported to modulate the tissue repair process tightly and chronologically by modulating fibroblast differentiation state. To mimic this process in vitro, we utilized THP‐1 human monocytic cells and a three‐dimensional (3D) collagen matrix as a biomimetic tissue model. THP‐1 cells were differentiated into macrophages and activated using IL‐ 4/IL‐13 (MIL‐4/IL‐13) and IL‐10 (MIL10). Both macrophages were characterized by both their cell surface marker expression and cytokine secretion profile. Our results demonstrated that surface markers and cytokines secretion profile of MIL‐4/IL‐13 and MIL‐10 is akin to M2a and M2c macrophages derived from human PBMC, respectively. To mimic the initial and resolution phases during the tissue repair, both activated macrophages were co‐cultured with fibroblasts and myofibroblasts. We showed that MIL‐4/IL‐13 can modulate tissue repair by controlled secretion of TGF‐β1 to induce fibroblast differentiation, while MIL‐10 macrophages secrete high amounts of IL‐10 to resolve inflammation and tissue repair processes. Besides, we demonstrate that IL‐10 can reverse myofibroblast into fibroblast phenotypes. By neutralizing IL‐10 with antibody in co‐culture with MIL‐10, no dedifferentiation of myofibroblast could be observed, emphasizing the role of IL‐10 in resolution of the tissue repair phase. Overall, our results pinpoint the importance of the co‐culture model of fibroblast and macrophages for biomimetic wound healing. In addition, our established biomimetic model can guide the development of well‐defined high‐throughput platforms for improving tissue healing and anti‐fibrotic drugs testing, as well as other biomedical studies.
Breast tissue restoration after the partial mastectomy using polycaprolactone scaffold
1Yonsei University
As breast conserving surgery increases in the surgical treatment of breast cancer, partial mastectomy is also increasing. Polycaprolactone (PCL) is a polymer used as an artifact in various parts of the human body, and it is also used to make PCL‐Collagen nanofibers through an electrospinning process. We made a defect in the mammary tissue of rat and placed three types of spherical PCL scaffolds. They were PCL only scaffold, PCL‐collagen scaffold, and the PCL scaffold pushing surrounding breast tissue into it. After 6 months of implantation, proliferation of adipocytes was significantly observed in the PCL‐only scaffold and the proliferation of collagen I in the PCL‐collagen scaffold. TNF‐α was significantly increased in PCL only scaffold, but no significant difference was observed in all of IL‐6. Through this, it showed the possibility of using it as a method to conveniently repair tissue defects after partial mastectomy of the human body.
Computational modeling of cells and extracellular vesicles for pediatric cell therapy
1Emory University
Congenital heart disease affects 9 out of every 1000 live births. Improvements in surgery have greatly aided survival of these patients, though now has created need for therapies that improve cardiac function. Many of these repairs result in abnormal physiology, putting extra strain on the heart and lead to failure. Hypoplastic left heart syndrome for example, places a large burden on the right ventricle (RV) and many of these patients will develop RV failure. In cases where RV function falls below 35%, the 18‐month transplant‐free survival falls to 30% or less. In addition to transplant, the reduced RV function can lead to cognitive and physical impairments due to end‐organ damage.
We have recently developed computational models based on pediatric progenitor cells and extracellular vesicles (EVs) that can accurately predict a variety of cellular functions related to cardiac repair. When applied to new data sets, the models can predict with >80% accuracy cellular proliferation, angiogenesis, and suppression of fibrosis and inflammation. We have now applied these to patients from our 2 ongoing clinical trials in patients with hypoplastic left heart syndrome. Using clinical data and patient multi‐omic sequencing data, we can make predictions regarding the efficacy of cell therapy, as well as identify potential mechanisms of EV‐ and cell‐mediated cardiac repair and regeneration.
Cardioprotective activity of bioinspired cell‐derived nanovesicles
1National University of Singapore, 2The University of Queensland
Cell‐derived nanovesicles (CDNs) have been recently developed as nanosized cell‐based drug delivery systems (DDS). CDNs have similar membrane composition as the cell of origin from which they are produced upon shearing cells through an extruder or spin cups fitted with membrane filters; hence, they preserve crucial features from their parent cells, which can be exploited to impart some extent of specificity towards target tissues. For example, CDNs produced from monocytes can specifically migrate and “recognize” inflamed cells at the injured myocardium after acute myocardial infarction through innate homing properties. However, the even higher accumulation in the liver reduces the effectiveness of CDNs for cardiac‐specific targeting. This study aims to incorporate an ischemic myocardium‐targeted peptide (Cardiac Homing Peptide) onto CDNs to improve the targeting specificity towards the myocardium. Monocytes can be functionalised by metabolic glycan labelling. These chemically engineered cells can then be used to produce functionalised CDNs. There is no significant difference in the size of the CDNs produced (< 200 nm) with and without metabolic labelling. The incorporation of azido‐functional groups onto CDNs provides the potential for specific labelling by fluorophores or various targeting moieties through bio‐orthogonal copper‐free click chemistry reactions. It is hypothesised that the addition of the cardiac homing peptide onto CDNs can improve the accumulation of CDNs in the heart. This will result in the improvement in the pharmacokinetic profile of CDNs (by decreasing accumulation in the liver) and broaden the potential to incorporate other forms of targeting moieties for a wider range of biomedical applications.
Extracellular vesicle therapeutics for treating inflammatory conditions
1The University of Queensland
Extracellular vesicles are naturally occurring nanoparticles that are released by all cells and serve important roles in physiological and pathological processes. Therefore, extracellular vesicles have promising applications as therapeutic agents and drug delivery vehicles. Although the medical use of extracellular vesicles is promising, clinical translation has been hindered due to inefficient, unreliable, and non‐scalable methods of isolation, for example, ultracentrifugation. To overcome these issues, my lab has developed improved techniques for isolation of extracellular vesicles, such as robust and scalable tangential flow filtration. These methods have opened up many opportunities for therapeutic uses of lipoaspirate‐derived extracellular vesicles for treating inflammatory conditions.
Ultraefficient exosome‐guided direct cell reprogramming technique
1Korea Institute of Science and Technology
Direct cell reprogramming has been hampered by some major hurdles such as meager conversion rates (∼ 10%) and the safety issue of the current major players, virus‐based methods. To implement in situ direct cell reprogramming, safe and efficient in vivo delivery of reprogramming‐inducing factors should be premised. Hence, one of our major research goals is to develop an entirely new platform technology to improve in situ direct cell reprogramming efficiency and safety. Since exosomes contain a variety of factors related to cell differentiation, growth, migration, and signal transduction, they have unlimited potential as cell conversion‐inducing factors and have versatility as their delivery vehicles with excellent biocompatibility. Rather than delivering one type of inducing factor each time, exosomes can deliver complex signals for direct cell conversion at once by transporting a group of miRNAs or other substances that can cause synergistic outcomes in direct cell reprogramming. Therefore, the exosome‐induced direct cell conversion strategy is radically distinct from virus‐based one. Exosome‐guided direct cell reprogramming could provide safe access without genetic manipulation and allow for the simple formulation of exosomal vesicles as drug carriers.
Therapeutic development of extracellular vesicles from adipose tissue‐ derived stem cells for inflammatory diseases
1Exostemtech Co., Ltd,, 2Exostemtech Co., Ltd, Department of Materials Science and Chemical Engineering Hanyang University ERICA
Mesenchymal stem cells (MSCs) derived from human tissues such as bone marrow, adipose tissue, and placental tissue are widely clinically used as therapeutic agents for tissue repair and regeneration. Human adipose‐derived stem cells (HASCs) are the most often used MSCs because of the ease of access by minimally invasive methods and ease of expansion by cell culture. Recently, it has been demonstrated that MSC‐derived extracellular vesicles (EVs) may contribute to the potential mechanisms of MSC‐based therapies. In this study, we investigated the therapeutic potential of human adipose‐derived stem cells EVs in alleviating osteoarthritis, liver fibrosis, and osteoporosis along with the mechanisms. HASCs are plastic and differentiate into various types of cells, including adipocytes, chondrocytes, osteoblasts, and neurons. For these reasons, stem cell–based therapy could be applied to induce lineage‐specific tissue regeneration by differentiating tissue cells at injection sites. Recently, we investigated whether EVs from white/beige adipogenic differentiating HASCs could provide biochemical cues for adipose tissue regeneration and browning.
Preclinical angiogenesis models for enhancing bone tissue engineering
1University of Leeds
The ability to vascularize engineered tissues is currently one of the greatest hurdles in tissue regeneration. Therefore, the research on vasculogenesis in vitro and angiogenesis in vivo is crucial for the generation of large‐scale functional tissues for clinical transplantation. This is to ensure the surgical integration of the engineered tissues into the patient's own vasculature after implantation and to increase the success rate for clinical translation. This presentation will review the different mechanisms between vasculogenesis, angiogenesis and arteriogenesis, as well as their importance on bone development, fracture healing and functional bone tissue engineering. Human dental pulp stem cells have been cultured in monolayers and in 3D scaffolds in vitro for enhancing the vasculogenesis gene expression and tube‐like structure formation, which were confirmed by the positive staining for angiogenesis markers (e.g. CD34, PECAM1 and VEGFR2) using immunocytochemistry and immunohistochemistry. Different preclinical angiogenesis models have been developed for promoting angiogenesis of tissue‐engineered constructs and enhancing functional bone tissue engineering. In a chorioallantoic membrane (CAM) culture model, the chorioallantoic membrane of growing chick embryos has been widely used for angiogenesis assay and as an explanted tissue/organ culture system providing a primary vascular invasion and similar microenvironment for the evaluation of human cells for innovative bone formation regimes. The subcutaneous implant model and bone defect model using immunodeficient nude mice/rats allow the test of human cells and provide suitable microenvironments for angiogenesis which is important for bone tissue engineering in vivo. The advantages and disadvantages of the preclinical models will be also discussed.
Tissue engineering approach to construct a vascularized 3D‐human tissue model
1Osaka University
The construction of a three‐dimensional (3D) human tissue model with blood vessels is expected to be beneficial in the evaluation of toxicity and drug efficacy in the pharmaceutical field since the interaction with blood vessels enhances cell function. We recently reported blood capillary arrays in 96 wells for the screening for blood vessel interactive molecules [1]. Interestingly, 3D‐capillary formation enhanced 10‐ 250 folds increase of gene expression of endothelial makers than those of monolayer culture of the endothelial cells. Such capillary formation has also been successfully achieved by live sinusoidal endothelial cells in vascularized liver tissues [2] and blood‐brain barrier (BBB) network formation [3], respectively, for toxicity assessments. One of the important roles of blood vessels is to deliver drugs and molecules in tubes. We developed perfusable blood capillary networks in 3D tissues by the formation of opening pores at the tissue surfaces [4]. Since the vascularized 3D tissues with opening pores were fabricated in the cell‐culture insert, 24 or 96 microwell was available for a high throughput screening assessment. Recently, this method has been applied to construct BBB networks with open pores to perform permeability assays for new modalities such as an antibody‐drug [5].
[1] M. A. Abdul Sisak et al., Small Methods
[2] Y. Naito et al., Acta Biomater.
[3] A Figarol et al. Biomed. Mater.
[4] D. Hikimoto et al., Adv. Healthcare Mater.
[5] M. Piantino et al., submitted.
Long‐term reversal of diabetes by subcutaneous transplantation of pancreatic islet cells and adipose‐derived stem cell sheet
1Asan Medical Center
Islet cell transplantation is considered an ideal treatment for insulin‐deficient diabetes, but implantation sites are limited and show low graft survival. Cell sheet technology and adipose‐derived stem cells (ADSCs) can be useful tools for improving islet cell transplantation outcomes since both can increase implantation efficacy and graft survival. Herein, the optimal transplantation site in diabetic mice was investigated using islets and stem cell sheets. We constructed multi‐layered cell sheets using rat/human islets and human ADSCs. Cell sheets were fabricated using temperature‐responsive culture dishes. Islet/ADSC sheet (AI sheet) group showed higher viability and glucose‐stimulated insulin secretion than the islet‐only group. In vivo, stable long‐term glucose control by the graft was achieved after subcutaneous transplantation of AI sheet due to enhanced capillary network formation around the sheet. Because of the adhesive properties of cell sheets, AI sheets were easily applied on the liver and peritoneal surfaces. Liver or peritoneal surface grafts showed better glucose control, weight gain, and intraperitoneal glucose tolerance test (IPGTT) profiles than subcutaneous site grafts using both rat and human islets. Stem cell sheets increased the therapeutic efficacy of islets in vivo because mesenchymal stem cells enhance islet function and induce neovascularization around transplanted islets. The liver and peritoneal surface can be used more effectively than the subcutaneous site in future clinical applications
Pre‐vascularization technique for the creation of 3D cardiac tissues
1Tokyo Women's Medical University
Direct transplantation of dissociated cells is already in clinical use as a method to regenerate myocardium and blood vessels and improve cardiac function, but it is sometimes difficult to control the shape, dimensions, and position of the transplanted cells. In an attempt to solve these problems, research to reconstruct functional three‐dimensional myocardial grafts by tissue engineering methods is currently being shown as a therapeutic approach. We proposed a unique tissue engineering technique cell‐sheet‐ based engineering, which reconstructs functional three‐dimensional tissue by layering cell sheets. A major obstacle to myocardial tissue engineering is inadequate oxygen supply to the engineered myocardium, which limits the thickness of the artificial myocardium to approximately 100um. Therefore, new techniques to control vascular growth are needed to create thicker and more functional myocardial tissue. We have developed a technique to fabricate myocardial tissue with perfusable vessels ex vivo. Using isolated tissue that can be connected to arteries and veins as a vascular bed, heart tissue was produced by stacking layered cardiac cell sheets, prepared by co‐culturing with endothelial cells, followed by supporting the tissue constructs with culture media perfused in a bioreactor. Co‐cultured endothelial cells were found to form lumens and connect to capillaries in the vascular bed to generate vessels capable of perfusing cardiac constructs. Besides, thicker engineered tissues can be produced by overlaying additional layered cell sheets. Currently, we are involved in the fabrication of 3D bio‐engineered tissue with circulatory support as an advanced therapeutic approach.
Decellularized tissue‐derived adhesive hydrogel with enhanced mechanical property for tissue regeneration
1Yonsei University, 2Dankook University
Decellularization is an attractive technique in the field of regenerative medicine because it provides tissue‐specific microenvironment by removing cellular contents for minimal immunogenicity whilst preserving the complex composition of extracellular matrix (ECM) and ECM‐associated proteins. The ECM derived from decellularized tissue can accurately mimic three‐dimensional tissue, which can be utilized in the development of hydrogels that support stem cell culture and transplantation for efficient tissue regeneration. However, conventional thermally induced hydrogels made by decellularized ECM have limitations such as long cross‐linking time and weak mechanical modulus, which have restricted their applications in tissue regeneration that requires structural integrity. Here, we developed a decellularized tissue‐derived hydrogel equipped with oxidative crosslinking chemistry. The modified ECM can be instantly crosslinked upon oxidation, leading to formation of ECM hydrogel with stronger mechanical properties and tissue adhesiveness compared to the unmodified ECM hydrogel. The hydrogel enhanced osteogenesis of stem cells and bone regeneration in a critical‐sized bone defect in mouse calvaria. Furthermore, the modified ECM can be altered into a lyophilized patch form, allowing for off‐ the‐shelf availability and long‐term storage. The patch facilitated wound healing by mediating efficient topical delivery of growth factors. These results altogether support the applicability of mechanically reinforced, adhesive ECM hydrogel for effective tissue regeneration.
Acknowledgement: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government, the Ministry of Science and ICT (MSIT) (No. 2021R1A2C3004262).
The modification of terminal end of thermal sensitive Pluronic for enhancing nasal drug deliver to brain
1National Taiwan University of Science and Technology, 2Hualien tzu chi hospital buddhist tzu chi medical foundation
Glioblastoma multiforme (GBM) is a common malignant tumor of the brain which has only 3‐4 months of medium‐term survival rate after standard care of treatments such as tumor resection, gliadel wafer and oral temozolomide. Although the anti‐angiogenic factors have showed a promising therapeutic outcome on GBM treatment, an intravenous administration is still not effective to penetrate blood‐brain barrier (BBB). However, nasal delivery of drugs is an effective option for bypassing the BBB, increasing bioavailability and reducing the obvious systemic adverse drug reactions. In this experiment, Butylidenephthalide loaded liposome was encapsulated in hydrogel for spraying in the nasal cavity and enabling to increase drug bioavailability and the chance of BBB bypassing. The hydrogel was prepared from modified temperature‐sensitive Pluronic F127 polymer. The end terminal of Pluronic F127 was chemically modified with functional groups for optimizing the mechanical stability and gelation temperature. The successful modification of the polymer was characterized by 1H NMR, FTIR and EA. The rheological test proved that the modified polymer exhibited an enhanced viscosity (1700 Pa.s) than the original one (1400 Pa.s), while the gelation temperature dropped from 33 °C to 24°C after modification. Thus, the modified hydrogel could easily transform into gel upon into the nasal cavity and effectively retained in it before being diffused to the brain. Animal nasal delivery experiments also revealed that the modified hydrogel could adhere to the nasal cavity and infiltrate the epithelial system of nasal cavity to the brain, an effective non‐invasive drug delivered for brain cancer treatment.
Preparation of doxorubicin‐liposomes loaded composite scaffolds of gelatin and gold nanoparticles for breast cancer therapy and breast tissue engineering
1Research Center for Functional Materials, National Institute for Materials Science; Sch. of Pure and Applied Science, University of Tsukuba, 2Research Center for Functional Materials, National Institute for Materials Science;
Surgical resection of breast cancers may suffer from existence of residual cancer cells and loss of adipose tissue. Elimination of remaining breast cancer cells and regeneration of the resected tissue is desirable after surgical resection. Implantable scaffolds having the capacities of anti‐cancer effect and adipose tissue regeneration in a controlled manner may solve the problems. In this study, photothermal composite scaffolds were prepared by hybridizing doxorubicin‐encapsulated thermosensitive liposomes and gold nanorods into porous scaffolds of gelatin and polyglutamic acid (Dox‐lipo/AuNR/Gel/PGA). The composite scaffolds showed porous structures with good interconnectivity. Hybridization with AuNPs rendered the composite scaffolds a good photothermal conversion property under NIR laser irradiation. Cooperating with doxorubicin encapsulated thermosensitive liposomes rendered the composite scaffolds a temperature‐dependent drug release property. The composite scaffolds could increase local temperature to ablate breast cancer cells and accelerate the release of doxorubicin to eliminate the breast cancer cells surrounding the scaffolds. In vitro cell culture and in vivo animal experiments demonstrated the anti‐ cancer effect of the scaffolds. Furthermore, after complete drug release, the composite scaffolds maintained the high viability of human bone marrow‐derived mesenchymal stem cells and promoted cell proliferation. Therefore, the photothermal composite scaffold not only produced synergistic PTT and doxorubicin therapeutic anticancer effects to efficiently kill breast cancer cells in the early stage of treatment but also served as a supporting scaffold to guide new tissue regeneration and recover tissue function in the late stage of treatment.
Delivery of a spheroids‐incorporated human dermal fibroblast sheet increases angiogenesis and M2 polarization for wound healing
1Sungkyunkwan University
Low cell engraftment is a major problem in tissue engineering. Although various methods related with cell sheets have been attempted to resolve the issue, low cell viability due to oxygen and nutrient depletion remains an obstacle toward advanced therapeutic applications. In this manner, cell therapy using fibroblasts is thought of as a good alternative due to the short doubling times of fibroblasts together with their immunomodulatory properties. Furthermore, three‐dimensional (3D) fibroblasts exhibit unique angiogenic and inflammation‐manipulating properties that are not present in two‐dimensional (2D) forms. However, the therapeutic effect of 3D fibroblasts tissue regeneration has not been fully elucidated. Macrophage polarization has been widely studied in wound healing, as it stimulates the transition from the inflammation to the proliferation phase of wound healing. Although numerous strategies have been developed to achieve better polarization of macrophages, the low efficacy of these strategies and safety issues remain problematic. To this end, we introduced a biocompatible flat patch with specifically designed holes that form a spheroids‐incorporated human dermal fibroblast sheet (SIS) to mediate the activity of inflammatory cytokines for M2 polarization and increase angiogenic efficacy. We further confirmed in vivo enhancement of wound healing with an SIS‐laden skin patch compared to conventional cell therapy.
3D printed isosorbide‐based polymer and gelatin methacrylate bilayer scaffold for osteochondral tissue engineering
1Division of Biomaterials and Tissue Engineering, Eastman Dental Institute, University College London, Royal Free Hospital, Pond Street, London, NW3 2QG, UK, 2UCL, 3Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 31116, Republic of Korea
3D‐printed airway model as a tool for studying SARS‐CoV‐2 infection and antiviral therapeutics
1POSTECH(Pohang university of science and technology), 2KRICT(Korea Research Institute of Chemical Technology)
The outbreak of the SARS‐CoV‐2 has caused the infection of numerous people, resulting in the majority of them suffering from respiratory disease. There is a need for an in vitro lung model in which antiviral drugs can be tested reliably and quickly against the novel coronavirus. A physiologically relevant respiratory model provides a drug screening platform to study SARS‐CoV‐2 infection. We recapitulated the multi‐layered human airway structure consisting of pulmonary endothelium, extracellular matrix, and airway epithelium through automated inkjet and microextrusion bioprinting. The 3D microarchitecture exhibits cell‐cell junction and mucus secretion which are the major respiratory barrier to viral infection, and also expressed ACE2 and TMPRSS2 which are known to be involved in SARS‐CoV‐2 cell entry. We investigated the response following infection with SARS‐CoV‐2 in the 3D airway model. The infection induced cytopathic effect and barrier destruction in the model over time. Virus replication was effectively inhibited when an infected 3D airway model was treated with remdesivir and molnupiravir, approved for the treatment of COVID‐19. Then the EC50 was determined for each drug in the model. The 3D‐printed airway model can be used as a tool for studying viral infection and validating the efficacy of therapeutics against other respiratory infection viruses as well as SARS‐CoV‐2.
Bioengineered mussel protein‐based multi‐layer dental implants for tooth‐mimicking interface construction
1POSTECH
Ti dental implant is directly integrated to alveolar bone without tooth‐supporting tissue including cementum and periodontal ligament (PDL). Due to the absence of periodontal tissue in peri‐implant sites, implants cannot perform biological functions and it even leads to implant failure. To overcome these limitations, periodontal tissue‐forming implants using PDL cells capable of differentiating into various cells such as cementoblasts and fibroblasts have been proposed as next‐generation technologies. In this study, we developed multiphasic scaffold, which is composed of biosilica‐coated Ti mesh and 3d‐printed porous membrane, based on bioengineered mussel adhesive proteins (MAPs) for construction of cementum and PDL tissue at dental‐implant sites. The effect of biosilica on the cementogenic differentiation of PDL cells was evaluated by the ALP activity, expression of cementum‐specific markers and alizarin red staining. The fibrogenic MAP and the well‐aligned 3d‐printed membrane induced fibroblastic differentiation of PDL cells based on expression of fibrogenic‐related markers and collagen production. Overall, the fabricated scaffold was characterized and evaluated for in vivo periodontal tissue generation using H&E and immunofluorescence staining, confirming the ability to establish natural tooth‐ mimicking structure around dental implant.
Fibroblast growth factor 2 enhances valvular interstitial cell growth on decellularised matrices
1University of Otago, 2University of Auckland
Tissue engineered heart valves are a holy grail of tissue engineering. However, the creation of a ‘living valve’ that supports a population of healthy valvular interstitial cells (VICs) has so far eluded researchers. One problem is that VICs are phenotypically unstable and will readily differentiation into contractile myofibroblasts if the biochemical and mechanical conditions aren't right. A healthy VIC population will remodel the scaffold over time, express structural matrix proteins, and avoid myofibroblast‐mediated contraction.
Recent research has shown that fibroblast growth factor 2 (FGF‐2) retains VICs in their healthy phenotype in vitro. We prepared decellularised matrices from bovine pericardium, and ‘functionalised’ the scaffold with FGF‐2 and/or heparin. We then seeded the scaffolds with valvular interstitial cells isolated from patients undergoing valve replacement surgery, and evaluated them for changes in gene expression, contraction, and cell migration. We found that ‘double’ heparin‐FGF‐2 functionalisation resulted in a pro‐remodeling gene expression profile, with significant upregulation in collagen 1, fibronectin 1, and integrin‐β1 genes when compared to non‐ or single‐functionalised scaffolds. The double‐functionalised scaffolds were significantly less contractile. Factorial analysis of gene expression also showed that neither FGF‐2 nor heparin alone was responsible for the changes, and that the synergy of the two peptides was responsible.
Overall, we show that heparin‐FGF2 functionalization of decellularised matrices creates an environment that supports proper behaviour of VICs, bringing us one step to closer to a true tissue engineered heart valve.
Cellulose nanofibers as a promising topical haemostatic agent
1Australian National University, 2The Graeme Clark Institute, The University of Melbourne
Unlike other medical technologies, the development of topical haemostatic agents has seen little progress in the last few decades, thus haemorrhage remains a major cause of death in trauma and complex surgery. Oxidized regenerated cellulose (Surgicel®), the most commonly used haemostat, have efficient biodegradability but they have poor haemostatic activity and inflammatory effect in the surrounding tissues. There is a need, therefore, for the design of superior haemostatic agents. We present cellulose nanofibers (CNFs) as a new haemostatic material that outperforms Surgicel®. CNFs were produced from cellulose in various forms (gel and sponge) using an economical chemical‐free method. In vitro thromboelastometry studies, demonstrated CNFs initiated coagulation via contact activation, reducing clotting time in healthy donors' blood (by 68 ±SE 2%), thrombocytopenic patients' blood (by 80 ±SE 2%) and heparinised blood (by 54 ±SE 2%). In an in vivo murine liver injury model, CNFs reduced blood loss by 38 ±SE 10%. In contrast to the Surgicel®, the pH‐neutral CNFs did not damage erythrocytes or impede proliferation of fibroblasts or endothelials. Although cellulose‐based materials are not degradable in the body due to the lack of cellulase enzyme, subcutaneous implantation of CNFs in mice showed slow degradation of CNFs over 8 weeks. Moreover, in contrast to Surgicel®, tissue scarring was not evident.
The lack of toxic bystander effects combined with excellent haemostatic performance, a variety of forms for topical application and the possibility of scalable environmentally‐friendly production, renders CNFs a new class of promising haemostatic materials applicable in wide‐ranging medical and veterinary situations.
Integrating extracellular matrix digest into microfibrous, elastic composite materials to provide mechanical support and redirect tissue remodeling
1University of Pittsburgh, McGowan Institute for Regenerative Medicine
Providing mechanically supportive, temporary scaffolds is a common approach utilized in surgical interventions directed across tissue types. In some cases, a sheet or patch of material is applied with the expectation that tissue healing will occur concurrent with scaffold degradation, resulting in the desired functional outcome. To improve the healing response, the controlled release of bioactive factors has been extensively studied with some notable successes. However, there can be economic, manufacturing, and regulatory disadvantages with this approach. An alternative is to leverage the inherent bioactivity of decellularized tissue. As a scaffold alone, biomaterials derived from decellularized tissue have been successfully adopted in many surgical repair scenarios. In some cases, however, the mechanics inherent to the biomaterial are limited. A hybrid approach is to physically integrate extracellular matrix digest with biodegradable elastomers to form microfibrous, elastic composite materials. These materials have tunable mechanics while still providing a scaffold with extracellular matrix components that have not been chemically crosslinked. These hybrid scaffolds were initially applied in the repair of the rat abdominal wall where processing methods to tune durability and tissue ingrowth were derived. Later, a series of studies were performed using this approach to provide temporary support to the rat left ventricular wall following a myocardial infarct. These studies showed improved functional and histological outcomes over scaffolds without extracellular matrix. In later studies the hybrid scaffold support recovered cardiac function when applied to chronic infarcts, even after extensive thinning and adverse remodeling of the ventricular wall had occurred.
Harnessing the MSC secretome for the treatment of osteoarthritis
1Inserm, Nantes Université
Osteoarthritis (OA), the most common inflammatory and degenerative joint disease, is a multifaceted rheumatic disease and a major socio‐economic problem in industrialized societies. OA is characterized by progressive cartilage erosion, subchondral bone remodeling, and synovial inflammation. Despite the disability and the significant impairment of quality of life, existing therapeutic solutions provide symptomatic relief of pain at best but fail to prevent joint damages.
The secretome of Mesenchymal Stromal Cells (MSCs), with its protective effect on chondrocytes, and its anti‐inflammatory and immunoregulatory properties, has been contemplated as a relevant therapeutic approach. Unfortunately, the issue of MSC long‐term persistence in an OA joint has been raised, considering the massive cell death after injection and cell leakage outside of the articular space. In this context, cell encapsulation in permeable hydrogels has been envisioned as a way to i) protect the MSCs and enhance their local retention, ii) provide a suitable microenvironment supporting their therapeutic potential in in a pro‐inflammatory environment, and (iii) extend the diseased tissue exposure to MSC‐ derived anti‐OA molecules. We will first review conventional microencapsulation approaches with natural polymers (hyaluronic acid, alginate) as well as droplet‐based microfluidics and micromolding ones. We will then present our recent studies where we demonstrated that alginate microparticles support human MSC ability to sense and respond to pro‐inflammatory signals (TNF/INF, pathological synovial fluids). This stimuli‐sensitive cell‐based system, able to provide an “on‐demand” release of biological factors, could pave the way of future developments for a wide variety of inflammation‐, age‐ and trauma‐ associated disorders.
Fragmented fibrous matrix directing cell and tissue fates
1Kangwon National University
Biomedical applications of electrospun nanofibrous meshes have been receive tremendous attentions because of their unique structures and versatilities as novel biomaterials. Fabricating tissue architecture‐ mimicking scaffolds is one of the major challenges in the field of tissue engineering. Electrospun nanofibers have been considered as potent techniques for fabricating fibrous scaffolds bio‐mimicking extracellular frameworks. We surface‐modified fragmented nanofibrils to control cell fate such as adhesiveness and water‐rich environments. To accomplish these task, these fibrous matrices were decorated with various ligands so that the chemically immobilized cues on nanofibrils can affect and manipulate cell fates according to the tissue and cells types. These nanofibrils induced self‐assembly of cell‐nanofibril complex or facilitated cell infiltration, that can be advantageous for 3‐D cultivation of cells in water‐rich and fibrous networks.
Epigenetic reprogramming fibroblasts through confined culture in the absence of exogenous factors
1The University of New South Wales, 2UNSW
The development of induced pluripotent stem cells (iPSC) revolutionized the fields of regenerative medicine and disease modelling in recent decades. However, the stochastic nature of the reprogramming process, its limited efficiency, and the usage of oncogenes and viral factors has hindered iPSC's clinical viability. Here, I will present a vector free approach to induce plasticity in fibroblasts with confined substrates and direct their subsequent 3D self‐assembly. An optimized combination of extracellular matrix (ECM) proteins was covalent conjugated onto polyacrylamide (PA) hydrogels of varied stiffness to control the cellular microenvironment. Pairing this ECM protein coupling with microcontact printing enabled defined interfacial topography which promoted epigenetic reprogramming of primary mouse embryonic fibroblasts (PMEF) into a pluripotent phenotype without exogenous factors. These stem‐like fibroblasts form a contractile outer layer to maintain tissue integrity while expressing both pluripotent and germ layer markers. Moreover, confined substrates primed PMEFs to detach upon confluency, forming spheroids that were capable of trilineage differentiation and were prone to 3D bio‐assembly in various biomaterial substrates. The onset of mechanically initiated epigenetic reprogramming correlated with elevated autophagy activities, which promoted cytoplasmic remodeling by reducing cell size and complexity. This work demonstrates a simple yet effective approach for reprogramming somatic cells in the absence of exogenous factors, providing insight into the correlation between autophagy and mechanically induced epigenetic reprograming.
Tonsil derived stem cells: The good, the bad and the weird
1Ewha Womans University College of Medicine
Recently stem cells are getting the spotlight in not only the cell therapy research but also almost field of medicine. The source of stem cell is roughly divided into the embryonic stem cells (ESCs) and the adult stem cells (ASCs). ESCs are derived from embryos. Therefore, there are ethical problems with getting and researching ESCs. ASCs have several advantages. Firstly, they are free from an ethical issue. Secondly the sources of ASCs are various. ASCs have been isolated from a number of adult tissues. However, mentioned ASCs have some drawbacks; donor morbidities, low cell yields, and limitation of differentiation property.
The tonsils are lymphoid tissues located in the pharynx. Tonsillar epithelium is derived from the second pharyngeal pouch (of endodermal origin) and during fetal development is invaded by lymphoid tissue (of mesodermal origin). Therefore, embryologically, tonsils could be a source of ASCs. Tonsil‐derived mesenchymal stem cells (TMSCs) with capacities to differentiate into mesodermal, endodermal and ectodermal lineages have been wildly addressed for their practical applications in many disease animal models.
Adult stem cells' differential and proliferation potency may be different according to the donors (donor dependency). The heterogeneity of each stem cells causes a decisive difference in the results from basic researches to clinical trials. However, no one have compared the stemness of each MSCs from donors with different ages or gender. In this presentation, I discussed that the heterogeneity of stemness of MSCs and consideration of its clinical aspects.
NIR fluorescence monitoring for stem cell‐based bone tissue engineering
1Wake Forest University School of Medicine, 2Ajou University, 3Harvard Medical School
Stem cell‐based tissue engineering has the potential to use as an alternative for autologous tissue grafts; however, the contribution of the scaffold degradation along with the transplanted stem cells to in vivo tissue regeneration remains poorly understood. Near‐infrared (NIR) fluorescence imaging has great potential to monitor implants while avoiding autofluorescence from the adjacent host tissue. To utilize NIR imaging for in vivo monitoring of scaffold degradation and cell tracking, we synthesized 800‐nm emitting NIR‐conjugated PCL‐ran‐PLLA‐ran‐PGA (ZW‐PCLG) copolymers with three different degradation rates and labeled 700‐nm emitting lipophilic pentamethine (CTNF127) on the human placental stem cells (CT‐PSCs). The 3D bioprinted hybrid constructs containing the CT‐PSC‐laden hydrogel together with the ZW‐PCLG scaffolds demonstrate that NIR fluorescent imaging enables tracking of in vivo scaffold degradation and stem cell fate for bone regeneration in a rat calvarial bone defect model. This NIR‐based monitoring system can be effectively utilized to study cell‐based tissue engineering applications.
Effect of tonsil mesenchymal stem cells‐derived exosomes for dry mouth after menopause
1Pusan National University Hospital, 2Pusan National University Medical Research Institute
Dry mouth that occurs after menopause significantly reduces the quality of life of the elderly. The exosomes derived from mesenchymal stem cells are being studied for application in various pathological conditions in the field of tissue regenerative medicine. This study is to investigate the therapeutic effect on salivary gland dysfunction occurring after ovariectomy using tonsil mesenchymal cells (T‐MSCs)‐ derived exosomes. The rats were acclimatized for a week and divided into the following groups: sham‐ operated rats (SHAM), rats that underwent ovariectomy (OVX), and rats that underwent OVX surgery and were simultaneously injected with T‐MSC‐derived exosomes (OVX+EXO). The rats were sacrificed 6 weeks after ovariectomy. Estradiol levels decreased in the OVX group compared with those in the SHAM group. Exosomes had no effect on estradiol levels or estrogen receptor β expression. The evaluation of pro‐inflammatory cytokines, TNF‐α and IL‐6, increased in the OVX group and decreased in the OVX+EXO group. The submandibular glands were investigated for collagen‐ and TGFβ‐associated fibrosis through immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR) analyses. The results showed that the expressions of collagen I and TGFβI increased in the OVX group but decreased in the OVX+EXO group. Moreover, to examine submandibular gland function, the levels of AQP5 and α‐amylase were determined using IHC and qPCR. AQP5 and α‐amylase expressions were downregulated in the OVX group, but improved upon exosome injection. In conclusion, T‐MSC‐derived exosomes are useful for the prevented submandibular gland dysfunction that occurs after menopause.
Vocal fold tissue engineering with stem cell spheroid, growth factor, ECM, and total artificial vocal fold
1Seoul National University Hospital
Glottic insufficiency is one of the most common contributing factors in patients who present with dysphonia. Glottic insufficiency results from the vocal fold immobility, vocal fold atrophy or soft tissue deficiency. Patients with these laryngeal disorders have symptoms of voice change, dysphagia, and aspiration pneumonia, which not only decrease the patient's quality of life, but also affects their lives. To compensate this glottis insufficiency, several materials have been devised and applied to clinical trials for several decades, including Teflon, calcium hydroxyapatite (CaHA), silicone, Gelfoam®, collagen, autologous fat, and others. But, these materials have limitations, such as inflammation, granuloma formation, migration, allergic reactions, and issue of expense. In addition, regeneration of laryngeal tissues or restoration of laryngeal intrinsic functions is not achieved with these materials.
In an effort to improve the conventional injection material, my lab developed several injection materials that have enhanced biocompability, cell attachment. Slowly released growth factor, and pDNA were incorporated for active vocal fold regeneration. Regeneration using mesenchymal stem cell spheroids were used to enhance the regeneration capacity of single stem cell. Lastly, I would like to present the hydrogel with self‐healing ability and strong tissue adhesion that can be applied as an artificial vocal fold implant
Regenerative medicine for the larynx and the trachea
1Kyoto University
Upper airway organs such as the larynx and the trachea play great roles in breathing, swallowing and voice production. As these functions directly affect human QOL, it is important to maintain these organs and their functions intact. Therefore, when these organs are damaged due to trauma or surgical injury, it is crucial how to reconstruct them.
In the reconstruction of the larynx and the trachea, both morphological and functional restoration is required. Although the conventional autologous tissue transplantation allows morphological reconstruction, it doesn't always result in functional recovery. Further, in a case with a large defect, even the morphological reconstruction cannot be completed, and patients suffer from sequelae for a long time.
In order to achieve morphological and functional reconstruction of the larynx and the trachea, various regenerative strategies have been tried all over the world. Our group have worked on the development of an artificial trachea composed of polypropylene and collagen sponge, and recently succeeded in applying the artificial trachea in a clinical setting. In this symposium, we will outline our research and introduce the remaining hurdles to overcome for ideal reconstruction of the larynx and the trachea.
Bone bioprocess mimicking materials
1Okayama University
Bone regeneration is an important research target in dentistry. So far, many treatment techniques have been developed for small bone defects, and their effects are being demonstrated. Under such circumstances, in future bone regeneration research, we are facing the questions how to improve the quality of bone tissue regeneration including ultra‐fast bone tissue engineering and acquiring regenerated bone with higher quality. We have been trying to mimic the bone development process in these decades. Here we do not follow the morphology of the bone but follow the process of bone development. Then we can observe, understand, and mimic the changes of reactions, morphologies, distributions, size, and structures of each component of bone in multiscale with more efficiency.
In our research, we have investigated the initial bone mineralization in secondary ossification center in mouse femur epiphysis and understood details of mineralization process as follows. 1) Rupture of hypertrophic chondrocytes, 2) Fragmentation of cell membrane by cell rupture, 3) Calcospherite formation at the surroundings of cell membrane nano fragments. 4) Fusion of calcospherites to enlarge the mineralized area. As a result of these understandings, it becomes possible to propose new bone fabricating methods. In order to reproduce these processes in vitro, we started to fabricate bone with a bottom‐up approach, and finally obtained mineralized tissue that is structurally almost the same as the actual trabecular bone in multiple layers. In this talk, I would like to introduce our approach of this bone bioprocess reproduction, achievements, and future goals.
Regeneration of periodontal tissue in oral and maxillofacial region
1Department of Periodontics, College of Medicine, The Catholic University of Korea
The regeneration of injured or destroyed supporting structures is one of the goals of periodontal treatment. To achieve this goal, regenerative treatment has been applied, including different bone grafts, guided tissue regeneration, use of growth factors or enamel matrix derivatives, or combinations of the aforementioned procedures. Bone graft material and a barrier membrane can be used for guided tissue regeneration. Enamel matrix derivative is made up of a variety of proteins, which are mainly produced by ameloblasts. It was discovered that an enamel matrix derivative could improve clinical attachment level and decrease probing depth. Several soft tissue grafting procedures, including connective tissue graft and free gingival graft, have been employed to achieve satisfactory root coverage. Cell therapy using stem cells can be applied for periodontal regeneration. Periodontal regeneration via various approaches will be covered in this presentation.
Scalable synthesis of whitlockite and its coated hydroxyapatite porous granules for bone regeneration in defect of rat calvaria
1Department of Oral and Maxillofacial Surgery, Dental School, Pusan National University
Hydroxyapatite (HA; Ca10(PO4)6(OH)2) is an excellent biocompatible alloplast and is known as a typical osteoconductive material. As the most abundant ceramic of bone, HA occupies 65 ∼ 75 wt % of the inorganic portion of bone. On the other hand, whitlockite (WH; Ca18Mg2(HPO4)2(PO4)12) is the second most abundant ceramic of bone, and possesses high osteogenic activity. Bone morphogenetic proteins (BMPs) are important mediators of bone regeneration involved in the proliferation of osteoblasts. This study presents a histological and radiological evaluation of the bone healing potential of WH coated HA granules (WHHAGs) in a 7 mm diameter calvarial bone defect in rats. At the 8th week after surgery, the bone volume fraction (BV/TV) of the WHHAG group was about 3.56 times higher than that of the nontreated (NT) group. In addition, the BV/TV of the WHHAGs with the bone morphogenetic protein‐2 (BMP) group was about 7.32 times higher than that of the NT group at the 8 weeks after surgery. Besides, histological evaluation confirmed that the WHHAGs promoted bone regeneration with the significantly higher expression in MTS (3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐ sulfophenyl)‐2H‐tetrazolium) assay and CCK‐8 (Cell Counting Kit 8) compared to the NT group without any expression. These results suggest that WHHAGs has considerable potential in bone regeneration.
Role of 17 β‐estradiol for the regeneration of the mandibular condyle in temporomandibular joint osteoarthritis
1Seoul National University Dental Hospital, 2Dental Research Institute, Seoul National University
Temporomandibular joint (TMJ) osteoarthritis (OA) is a degenerative joint disease caused by an imbalance of TMJ homeostasis. TMJOA is characterised by the progressive destruction of articular cartilage due to the loss and metabolic imbalance of chondrocytes, accompanied by subchondral bone destruction and synovial inflammation. Clinically, the progressive condylar resorption leads to mandibular retrusion, anterior openbite, shortened posterior facial height, and steep mandibular plane angle. TMJOA is reported to occur mainly in young women in their teens and twenties. The female dominance suggests that female hormones such as 17β‐estradiol (E2) may be potential modulators of TMJOA. However, the role of E2 in the pathogenesis of TMJOA has not been fully elucidated. In this presentation, the mechanisms of E2 for the pathogenesis of TMJOA and the regeneration of bone and cartilage of the mandibular condyle will be reviewed. The supplementation of E2 may be considered to treat TMJOA. E2 may restore bone mineral density in the mandibular condyle and increase the low resistance to masticating force. However, high doses of E2 have been reported to cause harmful effects such as inflammation in the TMJ. Therefore, it is necessary to elucidate the effect of E2 on TMJOA and to find an appropriate E2 concentration for the treatment of TMJOA. Experimental results to find the appropriate therapeutic concentration and treatment period of E2 will be reported together.
Trend of biomaterials in guided bone regeneration
1Yonsei University
Guided bone regeneration (GBR) combining different bone replacement grafts and barrier membranes has become the standard of care in the field of alveolar bone regeneration. However, there is no clear evidence for the ideal bone replacement graft or barrier membrane biomaterial. The most frequently used intervention was the combination of a deproteinized bovine bone mineral (DBBM) with a porcine natural non‐cross‐linked bioabsorbable collagen membrane; this combination is currently considered the standard of care for this regenerative approach.
Considering the characteristics of oral environment where various bacteria can easily inhabit and the reduced healing potential of elderly patients who require this procedure, GBR materials have been developed in the direction of maximizing their osteoinductive and antiinfective properties. In this lecture, these recent research trends will be presented.
DMOAD activity of ICM‐203 in canine OA model
1Yonsei University, 2ICM Co., Ltd.
While inflammation in osteoarthritis (OA) needs to be controlled to attenuate OA pathogenesis, maintenance of proper dynamics of chondrocyte physiology would also be critical to suppress OA. Due to the complicated etiology of OA, significant unmet medical needs exist for a. Nkx3.2 was initially identified as a pro‐chondrogenic factor promoting cartilage development. Our previous studies have revealed that reduced Nkx3.2 expression can be tightly linked with OA and compensation of Nkx3.2 expression can suppress OA in mouse models. In this study, ICM‐203, an AAV vector encoding a truncated form of Nkx3.2, was investigated for its DMOAD (Disease Modifying OA Drug) activity in a beagle OA model. Pre‐dose analyses of radiography revealed that beagle dogs' operated knee joint condition was comparable to KL grade 2 to 3 in human patients. After ICM‐203 administration, cartilage regeneration was evaluated by longitudinal MRI analyses. In addition, restoration of knee joint function was assessed by manual palpation, gait analysis, and SWB analyses. Current findings indicate that IA delivery of ICM‐203 is capable of regenerating functional cartilage, inhibiting synovial inflammation, and restoring normal joint functions. Considering these pre‐clinical observations, first in human trial using Nkx3.2‐based AAV gene therapy could potentially show function/pain and structural benefits for human OA patients.
Genome editing of therapeutic cells
1ToolGen, Inc., 2ToolGen
CRISPR/Cas9 is a revolutionary genome‐editing tool derived from a prokaryotic adaptive immune system. With its exceptional efficiency and specificity, CRISPR/Cas9 enabled the applications of genome editing in diverse fields of bio‐industries.
Current approaches to develop genome editing therapeutics combine CRISPR/Cas9 with existing gene therapy technologies and strategies. Thus, clinical trials ongoing worldwide mostly involve the use of CRISPR/Cas9 with proven viral gene delivery vector (such as adeno‐associated virus), and applications of CRISPR/Cas9 in proven therapeutic cell types (such as CAR‐T and hematopoietic stem cells). Types of genome editing used for therapeutic applications include gene knockout, gene correction, and therapeutic gene insertion. Among them, gene knockout is most frequently used because current genome editing technology is most suitable for introducing small indels.
In this talk, our approaches to translate the potential of genome editing in cell and gene therapy will be introduced.
Safe human genome modifications using novel retroviral vector system
1Sookmyung Women's University
Retroviral vectors offer significant benefits of efficient gene delivery and stable expression. However, their clinical use raises concerns about insertional mutagenesis and potential oncogenesis due to genomic integration preferences in transcriptional start sites (TSSs). To reduce the intrinsic genome integration preference of retroviral vectors, we attempted to perturb the structure of the viral integrase that plays a key role in determining integration sites. For this goal, we inserted DNA‐binding proteins into the internal sites of the integrase. This integrase engineering yielded multiple mutant vectors that showed significantly different integration patterns compared with that of wild‐type vector. Some mutant vectors did not prefer the key regulatory genomic domains of human cells, TSSs. Moreover, a couple of engineered vectors did not integrate into the genomic sites near the TSSs of oncogenes. Overall, this study suggests that structural perturbation of integrase is a simple way to develop safer retroviral vectors for use in clinical applications.
Preclinical safety assessments for cell and gene therapy products
1Korea Institute of Toxicology
Cell and gene therapy products are now widely developed and marketed in several countries including Korea. Their use for clinical application is increasing worldwide, where several regulatory barriers to drug approval exist. In this talk, we describe preclinical development of cell and gene therapy products for Korean market and discuss its regulatory considerations to aid successful product approval. Cell and gene based medicinal products are diverse, including cells or viral vectors, have been modified genetically or not, and so have safety concerns uniquely distinct from those of small molecule drugs and other macromolecule biologics. The regulatory body reviews how the product is produced, proof of potential for therapeutic efficacy, and then focus preclinical safety of the products before IND approval. Preclinical safety considerations for cell and gene therapy products include systemic toxicity, biodistribution, persistence and immunotoxicity in relevant animal species, and are the critical elements need to be addressed prior to intended clinical trials. In this review, we summarize cell and gene therapy products, discuss their preclinical experiences and safety concerns, and also describes regulatory frameworks for developers to support product development and IND application.
Gene delivery system for the treatment of type 2 diabetes mellitus
1Hanyang University, 2Korea national university of transportation
We investigated the effect of dose quantity on the therapeutic efficacy of oral GLP1 gene therapy. Using a multimodal gene complex (GLP1/PTCA), we showed glycemic improvement drawn for up to 1 week in three progressives diabetic mouse models. To the best of our knowledge, this is the first report of any dosage form of GLP1 agonist for the most extended period of glucose control using a minimal quantity of genes (< 0.5 mg human equivalent dose). Since Rybelsus® is the only FDA‐approved oral GLP1 agonist that needs to be taken with 7 or 14 mg daily, oral GLP1/PTCA may provide the first once or bi‐monthly oral treatment option for diabetic patients according to allometric principles.
Engineering cognitive biological machinery
1University of Illinois at Urbana‐Champaign
Modern machines and their components are assembled and man‐made. They cannot self‐ assemble, heal, or learn. With increasing advances in engineering of living cells, it is now possible to envision a future where machine components will consist of living cells and engineered scaffolds. The cells may include neurons for sensing the environment, storing and processing information, and learning with basic hallmarks of cognition. These machines may employ muscle cells for actuation. Such biohybrid machines may self‐emerge, heal, acquire homeostasis, and other feats that are no possible by solid state machinery, since the components of biohybrid machines will carry million years of evolutionary footprints. How can we design such machines? What are the rules of interaction between the cells, and between the cells and the engineered scaffolds? How long will they live? In this talk, we will discuss a few examples of biological machines consisting of neurons and muscles, assess their characteristics, evaluate their functional performance, study their emergent properties, and search for signatures of cognition. We will then highlight some of the lessons learnt from these examples and summarize challenges and opportunities in the field of cognitive biohybrid machinery.
Dual‐crosslinkable alginate hydrogel: use as a stem cell‐laden bioink and as a supporting slurry for generation of high resolution and fidelity engineered tissues with complex geometries
1University of Illinois at Chicago
Recently, 3D bioprinting has been explored as a promising technology for biomedical applications with the potential to create complex structures with precise features. Here, two unique biofabrication applications of a dual‐crosslinkable alginate hydrogel are presented.
Cell encapsulated hydrogels have been widely used as bioinks for 3D bioprinting. However, since most hydrogel‐based bioinks may not allow rapid stabilization immediately after 3D bioprinting, achieving high resolution and fidelity to the intended architecture is a common challenge in 3D bioprinting of hydrogels. We have utilized shear‐thinning and self‐healing ionically crosslinked oxidized and methacrylated alginates (OMAs) as a bioink, which can be rapidly gelled by its self‐healing property after bioprinting and further stabilized via secondary crosslinking. It was successfully demonstrated that stem cell‐laden calcium‐crosslinked OMA hydrogels can be bioprinted into complicated 3D tissue structures with both high resolution and fidelity. Additional photocrosslinking enables long‐term culture of 3D bioprinted constructs for formation of functional tissue by differentiation of encapsulated human mesenchymal stem cells.
In a second application of the OMA, we present a bioprinting strategy capable of printing a cell‐only bioink using a liquid‐like solid OMA microgel supporting bath. The microgel supporting bath initially allows 3D printing of cell‐only bioinks and maintains the 3D printed constructs after photopolymerization. The printed human cell‐only bioink can assemble together and differentiate down tissue‐specific lineages with precisely controlled microarchitectures. This bioprinting strategy permits printing of cell‐only bioinks and provides a platform to generate biomimetic cellular condensation‐based engineered tissues with defined geometries.
The effect of hydrophobicity to the cell growth and behavior for tissue regeneration and cell therapy
1KAIST
3D in vitro cancer models have emerged as a promising tool for various cancer‐related applications. Here, it is demonstrated that the proteins adsorbed on the culture substrate significantly influence the characteristics of the cancer cells. A series of polymers are prepared for the precise control of the surface hydrophobicity of the culture plate. Cancer cells cultured on the polymers exhibit distinct morphological transitions ranging from monolayer to spheroid with entirely different characteristics depending on the surface hydrophobicity. The poly (cyclohexyl methacrylate) surface of the highest hydrophobicity tested in this study strongly attracts albumin from the media and induces conformational changes, leading to the formation of spheroid with the most enriched tumorigenic properties. It is believed that this finding can provide new insights when selecting the experimental strategy to appropriately mimic the complex interplay between the cancer cells and proteins.
Hydrogel‐based direct contact pressing culture method to manipulate shape and function of adherent cell
1Korea Institute of Science and Technology
Cell‐manipulating technologies that selectively control cellular behavior in a specific region at the nano/micrometer scale provide a model system for studying cell behavior, such as cell‐to‐cell, cell‐to‐ surface, or cell‐to‐matrix interactions, to promote increased understand of cellular mechanotransduction. We developed a direct contact pressing culture method (DCP) that allows manipulation of the shape and function of adherent cells on commercially available tissue‐culture polystyrene (TCP) by leveraging enzymatically degradable hydrogels, which offer not only precise chemical/physical stimuli but also a temporal three‐dimensional environment. Here, alginate hydrogels with different chemistry and structures are used to manipulate cell shape for DCP treatment. As a proof of concept, targeted muscle cells adherent to TCP exhibited a reshaped structure when the physical stimuli were applied with the hydrogels. When human primary chondrocytes were stimulated by DCP for only two days, the original shapes and properties of chondrocytes were maintained, and recovery of intrinsic properties was observed even in aged cells which lost their original cell properties. These results indicate that the unique in vitro cell‐culture approach presented here might be useful for investigating relationships between cells and physiological extracellular matrix environments, which can closely mimic the native tissue microenvironment.
Empowering engineered muscle for pump‐bot
1University of Illinois at Urbana‐Champaign
In a multicellular engineered living system, cells perform their intended functions not individually but collectively by forming three‐dimensional structures to evolve. These structures are composed of clusters of cells, which may be thought of as biological machines. They exhibit complex functional behaviors as they interact with each other and their environment through active or passive cell‐cell and cell‐matrix interactions. Therefore, the aging triggered by DNA damage and reactive oxygen species in any cells of the multicellular system would propagate easily through the entire system. This talk will discuss the aging‐related muscle degeneration and neural denervation process using a neuromuscular tissue model in which motor neurons innervate skeletal muscle. In addition, we will introduce a method to retain homeostasis and, in turn, prevent neural denervation and muscle volume loss.
Scalable delivery of highly proliferative co‐cultured skin cells in 3D GelMA core‐shell microspheres
1School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore.
Split‐thickness autografting remains the gold standard in the management of burn wounds to date. However, this method is greatly limited in patients with extensive burn wounds where healthy donor sites are scarce for skin graft harvesting, resulting in delayed wound closure, detrimental wound infection and even death. This work thus entails the scalable delivery of highly proliferative co‐cultured skin cells in GelMA core‐shell microspheres toward large burn wound healing. Uniform GelMA core‐shell microspheres with an average diameter of 382 ± 26 μm were first successfully fabricated by co‐axial electrospraying 10% GelMA‐DS40 and 10% GelMA‐DS90/0.5% alginate as the core and shell solutions. Human dermal fibroblasts and human keratinocytes were then encapsulated in the core and shell compartment of the GelMA microspheres at a skin‐equivalent ratio of 1:5. Through in‐vitro studies, the platform was found to be able to support the growth of encapsulated cells over days in their respective core‐shell compartment, with high cell viability of 74%. It was then discovered that this confined co‐ culture system could significantly enhance the proliferation of keratinocytes, as compared to when mono‐ cultured alone. The skin cell‐laden GelMA core‐shell microspheres were found to naturally degrade after 7 days of co‐culture and the released cells were able to subsequently attach and expand into a large epithelial‐dermal sheet with their proliferative capacity retained. The scalability of electrospraying skin cell‐laden GelMA core‐shell microspheres was then confirmed, where a high yield of microspheres that can cover 70% of a 9cm dish was achieved in 3 minutes of fabrication time.
Supramolecular injectable controlled peptide delivery hydrogels for diabetic wound healing
1Pohang University of Science and Technology
Diabetic wound patients are often exposed to bacterial infections with delayed healing process due to the hyperglycemia in the damaged skin tissue. Antimicrobial peptides (AMPs) have been investigated for the treatment of infection induced diabetic chronic wound healing, but the low stability and toxicity have limited the further applications of AMPs for diabetic chronic wound healing. Here, we developed a precisely controlled AMP releasing injectable hydrogel platform (Gel‐AMP), which can respond to infection related materials, matrix metalloproteinases (MMP) and reactive oxygen species (ROS). Injectable hydrogels were prepared by the mixing of hyaluronic acid modified with cyclodextrin (HA‐ CD) and adamantane (Ad‐HA). AMP, KR‐12 peptide, and Ad‐HA were conjugated with a cyclic peptide linker composed of MMP and ROS cleavable sequence (Ad‐HA‐AMP). Remarkably, only when the Gel‐ AMP was exposed to both MMP and ROS simultaneously, AMP was released from the Gel, enabling the controlled release of AMP without causing cytotoxicity. In addition, we confirmed the enhanced serum stability of Ad‐HA‐AMP conjugate. The antimicrobial activity of Ad‐HA‐AMP was maintained much longer than that of the native AMP. Finally, we could demonstrate the greatly improved wound healing effect of Gel‐AMP with the enhanced safety of Gel‐AMP for the treatment of infection induced diabetic chronic wounds.
Phlorotannin‐incorporated nanofiber exhibits cytocompatibility and accelerates hyperglycaemic wound healing
1Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, 2School of Biosciences, Faculty of Health and Medical Sciences, Taylor's University, 47500 Selangor, 3Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000 Kuala Lumpur
The lack of effective diabetic wound dressings has been a significant problem in diabetic foot ulcer management. In this study, we aim to establish a phlorotannin‐incorporated nanofiber system and determine its potential in accelerating hyperglycaemic wound healing. Preliminary screening was done to determine the effective dose of phlorotannin on hyperglycaemic wound healing without compromising cell viability and proliferation. Different concentrations of phlorotannin (0.01wt%, 0.1wt%, 1wt%) were incorporated into the polyvinyl‐alcohol/polyvinylpyrrolidone (PVA/PVP) fiber matrices via electrospinning. The safety application of phlorotannin nanofiber was validated by 3‐(4,5‐ dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium‐bromide (MTT), Live/Dead assay, and Ki‐67 immunofluorescence. The ability of phlorotannin nanofiber to support wound closure was determined by 2D wound scratch assay and immunofluorescence staining of wound healing‐correlated proteins (Collagen‐I and Cytokeratin‐14). Our results demonstrated that administration of 0.01μg/mL phlorotannin significantly improved hyperglycaemic wound healing in both human dermal fibroblasts and human epidermal keratinocytes while maintaining their viability and proliferation. We also validated phlorotannin nanofiber as a non‐cytotoxic wound dressing as it supports the viability, attachment and proliferation in both cells. In parallel to the phlorotannin compound, administration of 0.01wt% phlorotannin nanofiber significantly ameliorated the impaired hyperglycaemic wound healing. Immunofluorescence staining of Collagen‐I expression in fibroblasts indicated their ability to synthesize matrix components on the phlorotannin nanofiber, whereas Cytokeratin‐14 expression in keratinocytes demonstrated their mitotically active and proliferating state on the phlorotannin nanofiber. Altogether, our work herein displays the potential of phlorotannin nanofiber as a diabetic wound dressing.
Type II collagen‐specific regulatory T cell‐inducing nanoparticle for osteoarthritis
1Seoul National University, 2The Catholic University of Korea
Local inflammation in the joint is considered to contribute to the progression of osteoarthritis (OA). Here, we describe an immune‐modulating nanoparticle for OA treatment. Injection of lipid nanoparticles (LNPs) loaded with type II collagen (Col II) and rapamycin (LNP‐Col II‐R) into OA mice effectively induced Col II‐specific anti‐inflammatory regulatory T cells, significantly increased anti‐inflammatory cytokine expression and reduced inflammatory immune cells and proinflammatory cytokine expression in the joints. Consequently, LNP‐Col II‐R injection inhibited chondrocyte apoptosis and cartilage matrix degradation and relieved pain, while injection of LNPs loaded with a control peptide and rapamycin did not induce these events. Adoptive transfer of CD4+CD25+ T cells isolated from LNP‐Col II‐R‐injected mice suggested that Tregs induced by LNP‐Col II‐R injection were likely responsible for the therapeutic effects. Collectively, this study suggests nanoparticle‐mediated immune modulation in the joint as a simple and effective treatment for OA.
Development of polymer therapeutics that selectively disrupts the cell membrane in the tumor microenvironment
1Osaka university
Drug delivery system (DDS) that realizes the selective accumulation of drug in the tumor site via enhanced permeability and retention (EPR) effect have been developed in cancer therapy [1]. However, DDS requires intracellular delivery with multi‐step process, resulting in an insufficient therapeutic intervention. To overcome this challenging, we have previously reported a 4‐arm poly(ethylene glycol) functionalized with deoxycholic acid (DCA), a drug‐free cancer therapeutics, named molecular block (MB).[2] MB formed hydrophobic self‐aggregates in the acidic tumor microenvironment (pH = 6.5) and induced the cell death by the cell membrane disruption. However, MB showed the considerable cytotoxicity even at neutral pH due to pH‐independent aggregation. In the present work, we aimed to improve the acidic environment selectivity of MB by optimizing the bile acid incorporated in MB. We screened bile acid candidates, DCA, chenodeoxycholic acid (CDCA), cholic acid (CA) and ursodeoxycholic acid (UDCA) based on their cytotoxicity under neutral and acidic pH. The results revealed that UDCA showed the selective cytotoxicity in the acidic tumor environment. Thus, we developed a novel MB composed of polyvinyl alcohol (PVA) conjugated with UDCA (PVA‐UDCA). The PVA‐UDCA showed more selective cytotoxicity at the acidic pH compared to PVA conjugated with DCA. The results would indicate that PVA‐UDCA functions as a polymer therapeutics with high selectivity for cancer cells.
[1] Davis, M. E. et al., Nat. Res. Drug Discov.
[2] Nakatsuji, H. et al., Mater. Horizons.
Delivering micro‐RNA (miRNA) via β‐peptide derived biomaterial to treat ischemic stroke
1Department of Pharmacology, Monash University
Stroke is the world's second leading cause of mortality accounting for 11% of all deaths and is the third biggest cause of disability worldwide. To date, the only therapeutic avenue for patients presenting with stroke is recombinant tissue plasminogen activator (rt‐PA). However, due to increased risk of hemorrhage, only less than 10% of patients are eligible for the treatment. Therefore, new therapeutic strategies that limit post‐ischemic damage and promote recovery are desperately needed.
There are numerous apoptotic signalling mechanisms that are initiated following a stroke. We aim to target these pathways by utilizing a miRNA antagomir. However, these molecules have poor pharmacokinetic profiles which make them unsuitable as a therapeutic. Therefore, we have tailored a hydrogel for the controlled release of this antagomir.
We have created bespoke β‐peptides that self‐assemble into injectable hydrogels with varying ratios of positive charge to enhance the electrostatic interactions between hydrogel and miRNA. Characterisation of miRNA release shows sustained and controlled delivery of genetic products. In vitro testing also confirmed that released nucleotides can be efficiently absorbed by neural cells. In vivo testing has been performed by intracerebral injection of antagomir loaded hydrogel in a mouse model of ischemic stroke. Immunostaining, apoptotic gene expression and motor functional test have been used to determine therapeutic efficacy.
Dual ligand functionalized nanosystem for prolonged and enhanced inflammation targeting in dermatitis
1Gwangju Institute of Science and Technology
Inflammation is associated with various diseases including auto‐immune disease and allergy. So, early diagnosis and detection of the inflammatory sites in the body are desired to prevent further progress into a fatal disease. Many studies have tried to develop more accurate and sensitive detection systems targeting inflammation. In general, surface functionalization of nanosystems with appropriate ligands is considered to be the most efficient way to enhance the target selectivity and sensitivity. We explored the efficacy of dual ligand functionalization of a polymer nanosystem for targeting inflammatory tissue. The dual ligand consists of an inflammation‐specific ligand and chitosan as a less sensitive but more general ligand. Using a skin hypersensitivity model caused by DNFB (1‐fluoro‐2,4‐dinitrobenzene), we systematically compared the in vivo targeting characteristics of a given nanosystem with no ligand, either one ligand or both ligands. Chitosan allowed longer circulation in vivo, and inflammation‐specific ligand endowed sensitivity and specificity to the inflammatory tissue. As a result, faster, stronger, and prolonged detection of the inflammatory site was possible by the dual ligand functionalized nanosystem compared to all other control groups. Also, dual ligand functionalization could reduce the non‐specific accumulation in other organs such as the liver. In conclusion, the synergistic role of both ligands was confirmed. The present system can be further applied as a multi‐functional drug delivery system that can have a therapeutic effect as well as a target for various inflammation diseases after loading proper drugs.
Engineering self‐assembling peptide scaffolds for controlled delivery of viral vector serotypes
1ACRF Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, ANU College of Health & Medicine, Australia. College of Engineering and Computer Science, School of Engineering, Australia, 2ACRF Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, ANU College of Health & Medicine, Australia. Research School of Chemistry, ANU College of Science, Australia. The Graeme Clark Institute, The University of Melbourne, Melbourne, Australia. Department of Biomedical Engineering, Faculty of Engineering and Information Technology, The University of Melbourne, Melbourne, Australia. Melbourne Medical School, Faculty of Medicine, Dentistry and Health Science, The University of Melbourne, Melbourne, Australia., 3iMPACT, School of Medicine, Deakin University, Waurn Ponds, VIC 3216, 4The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Melbourne, VIC 3010, Australia, 5Translational Vectorology Unit, Childrenu2019s Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia. Vector and Genome Engineering Facility, Childrenu2019s Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia. Military Institute of Medicine, Laboratory of Molecular Oncology and Innovative Therapies, 04‐141 Warsaw, Poland., 6School of Human Sciences, The University of Western Australia, and Perron Institute for Neurological and Translational Science, Perth, WA 6009, Australia, 7College of Engineering and Computer Science, School of Engineering, Australia
To date, adeno‐associated viruses (AAVs) have been employed in hundreds of clinical trials. Although many of these have demonstrated encouraging outputs, there are some significant limitations that reduce the efficacy of gene therapy. The major hurdles that must be overcome include, neutralisation by the host immune system, low transduction efficiencies, and virus distribution to the distant regions. This highlights the need to explore alternative approaches, such as biomaterials that are capable of programmed targeted delivery to achieve expression precisely at the site of therapeutic need 1‐3.
Here, were compared 3 rAAV serotypes (AAV2, AAV5, and AAVDJ) assessing their efficacy for deployment post brain insult. To achieve this, we tested all serotypes on primary cortical neurons and primary astrocytes, in addition to a human astrocyte cell line. We also tested all serotypes in the striatum of mice. Our in vitro and in vivo results demonstrated that the highest rates of transduction of all cell types (neuron and astrocytes) was achieved with the rAAVDJ. Next, we rationally designed a library of Fmoc‐ SAP hydrogels to demonstrate an ability to program the delivery of each serotype. We highlight an ability to reliably controlled the strength of secondary interactions between the vectors capsule proteins and the peptide fibres that composes the scaffold. We exploited this interaction to manipulate each of the viral vectors release kinetics, achieving burst, sustained and delayed release. Overall, we demonstrate a versatile hydrogel delivery tool that has the capacity to control the delivery genetic material, optimising it for different injury states.
Sustained local release of PPAR‐gamma agonist from 3D fabricated biodegradable nerve guide conduits
1University College London, 2Universities of Greenwich and Kent
Objective. The recognition that the Rho/Rho‐associated kinase (ROCK) pathway regulates neuronal growth has unlocked the potential for the treatment of peripheral nerve injuries (PNIs) through delivering drugs locally to activate the upstream receptors such as peroxisome proliferator‐activated receptor gamma (PPAR‐gamma). Utilizing ibuprofen as a potential agonist of PPAR‐gamma has shown to enhance neurite elongation in rats when administrated systemically. However, such necessity for sustained administration of the dose over the regeneration period remains a major challenge especially when the PNI is severe, and the condition requires a nerve guide conduit (NGC) to bridge between the injured nerve endings. Controlled local release of the drug through a biodegradable NGC could potentially allow sustained dosing over this prolonged time. Approach. In this study, a biodegradable ibuprofen‐loaded polycaprolactone/polylactic acid (PCL/PLA) NGC was fabricated using a customized 3D printer. The ratio of PCL to PLA of the NGC was varied and the physicochemical properties of the final products along with the consequent impact on drug release were studied. Main results. In‐vitro dissolution studies over 4 weeks show that the ratio of PCL to PLA particularly regulates the drug release performance and linear sustained release is achievable as long as the PCL contribution is kept below a threshold value that depends on the drug loading percentage. Significance. This study unveils the optimized 3D printing environment and material formulation that can locally deliver ibuprofen in a sustained manner over the regeneration period, which could serve as a desirable NGC candidate for the treatment of PNIs.
Treatment development research for tracheomalacia to promote the tracheal cartilages
1The University of Tokyo
Tracheomalacia is a condition that causes airway obstruction from neonatal period, and when sudden death is assumed, surgical treatment such as tracheotomy, outside stenting is done. In mild cases, medical conservative therapy is prioritized, which takes several years. The cause of the tracheomalacia is regarded as the immature cartilage. There is no therapeutic medicine which can promote the trachea growth and patency for tracheomalacia. We speculated that rapid growth of tracheal cartilage induced by basic fibroblast growth factor (b‐FGF) could act as a novel treatment for tracheomalacia. And, basic research were performed to reveal our hypothesis.
1. Slow‐release b‐FGF administration from outside of trachea enlarges the tracheal lumen and thickens the cartilage in a dose‐dependent fashion after 4weeks of administration in rat model. The growth‐enhanced trachea was more resistant to collapse, suggesting that slowly released b‐FGF might be useful in patients with severe tracheomalacia.
2. Intra‐tracheal injection of slowly released b‐FGF or b‐FGF in the enough one shot dose range used significantly promoted the growth of tracheal cartilage after 4weeks of administration in rabbit model. And, a single injection of b‐FGF at the inner side of trachea during infancy continues its artificially accelerated tracheal generation into adulthood with rabbit model.
The evidence presented herein indicates that b‐FGF may exert an effect on patients with tracheomalacia. Nowadays, drug industry develops the injectable b‐FGF formulation, and diagnostic quantitatively criteria and the tracheomalacia severity classification based on the endoscopic video image are going to be created in our laboratory.
Human microphysiological systems for disease modeling and drug screenin
1Johns Hopkins University
My laboratory research focuses on the mechanobiology of human diseases and the development of human organ/tissue‐on‐a‐chip platform technologies for disease modeling, drug screening, and precision medicine. In this talk, I will introduce human microphysiological systems developed in our laboratory, including microphysiological model of tumor invasion, micro/nano‐fabricated platforms for drug‐induced cardiotoxicity, and engineered heart tissue chip for disease modeling and space biology. Using our multi‐ scale biofabrication tools in combination with patient‐derived induced pluripotent stem cells and cancer cells, I will highlight how our biomimetic models helps to gain a better understanding of the structure‐ function relationship in complex 3D tissues and serve as emerging platforms for disease biology studies and biotherapeutic development.
3D bio‐printing systems for tissue engineering applications
1Sungkyunkwan University
The achievement of tissue engineering can be highly depending on the capability to generate complicated three‐dimensional (3D) micro/nano‐structures. So, various fabrication techniques that can be used to precisely design the architecture and topography of scaffolding materials will signify a key aspect of multi‐functional tissue engineering. Previous methods for obtaining scaffolds based on top‐down are often not satisfactory to produce complex micro/nano‐structures due to the lack of control on scaffold architecture, porosity, and cellular interactions. However, the 3D bio‐printing method can be used to design sophisticated 3D tissue scaffolds that can be engineered to mimic the micro/nano‐architecture of tissues, using computer aided approach. Also, in recent, the method has been modified and optimized to fabricate 3D scaffolds using various natural biopolymers (collagen, alginate, and chitosan etc.). Variation of the structure and polymer concentration allowed tailoring the physical and biological properties of the scaffolds. In this presentation, the 3D bio‐printing methods including a cell‐printing process for attaining highly bioactive and functional scaffolds for tissue engineering applications will be introduced. Moreover, various in vivo and in vitro results will show that the fabricated scaffolds can carry out their structural and biological functionality.
Development of microenvironmental conditioned biomimetic model in age realted macular degenerations
1School of Medicine, Kyungpook National University, 2Kyungpook National University Hospital
Eyes are one of the most important organs in the body, detect light and convert it into chemical signals. Several abnormal conditions are related to ocular diseases, including cataracts, glaucoma, and retinal degeneration. Visual impairment or loss is not a rare condition and significantly affects quality of life. Of the conditions, age‐related macular degeneration (AMD), is quite common and causes substantial vision loss in the elderly population. The development of experimental models of AMD has contributed to elucidating its pathophysiological processes and screening drug candidates. Currently, several in vivo experimental models have been suggested, but there is no appropriate in vitro model related to the development and progression of AMD has not been presented.
In this presentation, a newly developed in vitro 3D RPE‐BM‐CC complex biomimetic system through a nanofiber membrane will be presented. This in vitro biomimetic system was designed to be more physiologically similar to the human retina‐choroid environment. Microenvironmental stress using lipofucsin‐like material and blue light was adopted to induce similar conditions of AMD. The in vitro AMD model can be a standardized model for finding new treatment modalities and studying pathological progression of AMD
Bioimaging and nanomedicine in tissue engineering and regenerative medicine
1Harvard Medical School
Cell‐based therapies hold great potential in regenerative medicine to treat a wide range of human diseases, yet the mechanisms responsible for cell migration and homing are not fully understood. Emerging molecular imaging technology enables in vivo tracking of transplanted cells and their therapeutic efficacy, which together improves the clinical outcomes of cell‐based therapy. Particularly, optical fluorescence imaging provides highly sensitive, safe (non‐radioactive), cost‐effective, and fast solutions for real‐time cellular trafficking compared to other conventional molecular imaging modalities. This talk will be focused on recent progress and advances in optical fluorescence imaging for cell‐based therapy and regenerative medicine. Several types of fluorescent imaging probes and their labeling methods with a special focus on cardiovascular disease, cancer immunotherapy, and tissue regeneration will be discussed. Particularly, a new pharmacophore design strategy “structure‐inherent targeting” will be focused on where tissue‐ and/or organ‐specific targeting is engineered directly into the non‐resonant structure of a near‐infrared fluorophore, thus creating the most compact possible optical contrast agent for bioimaging and nanomedicine (Nat Med. 2015). The biodistribution and targeting of these compounds vary with dependence on their unique physicochemical descriptors and cellular receptors, which permit 1) selective binding to the target tissue/organ, 2) visualization of the target specifically and selectively, and 3) curing options such as image‐guided drug delivery and theranostics. Finally, the advantages and limitations of optical imaging‐based cell tracking strategies along with the future perspectives to translate this imaging technique for a clinical realm will be debated.
Bio 3D printing for tissue engineering
1Hallym University, Nanobio Regenerative Medical Institute
Tissue engineering is rapidly developing in the field of regenerative medicine as a method to solve problems related to tissue and organ damage caused by diseases, trauma and aging. 3D bioprinting enables fast and precise spatial patterning of cells, growth factors and biomaterials to create complex 3D tissue structures. However, many challenges still need to be solved before the complexity of a complete functional organ can be recreated by stacking biological elements within a 3D structure. Bioink is one of the main elements of 3D bioprinting, in which biomaterials can be 3D printed with living cells to imitate native tissue characteristics. To fabricate an implantable 3D hydrogel structure from bioink, it should have cell encapsulation capabilities and printability. After printing, the structure should have cytocompatibility, tissue‐like mechanical properties and biomimetic properties. To achieve these characteristics, hydrogel precursor solutions such as fibrinogen, agarose, alginate, collagen, gelatin, hyaluronic acid, Pluronic or poly(ethylene glycol) have been used as bioink materials. Cell‐laden 3D structures have been fabricated using various bioprinting strategies, including material jetting (e.g., inkjet, microvalve, laser‐assisted printing), material extrusion and polymerization (e.g., stereolithography, digital light processing (DLP), two photon polymerization based printing). And this lecture will show the new trend of bio 3D printing technology and fabrication method in Othorhinolaryngology field.
Maximum of 250 words in length and should be typed in English. No images/photos/figures/ tables.
Transnasal injection of mesenchymal stem cells with silk fibroin hydrogel for the treatment of traumatic brain injury
1Hallym University
Stem cell‐based therapy has shown promising outcomes in treating various neurological disorders beyond cure. Because of the reduced survival rate of transplanted cells, different biomaterials in hydrogels/scaffolds have been employed. In this study, a silk fibroin‐based hydrogel was fabricated to encapsulate engineered human mesenchymal stem cells (hMSCs) with brain‐derived neurotrophic factor (BDNF) that can be injected via a transnasal route to pass the blood‐brain barrier and ultimately to improve traumatic brain injury in a mouse model. The transnasal injection route and the silk fibroin‐based hydrogel offered a safe treatment method while maintaining the survival rate of the injected stem cells. The hydrogel consisted of 15% GelGMA +5% silk fibroin, exhibiting excellent mechanical properties and biocompatibilities. The enhanced neuronal regeneration activity of BDNF was well observed in the silk fibroin‐based hydrogel by the increased neuronal outgrowth in the N2a cells through immunohistochemistry. Immunofluorescence analysis, assessment of neurological deficits, and magnetic resonance imaging analysis showed enhanced neurological functional recovery and reduced neuronal death by the transplantation of the encapsulated BDNF‐hMSC in the traumatic brain injury mouse model compared to the control. This stem cell transplantation approach and BDNF‐hMSC embedded silk fibroin‐based hydrogel can present a potential therapeutic method for the clinical treatment of various brain disorders.
Treating sensorineural hearing loss using PRP(platelet‐rich plasma)
1Cheong Min Clinic
Sensorineural hearing loss has been considered permanent, especially in the chronic phase. Usually, people with sensorineural hearing losses try to use hearing aids. When the hearing levels are too low, even hearing aids are not helpful. So we need to find out any way to regenerate the inner ear cells to elevate hearing levels. Platelet‐rich plasma (PRP) has been widely known for its regenerative potential. However, the fixed idea that damaged human inner ear cells could not regenerate themselves has prevented most researchers from trying to use PRP to help revive the inner ear cells. The author injected PRP, derived from either blood or bone marrow, through the recipient's eardrum for acute and chronic sensorineural hearing losses, elevating the basal hearing level. The author even invented a device to guide to an appropriate point while injecting the PRP. Regardless of the duration of the illness, the hearing improved.
3D in vitro models of head & neck squamous cell carcinomas using patient‐derived cells
1University of Pisa, Dept. of Civil and Industrial Engineering, 2University of Pisa, Dept. of Surgical, Medical, Molecular and Critical Area Pathology, 3Instituto de Investigaciu00f3n Sanitaria del Principado de Asturias, Dept. of Head and Neck Oncology, 4University of Pisa, Dept. of Translational Research & New technologies in Medicine & Surgery
Taste regeneration by photobiomodulation
1Dankook Institute of Medicine and Optics, Dankook University, Cheonan, 2Department of Otorhinolaryngology, Head and Neck Surgery, Dankook University Hospital, Cheonan, 3Interdisciplinary Program for Medical Laser, Dankook University College of Medicine, Cheonan
Taste is one of the fundamental senses that allow us to distinguish nutritious food substances from toxic ones. However, the ability to taste decreases with age or can be lost due to some diseases such as COVID‐ 19, drugs, and disturbances in the molecular activities in taste homeostasis and renewal. Previous studies have shown the potential role of the Hedgehog signaling pathway in taste papillae homeostasis. Therefore, inhibition or stimulation of the Hedgehog pathway can be explored to address taste disturbances. This study investigated photobiomodulation effects on the Hedgehog signaling pathway after inhibition with Vismodegib of taste receptor and geniculate ganglion neuronal cells in vitro and in vivo. Laser treatment performed at 630 and 850 nm, with varying energies of 30, 60, and 120J resulted in the modulation of Hedgehog signaling proteins and genes. Photobiomodulation offers a noninvasive approach with deep penetration in tissue to restore taste by promoting tastebud regeneration and stimulating desensitized afferent nerves.
Odontoblast‐like cell differentiation process after exogenous tooth injuries and prospects for regeneration medicine in dentistry
1Niigata University
The crosstalk among tertiary dentin formation, reinnervation, and revascularization after exogenous tooth injuries is not well understood. Although dental pulp stem/progenitor cells are assumed to reside in multiple niches, including the central pulp, the subodontoblastic layer, and the apical pulp, the extent of external injury and the combination of artificial procedures such as root resection and pulp floor perforation during tooth replantation affect the odontoblast‐like cell differentiation process. In this presentation, I would like to discuss the process of odontoblast‐like cell differentiation in relation to the restoration of intrapulpal blood circulation and pulp stem/progenitor cells. We demonstrated the existence of slow‐cycling long‐term label‐retaining cells (LRCs), or putative adult stem/progenitor cells, which reside in the dental pulp. Using several tooth injury models, we have clarified the dynamics and differentiation capacity of LRCs postoperatively. Furthermore, we introduced our recent data regarding the pulpal healing following tooth injuries using Osteopontin (Opn) knock out and Nestin‐EGFP transgenic mice and the behavior of label‐retaining cells in the TetOP‐H2B‐GFP and BrdU‐labeled mice. On the basis of these findings, we proposed the new hypothesis that both progenitors and stem cells are equipped in the dental pulp and that the stem cells play crucial roles in the pulpal healing process following the exogenous stimuli in cooperation with the progenitors. Our understanding of pulpal healing mechanism after exogenous tooth injuries and characteristics of dental pulp stem cells would lead to the essential step for the realization of regenerative medicine in dentistry.
Vitronectin‐derived peptide promotes reparative dentin formation
1Seoul National University, 2Dankook University
Exposed dental pulp can maintain its vitality through a pulp‐capping procedure with biocompatible materials followed by reparative dentin formation. Our previous study demonstrated that a vitronectin‐ derived peptide (VnP‐16) promotes osteoblast differentiation, and concomitantly restrains osteoclast differentiation and resorptive function. In this study, we aimed to demonstrate that VnP‐16 promotes odontoblast differentiation, mineralization, and reparative dentin formation in a pulp exposure model using a rat tooth. VnP‐16 showed no cytotoxicity and promoted cellular behavior in human dental pulp cells (hDPCs), enhancing their differentiation into odontoblast‐like cells and mineralization, effects that are comparable to those obtained with vitronectin. In a rat pulp exposure model, VnP‐16 showed “none” to “mild” inflammatory responses at 2 and 4 weeks. Mineral trioxide aggregate (MTA) showed a tendency of early formation of reparative dentin at 2 weeks compared to recombinant human bone morphogenetic protein‐2 (rhBMP‐2) and VnP‐16. However, VnP‐16 induced reparative dentin formation similar to MTA and rhBMP‐2 without inflammation at 4 weeks. In addition, VnP‐16 showed a thicker and homogeneous reparative dentin formation compared to MTA and rhBMP‐2. Collectively, these results suggest that VnP‐16 can be a useful direct pulp‐capping agent for highly qualified reparative dentin formation by promoting cell behavior and odontoblastic differentiation of hDPCs.
Targeting class A GPCRs for pulp‐dentin complex regeneration
1Seoul National University
G protein‐coupled receptors (GPCRs) play an important role in various pathogenesis and physiological regulation. Based on the functional characteristics, GPCRs have been regarded as one of the main targets in the current pharmaceutical market. However, until now, the development of drug targeting GPCRs has not been established in regenerative dentistry. Dental pulp stromal cells (DPSCs) are key cellular component in the regeneration of pulp‐dentin complex. Functional recovery of the pulp‐detin complex is closely related to the maintenance of natural teeth. Here I propose a new approach for pulp‐dentin complex regeneration through the regulation of class A GPCR activity. The candidates of class A GPCRs was selected as primary drug targets, and the screening validated the effect of selected class A GPCR antagonists in the differentiation of DPSCs. Based on the screening result, I discovered a core class A GPCR candidate group, which have a potent effect on the biomineralization process. Through RNA sequencing (RNA‐seq), transcriptional changes related to phosphatidylinositol 3-kinase (PI3K)‐protein kinase B (AKT) signaling pathways were commonly identified in the core group. In particular, the expression of the p53 protein, a downstream signaling component of PI3K‐AKT pathway, was specifically increased upon the treatment with core class A GPCR antagonists. This study identified a key candidate group for inducing pulp‐detin complex regeneration by multidisciplinary approaches and a previously unknown mechanism underlying the regenerative process.
Bone reconstruction using two‐layer porcine‐derived bone scaffold composed of cortical and cancellous bones
1Department of Prosthodontics, School of Dentistry, Pusan national university, Korea
In this study, we aimed to investigate the bone regeneration efficiency of two‐layer porcine‐derived bone scaffolds composed of cancellous and cortical bones in a rabbit calvarial defect model. Four circular calvaria defects were formed on cranium of rabbit and were filled with block bone scaffolds of each group: cortical bone block (Cortical group), cancellous bone block (Cancellous group), and two‐layer bone block (2layer group). After 8 weeks, new bones were primarily observed in cancellous parts of the Cancellous and 2layer groups, while the Cortical group exhibited few new bones. In the results of new bone volume and area analyses, the Cancellous group showed the highest value, followed by the 2layer group, and were significantly higher than the Cortical group. Within the limitations of this study, the cancellous and two‐layer porcine‐derived bone scaffolds showed satisfactory bone regeneration efficiency; further studies on regulating the ratio of cortical and cancellous bones in two‐layer bones are needed.
3D platform to control orientations of periodontal ligament in periodontal defects in canine model: Pilot study
1Kyungpook National University
Periodontal ligaments (PDLs) generate systematical force‐responses with specific orientations and tissue integrations to both mineralized tissues. However, it is still challenging for regenerated PDLs to have periodontal‐functioning restoration, optimal position stabilities, transmission of stresses under biomechanical loadings, or appropriate tissue remodeling by mechanical stimulations. Here, we investigated the 3D microarchitectures, which can spatiotemporally organize PDLs and limitedly promote mineralized tissue formations.
Poly‐ɛ‐caprolactone (PCL)‐based, perio‐complex scaffold was computer‐designed with two parts; 1) PDL architectures had aligned surface patterns using the slicing procedure and 2) bone region with open‐ structures. Scaffolds were transplanted into 2‐wall periodontal defects in the canine model (N = 2) for in‐ vivo tissue formations. Micro‐CT and histology including immunohistochemistry (Periostin) were utilized for biological assessments of mineralized tissue formation, PDL fiber orientations, and inflammation.
Bone ingrowth could be limitedly controlled into the bone regions of perio‐complex scaffolds but there was statistically significant difference in bone parameters using micro‐CT. In histological analyses with H&E staining, PDL architecture surfaces in perio‐complex scaffolds can regulate orientations of fibrous tissues 4 weeks. Interestingly, individual bone and fibrous tissues were spatially compartmentalized but biologically integrated like bone‐PDL structures.
Although there was the critical time limitation for the canine tissue formation in this pilot study (time point: 4 weeks), 3D‐printed PDL architectures could regulate limited fibrous tissue orientations and bone formation with predictability and controllability of tissue formations. Therefore, the single perio‐complex scaffolds can provide spatial compartmentalization for oriented PDL regeneration in PDL regions and bone formation.
Exosomes as emerging nanomedicines for immune modulation: regulation of their in vivo fates for therapeutic applications
1Sungkyunkwan University
In recent years, exosomes have been demonstrated to be involved in a range of biological phenomena, implying their potential as the therapeutics and/or diagnostic markers. Particularly, exosomes have been considered key players in determining the immune microenvironments. In this regard, for therapeutic applications, innately therapeutic and exogenous drug‐loaded exosomes should be located at the target site, whereas pathological exosomes or their biogenesis pathways should be targeted to control them. Reflecting recent preclinical efforts in my research group to meet such needs, the related previous work history and initial accomplishments for regulating the in vivo fate of exosomes will be covered in this presentation.
High‐throughput platform for 3D in vitro tumor vasculature model to monitor real‐time Immune cell cytotoxicity
1Seoul National University, 2KIST, 3Sungkyunkwan University, 4Samsung Medical Center
Several advances in anticancer therapy have demonstrated significant improvements in clinical outcomes, and adoptive cell therapy has emerged as a type of immunotherapy that can modulate immune responses by transferring engineered immune cells. However, it remains a challenge because only a small percentage of respondents have responded. Three‐dimensional (3D) in vitro models of the tumor microenvironment (TME) have the potential to provide a platform for assessing and predicting responses to therapy. Here, we propose an in vitro 3D tumor model with clusters of colorectal cancer (CRC) cells around perfusable vascular networks to evaluate immune cell‐mediated cytotoxicity against cancer cells. A 3D injection‐molded co‐culture model consists of 28 microwells where identical vascularized cancer models can be formed in separate wells. It allows robust hydrogel patterning, resulting in high‐throughput experiments. Compared to the polydimethylsiloxane (PDMS)‐based microfluidic devices, our devices allowed a greater number of experiments to be conducted. A permeability test was also conducted to confirm the characteristics of the tumor vasculature. Primary natural killer (NK) cells were introduced into a tumor vascularized network and monitored using live‐cell imaging. The extravasation, migration, and cytotoxic activity of six types of CRC cell lines were examined. Based on the consensus molecular subtypes (CMS) of CRC with distinct immune responses, CMS1 cancer cells were most susceptible to NK cell cytotoxicity. This study indicates the potential of our vascularized tumor model in assessing the responses to adoptive cell therapy by understanding the various steps involved in the immune response.
Biomimetic platform for the study of T cell mechanosensitivity and exhaustion
1New York University Abu Dhabi, 2New York University
T cell activation is modulated by signaling molecules on the surface of antigen‐presenting cells (APC); however, in recent years, it has become increasingly clear that cellular forces have a crucial role in T cell activation and subsequent effector responses. Therefore, understanding mechanical modulators is critical in advancing current immunotherapy approaches. To address underlying questions, we engineered a biomimetic system to recapitulate the immune synapse, which is the interface of APC‐T cell interaction, using polyacrylamide hydrogels with a defined stiffness range comparable to APC stiffness. The hydrogels were functionalized with different ratios of immobilized anti‐CD3 (aCD3) and anti‐CD28 (aCD28) antibodies. Our results showed that T cell proliferation, cytokine secretion, and intracellular signaling were all reduced at lower gel stiffness. We observed similar results in our cells' models, in which APCs with reduced cell stiffness induced lower T cell activation. Overall, our biosystem allows the decoupling of biophysical and biochemical interactions in T cells activation in a physiologically relevant microenvironment.
Ex vivo natural killer cell surface engineering via lipid‐based multifunctional biomaterials for enhanced triple negative breast cancer therapy
1Department of Chemical & Biochemical Engineering, Dongguk University
Cell surface engineering techniques have been intensively developed for augmenting various functionalities of cells and enhancing signaling interactions between heterogeneous cell populations. However, conventional approaches including layer‐by‐layer deposition, chemical conjugation, or genetic/metabolic manipulation did not successfully facilitate cellular surface‐mediated biological interactions. Especially, in cancer immunotherapy, activation of cytotoxic lymphocytes and subsequent augmentation of immune‐synapse interaction, especially between immune cell and target tumorous cell populations, must be achieved. Therefore, patch‐style cell coating biomaterials with multiple functional moieties (i.e., (1) lipid for cell surface anchoring, (2) poly(ethylene glycol) blocker, (3) positively charged moiety, (4) cancer recognition moiety, and (5) stimuli‐responsive degradable linker) are developed for ex vivo natural killer (NK) cell surface engineering to treat triple negative breast cancers (TNBC). This coating material properly located onto NK cell surfaces via hydrophobic lipid interaction and significantly enhanced cancer recognition and killing efficacy of coated NK cells, without disturbing intrinsic properties of NK cells. Furthermore, the surface‐coated NK cells significantly infiltrated into a target tumor site, induced apoptosis/necrosis of cancer cell population, and suppressed tumor progression as well as metastasis in a TNBC mouse model. Hence, our material‐mediated NK cell surface engineering presented herein could be a promising platform technology to effectively enhance immune cellular players in cancer immunotherapy.
In vitro 3D tumour models: (R)evolution of the processing methods and biomedical applications
1University of Minho/3B's Research Group
Significant progresses have been made in tumour models design and reconstruction of the tumour microenvironment, and hydrogels capable of mimicking the native extracellular matrix (ECM), are largely responsible for these research advances. Accompanying these developments, we have also assisted to great evolution and advances of the commonly used methods for obtaining in vitro 3D tumour models, such as: i) Hanging‐drop culture, ii) Spinner flask culture, iii) Magnetic levitation of cells preloaded with magnetic nanoparticles, iv) Static suspension of cells in low‐attachment plates, v) Static suspension in round‐bottom 3D culture plates, vi) ECM embedding, and vii) 3D bioprinting. In particular, 3D bioprinting has introduced a new dimension to 3D in vitro tumour models by means of allowing the generation of a highly controlled and mimetic models as it allow to engineer the complex microenvironment that cancer cells are subjected to, disclosing important insights concerning cell‐cell and cell‐ECM interactions. The important 3B's research projects and tissue engineering approaches related to the processing of in vitro 3D tumour models and its applications will be discussed herein.
Acknowledgements: This work has the support of REMIX Project, funded by the European Union's Horizon 2020 Research and Innovation programme under the Maria Sklodowska‐Curie grant agreement n. 778078.
In vitro models: a new era in pre‐clinical researchIn vitro models: a new era in pre‐clinical research
1University of Minho/3B's Research Group, 2not applicable
In the last few years, the cell methods used in pre‐clinical research have been changing from two‐ dimension (2D) to three‐dimension (3D) studies since 2D approaches are not sustainable and poorly correlate with the human condition. In this context, emerging technologies involving tissue engineering, bioprinting, and advanced dynamic cell/tissue culture can provide exciting solutions to better engineer complex in vitro 3D models. Despite these advances, the establishment of the scientific validity of the in vitro 3D models in pre‐clinical research is still challenging and greatly dependent on many other progresses arising from underpinning technologies and biomaterials advances, in particular for being applied as artificial extracellular matrices (aECM). The challenges and recent reports related to the use of natural‐based biomaterials as aECM for developing mimetic in vitro 3D models are overviewed and discussed herein. Several examples of the integration of these models in different technological platforms for dynamic culturing, such as microfluidics and bioreactors are also provided. The 3B's Research Group (Univ. of Minho, Portugal) significant achievements involving the design and use of in vitro 3D models on chip and on bioreactors for testing of nanomedicines and drugs are also highlighted. In vitro models are in early stage of both technological/biological development requiring further standardization and validation, but the increasing research efforts are now enabled to reshape the preclinical steps and providing exciting prospects for its use in the clinics.
Acknowledgements: REMIX Project, funded by the European Union's Horizon 2020 Research and Innovation programme under the Maria Sklodowska‐Curie grant agreement n. 778078.
Biomaterials as an ECM for in vitro models
1University of Wollongong
The skin is richly innervated with a heterogeneous population of sensory neurons (SNs) that allow for a variety of somatosensory functions, including touch, temperature, and pain. Growing evidence suggests that the peripheral nervous system plays a major role in cutaneous health and wound healing. Tissue‐ engineered skin presents a promising solution for skin wound repair, however, the restoration of sensory perception remains a major challenge to date. To address this challenge, our laboratory has developed transgenic cell lines from human pluripotent stem cells (hPSC) that efficiently and reproducibly generate functionally mature SNs. These hPSC‐derived induced sensory neurons (iSNs) display molecular and functional properties akin to mechanosensory neurons that innervate the skin. Furthermore, we have demonstrated that the progenitors derived from the transgenic hPSC lines can be bioprinted and further differentiated within the 3D scaffolds to generate mature iSNs showing extensive neurite outgrowth. Building on this research, we propose the generation of innervated bioprinted human skin models, comprised of primary dermal fibroblasts, keratinocytes, and hPSC‐derived iSNs. The incorporation of human somatosensory neurons into tissue‐engineered skin will offer insights into the mechanisms of repair and regeneration of full‐thickness skin, thus advancing this promising technology aimed at improving graft patients' quality of life.
Microengineered systems to recapitulate the functional inner ear organoids with enhanced maturity
1Chonnam National University, 2Soonchunhyang University College of Medicine, 3Ajou University
Inner ear disorders (e.g., hearing loss) are common, but it is difficult to develop a therapeutic drug and to find a specific mechanism because of a lack of the research platforms. Inner ear organoids (IEO) are perceiving as an innovative research platform to reproduce the complex inner ear systems and to solve the previous problems. To improve uniformity and reproducibility of IEO, we develop a microengineered system that can collect cells and form aggregates rapidly. Using microengineered system, we can easily control the size of aggregates by the initial cell number and find the optimal condition to develop mature IEO. Compared with the traditional approach to develop IEO (i.e., IEO grown on U well plate; U‐IEO), it is possible to develop IEO with the mass production and more reproducible shape. In addition, IEO grown on microengineered system (M‐IEO) have an improved functions including the formation of mature kinocilia and the electrophysiological function than U‐IEO. Thus, we conclude M‐IEO may have great potential to overcome the limitations of the traditional approach, and it may be used as an advanced platform for inner ear disorder.
Human pluripotent stem cells differentiation into retinal pigmented epithelial cells on different ECM‐coated surface
1National Central University
Age‐related macular degeneration (AMD), which is the leading cause of irreversible visual impairment, is associated with the progressive dysfunction and death of photoreceptor cells and their supportive retinal pigment epithelial(RPE) cells. Transplantation of human pluripotent stem cell (hPSCs)‐derived RPE cells is considered as a promising approach to regenerate cell function and cure AMD. Human pluripotent stem cells (hPSCs), human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), can provide unlimited source of RPE because of indefinite self‐renewal. Moreover, microenvironment plays an important role in differentiation for hPSCs. Therefore, we investigated the effect of cell culture biomaterials on differentiation of hPSCs into RPE where Matrigel‐, Laminin‐521‐, Laminin‐511‐, Synthemax II‐ and Recombinant vitronectin‐coated surface were selected as the cell culture biomaterials in this study. hPSCs were differentiated into RPE on different extracellular matrix (ECM) protein‐coated surface by different protocol (NIC84 and Activin A protocols) in order to investigate the optimal differentiation conditions. We observed the polygonal morphologies of hPSCs (HPS0077)‐derived RPE, which expressed RPE specific marker (ZO‐1 and RPE65) by flow cytometry and immunostaining using both protocols. Finally, hiPSCs‐derived RPE showed brown color (pigmented cells). Matrigel‐, Laminin‐ 521‐ and Laminin‐511‐coated surfaces could support the differentiation of hPSCs into RPE efficiently, which were compared with Synthemax II‐ and Recombinant vitronectin‐coated surfaces. This is explained that RPE cells are ectodermal lineage of the cells where Laminin is preferably support ectodermal cells via integrin α6β1 rather than integrin αVβ5, which is the binding site of vitronectin.
Enzyme‐controlled, nutritive hydrogel for mesenchymal stromal cell survival and paracrine functions
1University Paris Cite, CNRS, INSERM ENVA, B3OA, F‐75010 Paris, France, 2Equipe de Recherche sur les Relations Matrice Extracellulaire Cellules (ERRMECe), Institut des Materiaux, Universite de Cergy‐ Pontoise, 95302 Cergy‐Pontoise Cedex, France, 3Lyon Neuroscience Research Center, AniRA‐Neurochem Technological Platform , Team TIGER , INSERM U1028, CNRS UMR5292 , 69675 Bron Cedex, France
Three‐dimensional hydrogel with biophysical modulation regulates cellular reprogramming into induced pluripotent stem cell
1Dongguk University, 2CHA University, 3Kangwon University
Biophysical modulations of stem cell behavior have shown controlled cellular responses, and these findings can be utilized to expand and isolate specifically characterized stem cell in need. Although many biophysical studies were done in two‐dimensional cell culture, the effect on three‐dimensional (3D) microenvironment system were largely unexplored. Here, we present new 3D microenvironment hydrogel system that promotes cellular reprogramming into induced pluripotent stem cells (iPSCs). Various biomaterials were prepared by methacrylation for photo‐crosslinkable 3D hydrogel system. Of all, hyaluronic acid (HA) hydrogel showed increased cellular expressions of mesenchymal‐epithelial transition, histone modification, and pluripotency, markers representing cellular reprogramming into iPSCs. Further analysis of biophysical changes in both ligands and stiffness show that initial upregulation of CD44 by HA ligand and low stiffness speed up the reprogramming process into iPSCs. In conclusion, presented HA hydrogel with soft microenvironment system accelerates reprogramming process and increase reprogramming efficiency of iPSCs, suggest its potential platform for translational tissue regeneration.
Single‐cell multiomic profiling identifies novel regulators of stem cell derived β‐cell differentiation and maturation
1Washington University in St. Louis, 2Washington University School of Medicine
The islets of Langerhans maintain homeostatic blood glucose levels by regulating insulin secretion. In type 1 diabetes, the β‐cells within the islets are loss leading to insufficient insulin release. There is no feasible long‐term cure for type I diabetes because cell‐based therapies are challenged with limited supply of donor human islets. Stem‐cell derived (SC) β‐cells make a promising alternative to donor human islets as cells can be generated in unlimited supply. We have developed a differentiation protocol to generate functional SC‐β cells capable of secreting insulin and restoring normoglycemia in diabetic mice after transplantation. However, these SC‐β cells have inferior performance when compared to primary human β‐cells because they have lower insulin release. Using multiomic single‐cell sequencing, we find that the SC‐β cells have transcriptional profiles that are immature and epigenetic settings that are ambiguous. By transplantation, we revealed that these SC‐β cells can mature and overcome epigenetic ambiguity to become more primary human β‐cell like. Through our comparative analysis, we identified ARID1B to be a key driver of epigenetic ambiguity. We used genetic engineering tools to modulate ARID1B expression, and unraveled major biological insights into improving β‐cell differentiation and maturation in vitro. Collectively, our study provides a comprehensive resource of transcriptome and epigenetic profiles in SC β‐cells and primary human islets. Our analysis reveals important regulators of SC‐β‐cell fate decision and maturation. Genetic engineering of target genes validates our ability to utilize this resource to improve SC‐β cell differentiations for diabetes cell replacement therapy.
Priming of FGF2/HGF combination restores the impaired osteogenic differentiation of adipose‐derived stem cells
1Kyunghee University, 2Kyunghee University East‐West Medical Research Institute
In the era of unavoidable aging, the importance of appropriate treatment for fractures in the elderly is being emphasized. Among the various treatment strategies, transplantation of adipose‐derived stem cells (ADSCs) is currently in the spotlight, but shows limited efficacy due to poor paracrine/differentiation capacity. Fibroblast growth factor2 (FGF2) and hepatocyte growth factor (HGF) are growth factors that promote bone formation, decrease in expression in the elderly, compared to the young ADSC (ADSC‐Y). Thus, we hypothesized that FGF2/HGF supplementation for ADSCs from the elderly would improve cellular function including differentiation and paracrine potential.
The deficiency of FGF2/HGF in elderly ADSCs (ADSC‐E) was confirmed during osteogenic differentiation. The supplementation of FGF2/HGF could enhance osteogenic differentiation ability of ADSC‐E, similar to ADSC‐Y. Priming of FGF2/HGF‐induced osteogenic differentiation was accompanied by an early shift of expression of osteogenic marker including Runt‐related transcription factor 2 (RUNX‐2), Osterix, and Alkaline phosphatase (ALP). Moreover, priming of FGF2/HGF created an environment favorable to osteogenesis by facilitating secretion of bone morphogenetic protein 2 (BMP‐2) and vascular endothelial growth factor (VEGF). Restoration of osteogenic potential of ADSC‐E by FGF2/HGF was proved by calcium deposition in vitro and ectopic bone formation in vivo, respectively.
Taken together, FGF2/HGF priming could enhance osteogenic differentiation in ADSCs. These results suggest that the growth factor‐mediated cellular priming can be used for transplantation of autologous ADSC in the elderly.
Exosomes secreted from fetal cartilage‐derived stem cells promote skeletal muscle regenerationvia the miR‐145a
1Department of Molecular Science and Technology, Ajou University, Suwon, Korea, Republic of, 2Department of Biomedical Sciences, School of Medicine, Ajou University, Suwon, Korea, Republic of, 3Department of Orthopedic Surgery, School of Medicine, Ajou University, Suwon, Korea, Republic of
Skeletal muscle is the most abundant tissue occupying about 40‐45% of the human body and playing an important role in maintaining body functions. Since skeletal muscle loss can lead to serious disability, tissue regeneration is considered an important part of muscle therapy. Recently, many studies have shown promise as an effective therapeutic agent for tissue regeneration of mesenchymal stem cells (MSCs)‐ derived exosomes. Treatment of MSCs‐exosomes increased the expression of myogenic‐related genes in the myoblast. In addition, the administration of exosomes induced muscle regeneration in animal models. However, despite various studies, a treatment method that can induce complete regeneration of skeletal muscle tissue has not yet been developed.
In this study, we induced the regeneration of skeletal muscle tissue using exosomes secreted from fetal cartilage‐derived stem cells(FCSCs). In the previous study, FCSCs showed higher proliferation and differentiation capacity than adult stem cells. In particular, it was confirmed that effective tissue regeneration was induced by secreting paracrine factors such as growth factors and cytokines in greater amounts than MSCs. As a result, exosomes isolated from FCSCs increased the expression of myogenic‐ related markers more in myoblast than MSCs‐exosomes. Also, in the skeletal muscle loss animal model, FCSCs‐exosomes effectively promoted the regeneration of skeletal muscle tissue. This result was confirmed to be due to a number of miRNAs contained in FCSCs‐exosomes. Interestingly, it was observed that FCSCs‐exosomes contained more miR‐145a involved in muscle regeneration than MSCs‐ exosomes. These results indicate that exosomes extracted from FCSCs have the therapeutic potential for skeletal muscle regeneration.
Manipulation of inherent niches in 3D MSC spheroids improves therapeutic potential
1Institute of Biomedical Engineering, National Tsing Hua University
Mesenchymal stem cells (MSCs) that derived from various human tissues have been widely used in cell therapy and regenerative medicine. Transplantation of MSCs not only serves as cell replenishment but also MSC secretome improves therapeutic potential during tissue regeneration. Compared to conventional culture, three dimensional (3D) multicellular spheroids configuration, MSCs exhibit native tissue‐ mimicking feature due to substantial cell‐cell and cell‐matrix interaction, thus considered as a potential implantable regenerative building blocks for stem cell therapy. Different inherent niches in 3D MSC spheroids will further improve their therapeutic potential. We found that 3D MSC spheroids size affect MSC paracrine molecules signaling and immunomodulatory enzyme level which have the most abundant level at 3D MSC spheroids prepared by 40,000 cells per well. Besides that, 3D MSC spheroids culture period affects their viability and therapeutic potential. After 3‐day cultivation, 3D MSC spheroids lead to cell death in the spheroid core and metabolic reconfiguration and autophagy are activated. However, we found that hypoxic niche development in 3D MSC spheroid is not the major component for its enhanced therapeutic potential. It is important for MSCs to be cultivated in a 3D niche for significantly enhanced therapeutic potential in terms of paracrine signaling and immunomodulatory activity. By optimizing cultivation parameters of 3D MSC spheroids, inherent niches could be modulated and further promote therapeutic capacity by activating the expression of pro‐regenerative paracrine molecules and immunomodulatory factors. In the present study, MSC spheroids that were assembled by 40,000 cells and cultivated for 2 days showed the best therapeutic potential.
Phototoxicity-free blue light for enhancing therapeutic angiogenic efficacy of stem cells
Eun Cheol Lee1, Sung Won Kim1, Yu Jin Kim1, Gwang Bum Im1, Yeoung Hwan Kim1, Gun Jae Jeong1, Sung Min Cho1, Hae Shin Lee1, Suk Ho Bhang*1
1Sungkyunkwan University
Low-level light therapy (LLLT) is a safe and noninvasive technique that has drawn attention as a new therapeutic method to treat various diseases. However, little is known so far about the effect of blue light for LLLT due to the generation of reactive oxygen species (ROS) that can cause cell damage. We introduced a blue organic light-emitting diode (bOLED) as a safe and effective light source that could generate a low amount of heat and luminance compared to conventional light sources (e.g., light-emitting diodes). We compared phototoxicity of bOLED light with different light fluences to human adiposederived stem cells (hADSC). We further explored molecular mechanisms involved in the therapeutic efficacy of bOLED for enhancing angiogenic properties of hADSC, including intracellular ROS control in hADSCs. Using optimum conditions of bOLED light proposed in this study, photobiomodulation and angiogenic properties of hADSCs were enhanced. These findings might open new methods for using blue light in LLLT. Such methods can be implemented in future treatments for ischemic disease.
Engineered 3D biomimetic skeletal muscle construct using induced myogenic progenitors that can self‐renew and differentiate
1Laboratory of Regenerative and Movement Biology, Department Health Sciences and Technology, ETH Zurich, 2Institute for Biomechanics, Department of Health Sciences and Technology, ETH Zurich
Skeletal muscle is a highly regenerative tissue that maintains its homeostasis via activation and differentiation of muscle stem cells, termed satellite cells. Due to its cellular complexity, mimicking the skeletal muscle tissue environment in vitro is challenging. Directly reprogrammed induced myogenic progenitors (iMPCs) represent a unique heterogenous myogenic cell culture that consists of self‐renewing Pax7+ stem cells, committed myoblasts and differentiated muscle fibers. Previous studies have shown the augmented potential of iMPCs in recapitulating a skeletal muscle differentiation program in a 2D culture. However, the capacity of iMPCs in capturing myogenesis in a 3D‐system, or potential alignment to mimic an in vivo muscle fiber organization, has not been investigated to date. Here, we developed a biomimetic model that is structurally and functionally akin to skeletal muscle tissue by seeding iMPCs on aligned polycaprolactone (PCL) substrates. Using electrospinning, we fabricated PCL scaffolds which allowed to align iMPCs on the substrates by controlling the orientation of the PCL fibers. These skeletal muscle bio‐constructs demonstrated presence of both Pax7+ stem cells and multinucleated myofibers. Furthermore, incorporation of Matrigel into the PCL scaffolds enabled enhanced activation and differentiation of iMPCs into highly multinucleated myofibers. Notably, when subjected to cardiotoxin injury, the iMPC‐derived bio‐constructs exhibited a highly regenerative response, manifested by stem cell differentiation. Taken together, we report on a novel engineered skeletal muscle model using aligned iMPCs which can be utilized to study skeletal muscle regeneration in a 3D‐environment, with further potential utility as a platform for disease modeling and drug screens.
Self‐organized insulin‐producing beta cells differented from human omentum‐derived stem cell
1Soonchunhyang Institute of Medi‐bio Science (SIMS), 2Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, 3Department of Otorhinolaryngology‐Head and Neck Surgery, Soonchunhyang University Cheonan Hospital, Cheonan, 4Department of Otorhinolaryngology‐Head and Neck Surgery Soonchunhyang University Bucheon Hospital, Soonchunhyang University College of Medicine, Bucheon
Human omentum‐derived mesenchymal stem cells (O‐MSCs) possess a great potential to differentiate into multiple lineages and self‐renewal capacity, allowing them to be utilized as patient‐specific cell‐ based therapeutics. Although various stem cells‐derived pancreatic beta cells or insulin‐producing progenitor cells have been proposed as a novel approach for treating diabetes mellitus, an efficient method for establishing functional insulin‐producing cells is still a daunting task. Here, we aim to develop a novel cell culture platform that could regulate important cell‐cell/cell‐matrix interactions by introducing a fibroblast growth factor‐2 (FGF2)‐immobilized matrix that can support the adhesion, proliferation, and differentiation of O‐MSCs into insulin‐producing cells. Our findings demonstrated that cells cultured on a FGF2‐immobilized matrix were able to self‐organize into insulin‐producing beta cell‐like progenitors, as shown by the upregulation of pancreatic beta cell‐specific markers, such as PDX‐1, Insulin, and Glut‐2. Furthermore, heparan sulfate proteoglycan, gap junction proteins connexin Cx36 and Cx43, cell adhesion molecule E‐cad and Ncam1 were upregulated, which promote maturation and insulin secretion in the pancreatic niche environment. Such a cell culture platform can offer novel strategies to achieve functional pancreatic beta cells from a patient‐specific cell source to treat diabetes mellitus.
Acoustic fabrication of living toroids and cardiomyocyte‐based hybrid biorobots
1Stanford University
Micro‐ and nano‐scale technologies can have a significant impact on medicine and biology in the areas of cell manipulation, diagnostics, and monitoring. At the convergence of these new technologies and biology, we research for enabling solutions to real‐world problems at the clinic. Emerging nano‐scale and microfluidic technologies integrated with acoustics in biology offer innovative possibilities for creating intelligent, mobile medical devices that could transform diagnostics and monitoring, soft micro‐robotics, tissue engineering, and regenerative medicine. We will present interesting applications of 3‐D acoustic bioassembly technologies in fluidics such as in tissue engineering constructs and soft microswimmers.
Sound programmable vasculature morphogenesis
1AO Research Institute Davos
Engineering vasculature is fundamental for the generation and sustenance of tissues and organs. Biofabrication technologies have been increasingly considered for creating functional tissues. Among them, the sound patterning approach allows for spatial arrange living materials such as individual cells or spheroids (1). Faraday waves‐driven hydrodynamic forces induce contactless aggregation of cells within hydrogels into large‐scale, defined, and reproducible patterns (2). We use this fast, mild, and simple process to assemble endothelial cells and human fibroblasts into shape‐controlled microcapillary networks.
An open fluidic chamber was developed for supporting sound pre‐patterning followed by self‐assembly vasculogenesis. By tuning the frequency, amplitude, and shape of the patterning chamber, we are able to generate several vascular morphologies, including centimeter scale concentric rings and honeycomb‐like cell distributions. The pattern's symmetry folds increase by raising the sound frequency, while initial cell concentrations impact the formation of periodical gradients of vessel sizes and densities.
Altogether, this simple approach represents a versatile tool to control on‐demand vasculature morphogenesis, paving the way to a better understanding of the influence of vascular network morphology in development, regeneration, and disease.
Acoustic bioassembly for constructing in vitro histotypic and organotypic models
1Wuhan University
Bioassembly is regarded as a critical alternative technical route to bioprinting for 3D biofabrication. Particularly, emerging acoustic assembly represents a promising bioassembly technique to build histotypic and organotypic constructs in a flexible and tunable manner. Currently, simultaneous assembly of heterogeneous cell types into a spatially‐defined heterocellular architecture remains a long‐lasting challenge for acoustic assembly, but it is essential to recapitulate native cell organization in natural tissues and organs. In this study, we develop an acoustic differential assembly method to solve this critical issue by assembling different cell types into complementary or sandwiched cytoarchitecture based on the cell's intrinsic physical characteristics. Specifically, cell‐encapsulating building blocks are driven by Faraday‐ wave patterned hydrodynamic drag force and spatially differentially located based on their sizes and buoyant densities. As a proof of concept, we demonstrate the bioassembly of functional hepatic lobule‐ like tissue constructs from hiPSC‐derived liver organoids and HUVEC encapsulating microgels. We expect this acoustic differential assembly method will enable the construction of human physiologically/pathologically relevant in vitro models with spatially‐defined heterocellular architecture and find wide applications in tissue engineering and regenerative medicine.
Stabilized cardiac differentiation by improving the homogeneity of epithelial‐mesenchymal transition in cell aggregates
1Department of Biotechnology, Graduate school of Engineering, Osaka University, 2Department of Biotechnology, Research Base for Cell Manufacturability, Graduate School of Engineering, Osaka University
Human induced pluripotent stem cells (hiPSCs) have capability to differentiate into cells of three germ layers, such as endoderm, ectoderm and mesoderm and following various cell lineages. We focused on the 3D cardiac differentiation culture of hiPSCs and proposed improved method to control the stability of hiPSCs fate commitment toward cardiomyocytes in batch cultures.
In cardiac differentiation culture, hiPSCs was forced to make cell aggregates under static culture in 4 days. Cell aggregates were further subjected to a 5 mL bioreactor to conduct suspension culture for 12 days. At the end of 3D cardiac differentiation culture, cell aggregates were dissociated and the cardiomyocytes marker cTnT was detected. E‐cadherin and N‐cadherin expression inside cell aggregates were investigated by cryosections and immunofluorescence staining.
cTnT positive ratios showed to be 0.49 ± 0.14 (AVG ±SD) in differentiation cultures, suggesting the processes prone to quality fluctuations. To understand the trigger for the fluctuation, Epithelial‐ Mesenchymal Transition (EMT) was investigated. Cell aggregates exhibit the localization of E‐cadherin (center) and N‐cadherin (periphery) formation, suggesting that the spatial heterogeneity of EMT occurred with and the non‐synchronized EMT caused the unstable differentiation, leading to quality fluctuation of cTnT in culture process. To make the synchronized EMT, we selected the specific component and add to the cultures. The 15 cultures revealed to be 0.49 ± 0.07 of cTnT ratio, providing much less SD in the same AVG, compared to those in conventional culture described above. This shows that the robust process in differentiation culture was performed, which is the critical for practical cell manufacturing.
Engineering human cardiac patch from human induced pluripotent stem cells derived from expanded CD34+ cells in mesenchymal stem cell coculture
1Cryocord Sdn Bhd, Bio‐X Centre, Persiaran Cyber Point Selatan, Cyberjaya, 63000 Cyberjaya, Selangor, Malaysia, 2Advanced Medical and Dental Institute, University of Science Malaysia, 3Department of Neurosciences, Universiti Sains Malaysia 16150 Kubang Kerian, Kelantan, Malaysia, 4Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman (UTAR), 43000 Kajang, Selangor, Malaysia, 5Henan Key Laboratory of Medical Tissue Regeneration, Xinxiang Medical University, Henan,China, 6Department of Neurobiology and Physiology, Xinxiang Medical University, Xinxiang 453003, Henan, China
Reprogramming cord blood (CB)‐derived cells could generate human induced‐pluripotent stem cells (hiPSCs) with comparatively higher proliferation capacity. Here we aim to expand CB with low CD34+ cells using mesenchymal stem cell (MSC) coculture, reprogram them to hiPSCs and differentiate cardiomyocytes for cardiac patch engineering.
Drivers of age‐dependent changes in angiogenesis: Role of pericytes and impacts on tissue engineering outcomes
1The University of Queensland
Successful integration of tissue engineered (TE) scaffolds into a host relies heavily on the environment being supportive of angiogenesis from the host vasculature. Angiogenesis requires a highly coordinated and complex interaction between endothelium and pericytes (PCs) to initiate, sustain and mature new vessel networks. PCs, our perivascular stem cells and the originators of our much‐celebrated mesenchymal stem cells, are critical to angiogenesis, however, little is understood as to what changes in these cells with ageing and how their ability to maintain and support tissue repair/regeneration through angiogenesis and morphogenesis is affected. This study investigated the intrinsic changes in primary human pericytes with donor age (16 – 81 years) and their impacts on their function within in vitro 2D/3D angiogenesis models. We showed that PCs exhibit minimal changes in gold‐standard phenotype markers or morphological traits with age, but show substantial changes in global gene expression, protein and growth factor production. Functionally, young PCs exhibit increased interactions with endothelial cells (ECs) compared to older PCs, whilst old PCs appear more 'angiogenic' than young PCs, based on their secretome profile. We show that the exogenous supply of pro‐angiogenic factors (as often utilised in TE strategies!) are not sufficient to overcome these age‐related functional deficits, and moreover, that these changes are a result of altered cell‐cell communication and mechano‐signalling. This study confirms that age‐related changes in pericyte function are important considerations in a TE framework, and that new therapeutic strategies are required to support improved angiogenesis/integration of TE scaffolds in older patient cohorts.
Tissue‐engineered vascular microphysiological platform to study immune modulation of xenograft rejection
1Korea Institute of Science and Technology
Most of the vascular platforms currently being studied are lab‐on‐a‐chip types that mimic capillary networks and are applied for vascular response analysis in vitro. However, these platforms have a limitation in clearly assessing the physiological phenomena of native blood vessels compared to in vivo evaluation. Here, we developed a simply fabricable tissue‐engineered vascular microphysiological platform (TEVMP) with a three‐dimensional (3D) vascular structure similar to an artery that can be applied for ex vivo and in vivo evaluation. Furthermore, we applied the TEVMP as ex vivo and in vivo screening systems to evaluate the effect of human CD200 (hCD200) overexpression in porcine endothelial cells (PECs) on vascular xenogeneic immune responses. These screening systems, in contrast to 2D in vitro and cellular xenotransplantation in vivo models, clearly demonstrated that hCD200 overexpression effectively suppressed vascular xenograft rejection. The TEVMP has a high potential as a platform to assess various vascular‐related responses.
Extracellular matrix properties regulate age‐related cardiac function
1National University of Singapore
As the heart ages, disruption of the extracellular matrix (ECM) occurs, manifesting in tissue stiffening and disorganized architecture. While ECM stiffening often compensates for a weakening heart, these altered matrix properties can lead to further complications. It is well known that cardiac cells are mechanosensitive, but a precise investigation into cellular function based on altered ECM properties representing an aged state is lacking. Thus, we employed parallel strategies to interrogate the influence of matrix properties, stiffness and ligand presentation, on age‐related function. To investigate mechanical properties, polyacrylamide stiffness gradient hydrogels were fabricated utilizing a double polymerization method, in which stiffness regimes spanning ∼10‐50 kPa were created. On these hydrogels, both young (∼1 month) and aged (∼2 years) cardiac cells exhibited age‐ and stiffness‐dependent responses in terms of morphology and mechanosensitive marker expression. Using AFM, cell stiffness was also found to increase with substrate rigidity, indicating a robust cytoskeletal rearrangement due to mechanical cues. To study the role of ligand spacing, we employed Block Copolymer Micelle Nanolithography to create highly ordered nanoparticle arrays with precise control of inter‐particle spacing (35 – 70 nm). Nanoparticles were then functionalized with fibronectin or collagen mimicking peptides to understand the contribution from different matrix components. Cardiomyocyte spreading was found to decrease as ligand spacing increased, which coincided with mechanosensitive marker expression. Taken together, these results indicate that two important ECM properties, stiffness and ligand presentation, play vital roles in cardiac cell function, and therefore can inform future ECM‐based treatment strategies for mitigating age‐ related heart disease.
Sourcing of blood‐derived angiogenic cells (BDACs) under serum‐free and xeno‐free conditions for the treatment of critical limb ischemia
1Institute for Tissue Engineering and Regenerative Medicine, School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China, 2Institute for Tissue Engineering and Regenerative Medicine, School of Biomedical Sciences, and Department of Orthopaedics & Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
Critical limb ischemia (CLI) is the most severe form of peripheral artery disease, which could result in amputations of the lower extremity. The therapeutic potential of mesenchymal stem cell (MSC) transplantation for the treatment of ischemic conditions has been explored in animal models and early‐ phase clinical trials. MSCs have an extensive historical background in being characterized and explored for the treatment of various pathologies. However, MSCs may not be the solution for the treatment of all pathologies. Human peripheral blood‐derived angiogenic cells (BDACs) have demonstrated pro‐ angiogenic potential in vitro and in vivo, where they were observed to take perivascular location at sites of active angiogenesis, thereby resembling hematopoietic pericytes in angiogenesis. As standard BDAC sourcing techniques involve the use of animal‐derived serum, we first established a xeno‐free protocol to pave their way towards clinical application. When these BDACs were injected intramuscularly in a murine model of CLI, they enhanced revascularization and rescued the affected limb from extensive necrosis, exceeding the therapeutic potential of bone marrow MSCs. As BDACs are derived from an easily accessible tissue and can be sourced in clinically relevant numbers and timeframe, they may represent an alternative and advantageous cell type for therapeutic angiogenesis.
The green lane graft: A novel decellularised scaffold for heart valve tissue engineering
1University of Auckland, 2Auckland District Health Board, 3University of Otago
Heart valve tissue engineering is being explored as a novel means of overcoming the limitations of valvular conduits in current clinical use. This tissue engineering process firstly involves decellularisation in which xenogenic collagen scaffolds are rendered non‐immunogenic by applying a proprietary detergent‐enzyme treatment protocol. The immunogenic response to the experimental scaffold may then be assessed in vivo using a surgical subcutaneous rat model.
A novel decellularisation protocol, comprising enzymes and detergents, was optimised using adult bovine pericardium and porcine aortic valve leaflets. Decellularisation was measured histologically by assessing cell nuclei counts and by quantifying dsDNA concentrations. Samples were subcutaneously implanted in rats and then harvested at 4‐ and 8‐weeks for immunohistochemical examination.
In decellularised tissue samples, a complete lack of cell nuclei was observed, and concentrations of dsDNA were significantly reduced, when compared with native, and industry control, samples. None of the grafts generated clinical signs of infection. There was a heterogenous, sub‐clinical, immune response when assessed immunohistochemically.
The Green Lane Graft is our first prototype of a successfully decellularised, xenogenic, collagen scaffold. We observed no acute signs of rejection in our first experience with a surgical subcutaneous rat model. The heterogenous, immunohistochemical findings will be addressed by refining the animal model in future work.
Advanced stem cell therapies for CNS repair using bioinspired nanotechnology
1Rutgers University
This presentation will focus on the interface between nanomedicine and stem cell therapeutics. Neurological disorders typically result in cellular dysfunctions that may cause severe and permanent paralysis. Given the central nervous system's (CNS) intrinsically limited regenerative abilities and the highly complex inhibitory environment of the damaged tissue, gene‐ and stem cell‐based therapies have great potential for CNS repair by regenerating a robust population of functional neural cells and modulating the neuroinhibitory microenvironment.
To this end, we have overcome the aforementioned challenges in current stem cell therapies by developing three different methods: i) to develop a nonviral and effective genetic manipulation method by mimicking the function of natural transcription factors (TFs), ii) to develop a multifunctional nanomaterial‐based bio‐scaffold method for the controlled delivery of therapeutic molecules in vivo, and
iii) to incorporate a nanomaterial‐based bio‐scaffold to enhance stem cell transplantation.
Since many neurodegenerative disorders and nerve injuries are characterized by disrupted neuronal circuits and neuro‐inflammation, the approach that we have developed has the potential to be extensively applied to a wide variety of different indications. In this presentation, a summary of the most updated results from these efforts and future directions will be discussed.
Liquid biopsy for spinal cord injury biomarker: miRNA biopsy
1Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea, 2The Spine and Spinal Cord Institute, Department of Neurosurgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea, 3Department of Neurosurgery, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, Seoul, Republic of Korea
Liquid biopsy has received tremendous attention as analysis tool of tumors using biomarkers circulating in fluids such as the blood. But the technique is isolated non‐coding RNA (miRNA), circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and exosomes, and analyzed them through an amplification process based on genetic information. It takes a considerable time and has a problem that can lead to non‐specific results, and liquid biopsy has not yet been commercialized
It is especially spinal cord injury (SCI) for change effects in direct damage is important to monitoring/investigating the acute and urgent characteristics of treatment management in real time or as soon as possible. Aims of this study is that observing the progress and monitoring of disease developing with three‐dimensional imaging analysis in blood‐based stable molecular biomarkers; miRNA without gene amplification via tissue clearing technique based liquid biopsy. Newly liquid‐based MAP technique using peripheral blood, provides an ideal tool for diagnosis and prognosis prediction for diseases of the central nervous system, including the brain and spinal cord as well as various biological fluids. Using miRNA liquid biopsy technology newly developed by this researcher, it was confirmed that miRNA 206 could be used as a new technology for diagnosis and prognosis prediction in patients with SCI and myelopathy, and briefly report the results.
Effect of electrical stimulation on spinal cord injury: in vitro and in vivo analysis
1Kyungpook National University
Electrical stimulation influences neural stem cell neurogenesis. We analyzed the effects of electrical stimulation on neurogenesis in rodent spinal cord‐derived neural stem cells (SC‐NSCs) in vitro and in vivo. Additionally, we evaluated functional recovery and neural circuitry improvements with electrical stimulation using a rodent spinal cord injury (SCI) model. Biphasic electrical currents (BECs) were applied to rodent SC‐NSCs under various conditions. Total SC‐NSCs and differentiated neurons were counted, and morphological changes in differentiated neurons were evaluated. Rats were divided into the following groups (20 rats/group): sham (Group 1), SCI only (Group 2), SCI+electrode implant without stimulation (Group 3), and SCI+electrode with stimulation (Group 4). In Group 4, daily electrical stimulation was delivered for 28 days. We analyzed Basso, Beattie, and Bresnahan scores and monitored the motor and somatosensory evoked potentials in all rats. After harvesting rat spinal cords, we analyzed Wnt3, Wnt7, and β‐catenin protein level and counted SC‐NSCs and differentiated neurons. In vitro, BECs increased SC‐NSC proliferation and neuronal differentiation and caused qualitative morphological changes in differentiated neurons. In rats, electrical stimulation promoted SC‐NSC proliferation and neuronal differentiation and improved functional outcomes and neural circuitry in SCI models. Increased Wnt3, Wnt7, and β‐catenin protein level were also observed after electrical stimulation. In conclusion, our study proved the beneficial effects of electrical stimulation on SCI. We guess that Wnt/β‐catenin pathway activation may be deeply related to this relationship between electrical stimulation and neuronal regeneration after SCI.
Lubricant skin on diverse biomaterials with complex shapes via polydopamine‐mediated surface functionalization for biomedical application
1Yonsei University
Implantable biomedical devices require an anti‐biofouling, mechanically robust, low friction surface for a prolonged lifespan and improved performance. However, there exist no methods that could provide uniform and effective coatings for medical devices with complex shapes and materials to prevent immune‐related side effects and thrombosis when they encounter biological tissues. Here, we report a lubricant skin (L‐skin), a coating method based on the application of thin layers of bio‐adhesive and lubricant‐swellable perfluoropolymer that impart anti‐biofouling, frictionless, robust, and heat‐mediated self‐healing properties. We demonstrate biocompatible, mechanically robust, and sterilization‐safe L‐skin in applications of bioprinting, microfluidics, catheter, and long and narrow medical tubing. We envision that diverse applications of L‐skin improve device longevity, as well as anti‐biofouling attributes in biomedical devices with complex shapes and material compositions.
Development of a three‐dimensional blood‐brain barrier network with opening capillary structures for drug transport screening assays
1Department of Applied Chemistry, Graduate School of Engineering, Osaka University
The blood‐brain barrier (BBB), a highly selective barrier between the circulatory system and the central nervous system is composed of brain microvascular endothelial cells (BMEC), pericytes and astrocytes. It prevents therapeutics to penetrate the brain. Current models fail to replicate the structural complexity of the native BBB and do not exhibit enough functionality of specific transporters for drug assessment. For example, the transferrin receptor (TfR), mediating iron transport in the brain, shows great potential for drug transport but is poorly investigated in the current BBB models.
We developed a three‐dimensional self‐assembled microvascular network formed by BMEC, pericytes and astrocytes in a fibrin gel [1]. It exhibited perfusable capillary opening structures on the bottom of the hydrogel, as originally reported for 3D blood‐/lymph‐capillary networks [2]. It demonstrated size‐ selective permeation of different molecular weights of dextran, which highly correlated (R2 = 0.973) with the permeability values found with in vivo rodent brain. The TfR functionality was also confirmed by permeability assay. Efficient permeability coefficient (Pe) of transportable anti‐TfR antibody (MEM‐189) was about 4.77 x 10‐6 cm/s, being seven‐fold higher than those of isotype antibody and low transportable anti‐TfR antibody (13E4), suggesting a higher TfR functionality than previously reported [3]. This BBB model could be useful for the screening of therapeutics based on the TfR‐mediated transport efficiency.
[1] M. Piantino et al., submitted. [2] D. Hikimoto et al., Adv. Healthc. Mater.
Diversity in tissues engineering —from regenerative therapy to sustainable food production—
1Tokyo Women's Medical University
Various types of tissue engineering have developed last two decades. Tissue engineering has contributed to regenerative therapy and 3D tissue model fabrication. Recently, research and development for cultured meat production have been carried out, where tissue engineering has also been applied. As scaffold‐free tissue engineering, we have developed cell sheet‐based tissue engineering. Cell sheets are harvested from temperature‐responsive culture dishes by only lowering temperature. Cell sheets have been clinically transplanted for several types of diseases including heart failure and cornea damage. By stacking cell sheets, functionally beating cardiac tissues have been successfully fabricated. Contraction of beating cardiac tissues have been successfully measured as in vitro heart model. Furthermore, we have produced cultured meats by stacking bovine myoblast sheets. Thus, the applications of tissue engineering are becoming more diverse. The processes for producing transplantable tissues, tissue models and cultured meats are essentially equivalent. Common techniques are used for cell isolation, cultivation and three‐ dimensional tissue fabrication. On the other hand, completely different materials and production methods may be required from the point of view of safety, reproducibility, cost and sustainability. Safety is requested for regenerative therapy and cultured meat. Reproducibility is most requested for tissue models. Cost and sustainability are strongly requested for cultured meats. These diversity of tissue manufacturing will be compared and discussed.
Recent technology for large‐scale cell manufacturing
1Department of Biotechnology, Osaka University; Research Base for Cell Manufacturability, Osaka University, 2Department of Biotechnology, Osaka University, 3Research Base for Cell Manufacturability, Osaka University
Regenerative medicine and cell therapy use the cell transplants which are manufactured, requiring interdisciplinary activities not only from medicinal and biological fields but also from manufacturing field. The ‘cell manufacturability’, is the critical concept to realize the high‐performance manufacturing by bridging between biological and process aspects.
The technological developments for large‐scale system including the culture (cell expansion, upstream) and dispense to freeze the cell products (filling, downstream) are the critical to perform the cell manufacturing.
Recently we constructed the 10‐L suspension cultures of human iPSC aggregate culture and to perform the production of more than 10 billion iPSC in one batch after establishment of protocols for passage and medium change. In addition, the cell dispensing and freezing by considering the time‐dependency of cell quality. These knowledges will support cell manufacturer to make social implementation for large‐scale production of cells.
Development of support‐free bioceramic 3D printing technique
1Korea Institute of Materials Science
Recently, bioceramic 3D printing technology enables to fabricate various types of scaffolds for bone tissue engineering using bioceramic materials with high osteoinductivity. However, human bones have complex geometries, and in order to replicate them through bioceramic 3D printing, it is necessary to fabricate the supports together with scaffolds. The addition of supports allows the fabrication of overhang structures that are difficult to 3D print. However, this can increase overall printing time and material consumption. Moreover, there is a risk of cracking and breakage due to the brittleness of the bioceramic when removing the support from the 3D printed scaffolds. In this study, support‐free bioceramic 3D printing technique was developed to overcome these limitations. A supportive bath was prepared using a poloxamer‐based hydrogel with temperature‐sensitive properties, and the ceramic ink was extruded through 3D printing in the supportive bath. The supportive bath promotes the cementing reaction of the bioceramic as soon as the ink is extruded, facilitating the fabrication of various types of bone scaffolds. The bioceramic scaffolds manufactured using the supportive bath showed similar cell viability, proliferation, and osteogenic differentiation properties to those of the scaffolds fabricated through the conventional bioceramic 3D printing process. This study demonstrated that bioceramic 3D printing using supportive bath can reduce the overall process time and material consumption due to supports production, and can fabricate bone scaffolds with various and complex shapes.
Innovative advancement of random positioning machines for tissue engineering applications
1New York University Abu Dhabi
Random Positioning Machines (RPM) are commonly used for simulating microgravity for space biology investigations to determine the effects of less than 1 g environments on extraterrestrial organisms. Typically, cell culture flasks are used for experiments, limiting the complexity of in vitro models, capping number of study repeats and conditions per experiment, and restricting experiments to end‐point assays only. Recently, there is a surge in applying RPMs for tissue engineering and regenerative medicine research, including creating organoids and inducing phenotypic changes in cells of interests. Concurrently, tissue engineering technology are applied to improve in vitro models for space biology research, which have been limited to 2D models at best. To this end, we developed enabling technologies to increase throughput of RPM‐based experiments. Our engineered innovations allow for simultaneous testing of multiple samples, continuous image‐based real‐time feedback, and facilitate the use of 3D cell culture scaffolds, whilst maintaining microgravity quality. In addition, lesser amounts of biological resources are required, which allows novel small molecules and drugs to be tested. We present our work, which advanced RPM technology and also demonstrate its use in assessing the effects of microgravity on immune cells.
Label‐free and high‐throughput removal of residual undifferentiated iPSCs from their differentiated progenitor cells by inertial microfluidic cell sorter
1School of Chemical and Biomedical Engineering, Nanyang Technological University (Singapore), 2Institute of Molecular and Cell Biology, A*STAR Research Entities (Singapore), 3Critical Analytics for Manufacturing of Personalized Medicine, Singapore‐MIT Alliance for Research and Technology (Singapore), 4Department of Electrical Engineering and Computer Science, Biological Engineering, Massachusetts Institute of Technology (USA); Critical Analytics for Manufacturing of Personalized Medicine, Singapore‐MIT Alliance for Research and Technology (Singapore), 5Department of Electrical Engineering and Computer Science, Biological Engineering, Massachusetts Institute of Technology (USA), 6School of Chemical and Biomedical Engineering, Nanyang Technological University (Singapore); Lee Kong Chian School of Medicine, Nanyang Technological University (Singapore); School of Materials Science and Engineering, Nanyang Technological University (Singapore)
The presence of residual iPSCs amongst differentiated progenitor cells poses a high risk as it can result in teratomas and other forms of tumors after the transplantation. Yet, it is challenging to find the safe and specific cell surface markers for residual iPSCs, and implement the elimination using Fluorescence‐ activated cell sorting (FACS) or Magnetic‐activated cell sorting (MACS) at manufacturing scale cell production. In this study, iPSCs derived from Cord Lining Endothelial cells (CLECs) were differentiated into Spinal‐cord Progenitor Cells (SCPCs) through a 10‐day differentiation process. By profiling the size of SCPCs, we found that the large‐sized group contains largely cells with residual pluripotent markers (i.e., Oct4) while the small‐sized group has relatively fewer such cells. A sized‐based, label‐free separation using an inertial microfluidic‐based device was exploited to remove Oct4 positive cells. This simple device takes advantage of label‐free, non‐contact, and high throughput (i.e., up to 3 million cells per minute and even more when stacking devices) without affecting cell viability and functions. The sorted cells were verified with immunofluorescence staining, flow cytometry analysis, and other function culture‐based tests. In our preliminary sorting experiment, approximately 33 million SCPCs were sorted in 30 minutes into small‐sized and large‐sized groups. A following flow cytometer analysis showed that 50% of total Oct4 positive cells were sorted into the large‐sized group which occupies ∼16% of the original cell number. Our technology is promising for downstream processing of cell manufacturing workflow, ensuring the generation of high‐quality progenitor cells derived from iPSCs and safe therapy.
3D bioprinted cardiac tissues for heart repair
1University of Technology Sydney, 2The University of Sydney
Cardiovascular disease, including myocardial infarction (MI) and heart failure (HF), is a leading cause of death globally. Oiur laboratory has recently demonstrated that 3D bioprinted cardiac tissues containing human cardiac spheroids (hCSs) using alginate/gelatin (Alg/Gel) hydrogels are characterised by morphological, biochemical and pathophysiological features similar to the ones of the in vivo human cardiac tissues. Given their unique ability to mimic the human heart microenvironment, we tested if bioprinted hCSs in Alg/Gel patches could be used to improve impaired cardiac function in an in vivo mouse MI model. Infarcted MI mice received either (i) AlgGel acellular patches, or (ii) AlgGel patches with freely suspended cardiac cells, or (iii) AlgGel patches with hCSs. Control groups included (iv) mice that underwent a sham (SHAM) procedure and (v) infarcted mice that did not receive any patch (MI). We performed cardiac function measurements via ultrasound imaging up to 28 days. Epicardial transplantation of AlgGel patches containing hCSs was the only treatment to significantly (p = 0.010) improve left ventricular ejection fraction (LVEF%) from 41% (MI mice) to 64%. The improvement in LVEF% was not statistically significant for AlgGel patches with freely suspended cardiac cells (59%, p = 0.106). Bulk RNAseq analyses of heart tissues demonstrated similar gene expression profiles between SHAM and MI mice receiving AlgGel patches with hCSs. Altogether, our findings support the novel use of cardiac Alg/Gel patches containing hCSs to protect against MI‐induced HF.
Engineering skeletal muscle and neural tissues
1Biomedical and Electrical Engineering, School of Engineering, RMIT University, Melbourne, VIC 3001, 2School of Medicine, Deakin University, Waurn Ponds, VIC 3216, 3Graeme Clark Institute for Biomedical Engineering, University of Melbourne, Parkville, VIC 3052, 4Department of Surgery, The University of Melbourne, St Vincent's Hospital, Melbourne, VIC 3010
Bioengineering of active tissue constructs for tissue engineering or for disease modelling, requires the fabrication of structures that allow appropriate cellular development and maturation. This requires providing both the appropriate temporal, mechanical and biological cues to direct appropriate cellular behaviour. Our laboratories are investigating the use of hydrogels and biofabrication techniques for synthesis of cell laden constructs designed for the engineering of disease models and for tissue engineering. In particular, we are investigating the fabrication of constructs that allow the development of electrically active tissues such as skeletal muscle and the nervous system.
We have investigated the use of a number of different hydrogel formulations for the synthesis of neural and muscle 3D constructs ranging from naturally derived hydrogels, such as collagen, to synthetically derived peptide‐based hydrogels. We have used hydrogel based materials to create electrically active 3D neural structures that show marked differences in network activity compared to 2D cultures. In addition, we have used hydrogel based biofabrication methods to create 3D bioprinted skeletal muscle constructs that can support appropriate muscle differentiation and maturation as well as innervation and vascularization of de novo muscle on implantation into animal models.
In conclusion, biofabrication techniques using hydrogels that provide appropriate biological and mechanical cues can be successfully to engineer functional 3D neural and muscle tissues for disease modelling or for tissue engineering.
Biofabrication within cell‐laden gradient microgel suspensions for spatial control of differentiation
1UNSW Sydney
During tissue development, progenitor cells form functional tissue with high cellular diversity and intricate micro and macro architecture. Previous approaches have attempted to replicate this process with materials cues or through spontaneous cell self‐organization. However, cell‐directed and materials‐ directed organization are required simultaneously to achieve true biomimetic structure and function. Here, we show how integrating live stem cells with gradient microgel suspensions steers divergent differentiation outcomes. By tuning microgel stiffness and combining small and large microgels, complex gradients can be formed to steer cell behavior with spatiotemporal control over differentiation. Freeform printing of high‐density cell suspensions demonstrates the potential for hierarchical tissue biofabrication. Using a sacrificial ink, hollow channels can be formed for subsequent perfusion with endothelial cells, thus providing prototype vessel structures. Overall, jammed suspensions of microgels provides a tunable scaffolding for directing differentiation, where concurrent direct‐write printing paves the way to fabricate tissue gradients at multiple scales through integrated and printed cell populations.
Bioprinting of grafts with micro‐vascular patterns for rapid vascularization and therapeutics of ischemic disease
1UNIST
Vascularization of implanted artificial tissues remains an unresolved challenge in tissue engineering. Angiogenesis of host micro‐vessels and infiltration into implanted grafts are major mechanisms of in vivo vascularization. Various studies demonstrated that the transplantation of pre‐vascularized constructs with vascular patterns can induce rapid angiogenesis and anastomosis with the host vasculature. Also, several recent studies have shown that patterned vascular grafts facilitate the recovery of blood perfusion within ischemic tissues. However, although various approaches have demonstrated the critical factors inducing the vascularization by implanting the pre‐vascularized grafts, the optimal design of patterned vascular grafts for rapid infiltration of host blood vessels is still unknown. In this study, we engineered the functional grafts with pre‐designed microvascular patterns with co‐printing and culturing of endothelial cells and supporting cells. The matured micro‐vasculature within the bridge patterns were generated by the guidance of paracrine secretions in supporting cell patterns during in vitro culture process. We investigated the in vivo vascularization according to different designs of pre‐vascularized construct by implanting the printed graft into a murine femoral bundle model. Finally, the murine hindlimb ischemic model assay was performed to examine the therapeutic effects of grafts with designed micro‐vascular patterns. We believe that our technique may contribute to the development of therapeutics for ischemic disease and engineering various vascularized tissue grafts.
Recent advances in bioengineering for the fabrication and study of thick living tissues
1Harvard Medical School, 2Khalifa University
Recapitulating inherent heterogeneity and complex microarchitectures within confined print volumes for developing implantable constructs that could maintain their structure in vivo, has remained challenging. We present a multimaterial and embedded bioprinting approach to fabricate complex functional living tissues that can be implanted post‐printing, and retain their 3D shape in vivo for proper functioning. Our microfluidics‐based single‐nozzle 3D bioprinting platform enables laminar flow‐based voxelation of up to seven individual bioinks. Rapid switching between multiple bioinks minimizes time consuming processes such as switching nozzles and the alignments thereof. To improve spatial organization, precision, and printing speed of the complex geometries within clinically relevant sized constructs, self‐healing biodegradable colloidal gels as support baths are introduced. Upon crosslinking, they create intricate networks to support complex geometries and promote constructive remodeling of 3D printed tissues and organs. Successful fabrication of vascularized liver and skeletal muscle tissue constructs show albumin secretion and bundled muscle mimic fibers, respectively. The interconnected microporous networks of colloidal gels result in maintaining printed complex geometries while enabling rapid cell infiltration, in vivo.
Functionalized PLLA membrane and tubular scaffold for vascular grafts: Combining electrospining and 3D bioprinting techniqueper
1Startup 3D Biotechnology Solutions, 2Startup 3D Biotechnology Solutions ‐ 3DBS, 3Sao Leopoldo Mandic Faculty/Campinas, 4BARONE COMPANY
Tissue engineering approaches, such as electrospinning (ES) and 3D bioprinting, have been explored for manufactured vascular graft, in order to solve some challenges and limitations of conventional nonbiodegradable materials because of their thrombogenicity and inability to grow, while autologous vascular grafts involve invasive procedures. This project aims to seed cells by an automated platform, using a 3D bioprinting with a rotary axis. Therefore, our goal is to use a hybrid system combining the ES for the generation of a tubular bilayer scaffold with the 3D bioprinting to insure homogeneous cells deposition. The fiber was fabricated using PLLA (control groups) and PLLA/Gelatin (functionalized group) in a 6‐8mm rotary axis. The ES process and solution parameters were optimized to obtain fibers based on PLLA and PLLA/Gelatin, in which the inner layer for Endothelial Cells (ECs) proliferation, was composed of random nanofibers and the outer layer, for Smooth Muscle Cells (SMCs) infiltration, of aligned microfibers. Two methods of cell seeding were compared: a manual and an automated approach, using a 3D bioprinter. Methods were analyzed by cell viability and histology. It was observed a difference in viability and cell growth in functionalized fibers. Otherwise, there are challenges related to the fibers reproducibility, the bilayer electrospinning in the rotary axis of 6 ‐ 8 mm diameter, cell infiltration and migration for the scaffolds. However, taking advantage of the electrospinning mechanical and porous scaffold formation and the 3D bioprinting hierarchical cell deposition, the use of this hybrid system is promised for vascular graft development.
Constructing a human liver tissue model that mimics the liver sinusoid
1School of Life Science and Technology, Tokyo Institute of Technology, 2Shimadzu Corporation
Liver is a vital organ that takes a part in drug metabolism, detoxification, protein production. It is difficult to construct hepatic histological structure having cellular polarities to express its specific and multiple functions. In recent, we invented a hepatic tissue on chip to co‐culture hepatocytes and endothelial networks on EHS‐gel for drug metabolism study (Tamai, M., et al., Tissue Engineering, 2020). However, construction of human iPS cell (hiPSC)‐derived hepatic tissue was impossible on EHS‐gel. Here, we aimed to make a liver sinusoidal culture model derived from human iPS‐derived hepatocytes and liver sinusoidal endothelial cell (LSEC)s by microfluidic device technology. Hepatocyte lineage cells and LSECs were generated from hiPSCs by each adequate method. HiPSC‐derived LSEC and hepatic lineage cells could be confirmed by each specific‐marker gene expression. Especially, the expression of CD32B, an LSEC‐specific marker, reached its peak Day 15 in LSEC‐differentiation process from undifferentiated stage. LSEC‐specific functions such as high scavenger function and immune tolerance were observed by absorption of AcLDL‐488 and stimulation of LPS in human iPS‐derived LSEC, but not in HUVEC. For constructing a sinusoidal structure model, microfluidic device was designed as the following. The device has two channels to supply each specific culture medium on the upper/lower side of the cell culture porous membrane. To reconstruct sinusoidal structure, endothelial cells and hepatocytes were attached to the front and rear side of the membrane, respectively in our microfluidic system. This sinusoidal chip is expected to be applies in drug metabolism study and screening for anti‐viral drug.
Tissue‐derived extracellular matrix hydrogel for gastrointestinal organoid culture
1Yonsei University, 2Yonsei University College of Medicine, 3Soongsil University
Various types of organoids, which recapitulate cell composition and function of actual organs, have been developed as innovative in vitro models to predict human physiological response and investigate disease development. The organoid transplantation as regenerative medicine has also emerged in recent years. In general, 3D extracellular matrix (ECM) hydrogels are required to culture organoids. In this study, we developed ECM hydrogels derived from stomach and intestinal tissues to culture the gastrointestinal organoids. ECM hydrogels contained tissue‐specific proteins present in actual gastrointestinal tissue, suggesting that these hydrogels can provide more suitable ECM environments for gastrointestinal organoid culture. We confirmed that gastrointestinal organoids exhibiting various cell types and functional phenotypes could be produced in tissue‐derived ECM hydrogels. Moreover, we confirmed the tissue‐specific and age‐related effects of tissue ECM hydrogels on organoid development. The applicability of ECM hydrogels for organoid transplantation was tested in injured epithelium of stomach and intestine. In summary, tissue‐derived ECM hydrogel could be utilized as organoid culture platform studying tissue development and diseases as well as regenerative medicine. This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government, the Ministry of Science and ICT (MSIT) (No. 2021R1A2C3004262).
A novel large‐scale production method for homogeneous cardiac organoids for regenerative therapy and drug discovery
1Keio University
Organoid technology is one of the most promising tools for regenerative therapy and drug discovery. However, there are challenges with producing a large number of homogeneous cardiac organoids (COs) derived from human pluripotent stem cells (hPSCs). Here, we describe
Bimatoprost ameliorates colistin induced nephrotoxicity
1Department of Microbiology, CHA University School of Medicine, Seongnam, Republic of Korea., ORGANOIDSCIENCES Co., Ltd., 406, 560, Dunchon‐daero, Jungwon‐gu, Seongnam‐si, Gyeonggi‐do, Republic of Korea, CHA Organoid Research Center, CHA University, Seongnam, Republic of Korea., 2Department of Microbiology, CHA University School of Medicine, Seongnam, Republic of Korea., 3Department of Nephrology, Department of Internal Medicine, CHA Bundang Medical Center, CHA University School of Medicine, Seongnam, Republic of Korea
Colistin is effective against multidrug‐resistant Gram‐negative bacteria. However, the high rates of nephrotoxicity caused by colistin limit its clinical use. but it causes nephrotoxicity, which limits its clinical use. To find a new compound, which can ameliorate colistin‐induced nephrotoxicity, 1,707 compounds were obtained from Korea chemical bank. Using a High‐content‐screening image‐based assay, we unveiled bimatoprost (prostaglandin F2α) that could ameliorate colistin‐induced nephrotoxicity. According to the in vitro and in vivo tests, bimatoprost effectively ameliorates colistin‐induced nephrotoxicity. In vitro, Colistin induced cell cytotoxicity in cultured human proximal tubular cells (HK‐ 2) in dose‐dependent manner. In vivo study, using Female C57BL/6 mice (n = 5 in each group) were randomly divided into the four groups and injected intraperitoneally for 14 consecutive days. Colistin injection increased the serum levels of blood urea nitrogen and creatinine. However, bimatoprost prevented deterioration of these serum levels. And Kim‐1(kidney injured marker‐1) markers were observed less frequently for the bimatoprost treated groups. To validate the protective effects of this drug on colistin‐induced nephrotoxicity, its protective effects were investigated in cellular, animal, and kidney organoid models using human embryonic stem cells (hESCs). we present a novel chemical, bimatoprost that ameliorates colistin‐induced nephrotoxicity.
Screening niche factors favoring hair lineage differentiation using a multiphoton microfabrication and micropatterning (MMM) technology‐ based cell niche factor biochip (CNFB)
1Tissue Engineering Laboratory, Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong Special Administrative Region, China
Engineering hair follicles using hair follicle stem cells (HFSC) is hindered by the low availability of cell source and easy loss of cell phenotype upon in vitro culture. Neonatal human epidermal keratinocytes (HEKn), an alternative cell source to HFSCs, have been employed to recreate in vitro hair follicle by differentiating to hair lineage upon exposure to appropriate inductive signals. However, factors that drive such epithelial cells to hair lineage differentiation have not been fully elucidated. A well‐ established Multiphoton Microfabrication and Micropatterning (MMM) platform enables the fabrication of arbitrary 3D protein microstructures with surface functionalization of niche factors (e.g., extracellular matrix (ECM) proteins, soluble factors) at the micron scale. Herein, we developed a cell niche factor biochip (CNFB) using such technology to screen the individual or combinational niche factor(s) favoring the hair lineage differentiation of HEKn. Specifically, different cell niches including ECM proteins, soluble factors, topological features, and elastic modulus were engineered on the glass surface in each biochip well. After culturing HEKn for 48 hours, the levels of hair lineage differentiation and epidermal stemness were evaluated by immunofluorescence staining of Krt75 and Cytokeratin 5 (C5), respectively. Results showed that fibronectin and BMP2 significantly enhanced Krt75 while reduced C5 expression, demonstrating their positive roles in inducing hair differentiation. Investigating the effect of combinational niche factors in different categories is underway. This study exemplified the potential application of CNFB in studying cell differentiation, provided useful insights for manipulating in vitro hair lineage differentiation of epithelial cells, facilitating future design of bioengineering products.
Accurate drug screening by design of antifouling channel wall microfluidic platform
1School of Electrical and Electronic Engineering, Yonsei University, 2School of Life Sciences, Ulsan National Institute of Science and Technology, 3yonsei
After the concept of the 3D cell culturing technique was introduced, various attempts have been made for accurate drug screening. Among materials used for the microfluidic devices in the laboratory, PDMS has been the most popular because of advantages such as flexibility, biocompatibility, and gas permeability. However, the permeability of PDMS promotes the non‐specific adsorption of small hydrophobic molecules which is a major issue in drug screening. Therefore, we developed an anti‐fouling microfluidic device with a perfluoro‐polymer lubricant coating. This hydrophobic lubricant barrier possesses intrinsic advantages in resistance to absorption of organic solvent, and bacterial infection, and also fully repels the adhesion of various liquids, including drugs. In this study, we evaluated cell culture in the anti‐fouling microfluidic device with Caco‐2 and BBB and observed organ‐like structures. About 30% of O2 and CO2 gas permeability of anti‐fouling microfluidic device was decreased by lubricant coating. However, we confirmed that decreasing gas permeability caused no damage to cell culture which was evaluated by PCR and fluorescence staining imaging. Real‐time RT‐PCR analysis revealed that the hypoxia‐inducible genes hypoxia‐inducible factor(HIF)‐1α, VEGF, SOX2, and CD42 were no significant difference between the PDMS based microfluidic chip and the anti‐fouling microfluidic device. Drug compound quantification was performed via HPLC. Compared to anti‐fouling microfluidic device group, PDMS based microfluidic group drops the amount of drug by about 27% in various flow rate conditions. Our strategy enables the evaluation of the accurate cellular uptake of target drugs into the cytosol by blocking the drug adsorption of PDMS.
Development of aging‐induced neurovasculature‐on‐a‐chip to study the aging‐mediated neurodegenerative disease
1KIST
Aging is a critical risk factor for developing neurodegenerative diseases, including Alzheimer's disease (AD). Abnormal deposits of aggregated proteins such as amyloid ß (Aß) and hyperphosphorylated tau (p‐ Tau) were observed in the brain tissue of older individuals with increased expression of advanced glycation end products (AGEs); however, it remains unclear whether the levels of these deposits are linked with the degree of cognitive impairment. While the prevalence of aging and AD has improved in the last decades, the mechanism of age‐related AD remains poorly understood. To clarify the aging‐ induced AD pathogenesis, we developed a neurovasculature‐on‐a‐chip (NV chip) that mimics the aged microenvironment in the brain. We reconstructed the advanced glycation end‐products (AGEs)‐ accumulated 3D glycated matrix for the aged brain model. Human brain microvascular endothelial cells (HBMEC) and neural progenitor cells (ReN cells) were co‐cultured in the NV chip. In an aged NV chip, the vessel permeability increased, and accordingly Aß and pTau accumulated in the 3D matrix in the NV chip, causing the dysfunction of neuron cells. Furthermore, the neurodysfunction was attenuated by treating chemical inhibitors targeting AGE receptor (RAGE) signalings. Our aged NV chip would be an impactful platform to clarify the axis of aging and AD and contribute to discover various AD drug candidates.
Effects of hydrogel charge and LCST on tissue integration for cartilage tissue engineering
1Rice University
Thermogelling hydrogels are a strong platform in cartilage tissue engineering due to their injectability, ability to space‐fill and thermogel rapidly within a defect and be combined with a chemical crosslinker to lock the network into place. In this current work, a study was designed to assess the role of hydrogel LCST and charge on the infiltration of native cells and integration with the native tissue in a cartilage explant model. P(NiPAAm)‐based hydrogels possessing 0, 5, and 10 mol% dimethyl‐γ‐butyrolactone acrylate (DBA) and crosslinked with a poly(glycolic acid)‐poly(ethylene glycol)‐poly(glycolic acid)‐ di(but‐2‐yne‐1,4‐dithiol) (PdBT) crosslinker bearing charged peptide sequences to possess positive, neutral, or negatively charge were utilized. Using this cartilage explant model, a relationship between hydrogel LCST and charge was established in promoting native cell infiltration and integration with the tissue, assessed via mechanical pushout testing and histology. Importantly, only negatively charged hydrogels demonstrated cell infiltration and integration. Moreover, the negatively charged hydrogels with changing LCST (5 and 10 mol% DBA) demonstrated cell infiltration and remodeling of the hydrogel as observed via histology. Indeed, significantly greater integration with the native tissue was established for the 10 mol% DBA groups possessing negative charge via mechanical pushout testing compared to the neutral and positively charged hydrogels, demonstrating the importance of both increasing hydrogel hydrophilicity and hydrogel charge in promoting integration with the native tissue. These data demonstrate the importance of understanding how the physicochemical properties of hydrogel materials influence behavior of the native tissue and can ultimately inform cartilage tissue engineering strategies in vivo.
Engineering multi‐cellular spheroid using bioinspired materials for tissue engineering
1Hanyang University
Recently, technologies to culture one or more cell types in 3D conditions have attracted a great deal of attention in tissue engineering. In particular, stem cell spheroids have been reported for improved viability, self‐renewal capacity, and differentiation potential. However, for the use of multi‐cellular spheroids in tissue engineering, modulation of spheroid functions with instructive signals is critical. We have been developing ECM‐mimicking fibrous materials decorated with cell‐instructive cues, which were incorporated within 3D stem cell spheroids to tune their functions as modular building blocks for bottom‐ up tissue‐engineering applications. In this presentation, engineering ECM‐mimicking materials and their use in 3D spheroid fabrication would be discussed with utilizing human‐adipose‐derived stem cells (hADSCs) with aims of enhanced bone regeneration.
Extrusion base 3D printing fidelity refinement of Kagome structure scaffolds for bone regeneration by design for additive manufacturing
1Wonkwang University
An open‐pore porous structure with interconnection as well as mechanical robustness is a typical characteristic required for a scaffold for bone regeneration. In order to control these characteristics, various types of structural designs have been performed, and 3D printing technology has been used to implement them. Although the extrusion‐based 3D printing technology is the most advantageous when considering the clinical accessibility of the scaffold for bone regeneration, there was a difficulty in fully implementing the scaffold with a complex microstructure as designed. Therefore, in order to improve the 3D printing fidelity of the scaffold, the existing scaffold design was modified in consideration of additive manufacturing, and the 3D printing fidelity evaluation was performed on the modified scaffold. As a result, the implementation of bridge strands that occur when fabricating scaffolds through an extrusion‐ based 3D printing system was stably improved through design for additive manufacturing (DfAM). In particular, DfAM improved printing fidelity thanks to a design in which the length of the bridge strands was induced to decrease gradually, even when the stack thickness was changed. Therefore, the printability of the theoretical predicted values of the Kagome scaffold compressive stiffness could be improved from 76.6% to 95.2% through DfAM. Moreover, since the cell proliferation ability of the Kagome structure, which is promising for bone regeneration ability, is similar to that of the conventional Kagome scaffold, it suggests that the DfAM ensured the stable fabrication and implementation of the Kagome scaffold.
Developing next generation adipose tissue grafts for soft tissue reconstruction
1University of Otago
Autologous fat grafting has favourable potential as a regenerative strategy to repair large contour defects (e.g. breast reconstruction or scar/burn repair). Clinically, there is a limit on the volume of lipoaspirate which can be utilised to repair a soft‐tissue defect. Surgical complications are the result of poor graft vascularisation and are hindered further by the poor structural fidelity of lipoaspirate as a filling material. This study aims to engineer lipoaspirate‐derived adipose tissue grafts with biologically and clinically‐ admissible structural and functional properties.
Patient‐derived lipoaspirate was crosslinked by establishing bonds between ECM proteins within the material. The degree of crosslinking was tuned and covalent bond formation measured using mass spectrometry. To predict patient response, SWATH‐MS was used to identify differences in patient ECM and crosslinked grafts were implanted in vivo using a subcutaneous mouse model. Functional vessel formation and resorption were quantified using micro‐CT and tissue‐remodelling assessed via histology.
There was an increase in the relative abundance of covalent bonds present with increasing degree of crosslinking (<10‐fold increase). When extruded, crosslinked lipoaspirate had better shape fidelity compared with native lipoaspirate – demonstrated by a 60% reduction in fibre diameter (p < 0.05). Crosslinked lipoaspirate remained viable over long‐term culture and resulted in more predictable resorption profiles (standard deviation reduced by 52%) when implanted in vivo.
This crosslinking approach is tunable and functional across different patient samples. Improving the structural properties of lipoaspirate through minimal manipulation has clinical utility for the delivery of grafts with higher shape‐fidelity and therefore increased graft survival when implanted.
Functional chitosan hydrogels for intra‐urethral stents: processing, in vitro and in vivo evaluations
1Laboratoire Ingenerie des Materiaux Polymeres, Universite Claude Bernard Lyon 1, INSA de Lyon, Universite Jean Monet, CNRS UMR 5223, Univ Lyon, 15 bd A. Latarjet, 69622 Villeurbanne Cedex France, 2Interaction Cellule‐Environnement, Vetagro Sup, Univ Lyon, 1 avenue Bourgelat, 69280 Marcy‐ l'Etoile France, 3Institut Lumiere Matiere, Universite Claude Bernard Lyon 1, CNRS, UMR 5306, Univ Lyon, 2 rue Victor Grignard, 69100 Villeurbanne Cedex France/Nano‐h, 2 Pl. de l' Europe, 38070 Saint‐ Quentin‐Fallavier, 4Departement of de radioterapie oncologie, centre hospitalier et universitaire Lyon Sud, Univ Lyon, 165, chemin du Grand‐Revoyet, 69495 Pierre‐Benite, France, 5CNRS@CREATE Ltd, CREATE Tower, #08‐01, 1 Create way, Singapore 138602/Laboratoire Ingenerie des Materiaux Polymeres, Universite Claude Bernard Lyon 1, INSA de Lyon, Universite Jean Monet, CNRS UMR 5223, Univ Lyon, 15 bd A. Latarjet, 69622 Villeurbanne Cedex France, 6CNRS@CREATE Ltd, CREATE Tower, #08‐01, 1 Create way, Singapore 138602 / Laboratoire Ingenerie des Materiaux Polymeres, Universite Claude Bernard Lyon 1, INSA de Lyon, Universite Jean Monet, CNRS UMR 5223, Univ Lyon, 15 bd A. Latarjet, 69622 Villeurbanne Cedex France
Patients with prostate cancer may undergo prostatectomy, hormonal therapy and/or radiotherapy. Radiotherapy can result in side effects, such as urethral stenosis. The overall incidence of urethral stenosis after prostate cancer is high and requires the insertion and later removal of a urinary catheter. This study proposes a resorbable and imageable intra‐urethral stent based on chitosan hydrogel to treat the urethral stenosis, with the advantage of not being necessary to remove the device in the end of the treatment. A hydrogel formulation associated with two contrast agents were tested: (1) superparamagnetic iron oxide nanoparticles coated with chitosan (SPION‐Ch) and (2) Chitosan grafted with DOTAGA complexed with gadolinium. The stents were processed by interrupted coagulation of high concentration of chitosan/contrast agent solutions. In both cases, the amount of contrast agents was optimized for MRI. In vitro tests showed the resorption of the material in artificial urine. Moreover, the mechanical properties of stent prototypes are comparable to conventional urinary catheters. Both formulations of the loaded hydrogel showed cytocompatibility. In vivo (rat and pig models), these loaded hydrogels allowed to localize the device in the body and to follow resorption in vivo using MRI. Further work is on‐going to analyse tissue reaction after implantation in order to validate safety and therapeutic approach.
A new way of producing natural antibacterial peptides from probiotics through intracellular stimulation by internalized polysaccharides nanoparticles as prebiotics
1Seoul National University
Recently, the gut microbiota has been attractive because they affect many human diseases such as cancer, Alzheimer's, aging, obesity, and immune activity. Among them, probiotics interact with the gut microbiota and provide health benefits by enhancing their antimicrobial activity against pathogens. We have studied the effect of polysaccharides (inulin, dextran, pullulan, and registered starch) nanoparticles (PSNPs) prepared by self‐assembly through hydrophobic interaction after conjugation of hydrophobic groups into hydroxyl groups in the PS as a new type of prebiotics on the inherent microbial activity of probiotics compared with PSs themselves. The PSNPs were internalized into probiotics when checked by confocal microscopy and FACS. The internalization of PSNPs was dependent on the sizes of PSNPs, energy, and sugar transporter. The PSNPs‐internalized probiotics showed higher antimicrobial activities against Gram‐positive and Gram‐negative pathogens than PS‐treated ones. Interestingly, by the transcriptional analysis, PSNPs‐internalized probiotics significantly showed a higher stress response to several heat shock proteins than probiotics, suggesting that the nanoprebiotics as a new type of intracellular inducer to the probiotics pave a way for the production of natural antibacterial peptides in vitro and in vivo. Also, the composition of the gut microbiome was clustered by the administration of the PSNPs. Furthermore, the administration of the PSNPs in the probiotics contributed to decreasing the number of pathogens and increasing the beneficial bacterial species in mice. More interestingly, the anti‐ inflammatory effects of probiotics internalized with the immune‐modulating agent were shown in the intestine of the DSS‐induced colitis mouse model.
Injectable nano‐complex hydrogels for immune modulation‐mediated tissue regeneration
1Korea Institute of Science and Technology
The immune system plays a central role in tissue regeneration, however, chronic inflammation disrupts the formation of the original architectures and restoration of the biological functions of the tissues. Anti‐ inflammatory drugs to reduce the inhibitory effects of chronic inflammation for balancing the immune system have been restrictively used because the drugs could show side effects via off‐target endocytosis and several administrations of high concentration. For this reason, the development of a new strategy for the ultimate regenerative system to adjust inflammatory responses was required to induce tissue regeneration. Here, we suggest an injectable nano‐complex hydrogel system as an anti‐inflammatory drug depot system for sustained drug release that provides long‐term effective therapeutic advantages. Amphiphilic poly(organophosphazene), which has temperature‐dependent nanoparticle forming and sol‐ gel transition behaviors when dissolved in an aqueous solution, was synthesized for anti‐inflammatory drug delivery. The polymer solution incorporating anti‐inflammatory drugs showed nano‐sized spherical complexes and turns into a hydrogel type after local injection within the target site. The nano‐complex was slowly released from the injected hydrogel and delivered the drugs to the target cells for an extended time with a single injection. Effective prevention and long‐term anti‐inflammatory disease treatment with fewer side effects were observed in in vitro and in vivo model
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Promoting neural regeneration on biomimic surface‐modified scaffolds by functionalized carbon nanotubes
1Korea Carbon Industry Promotion Agency, 2Dankook University
Carbon nanotubes (CNTs) offer attractive attributes that are useful in innovative biomaterial design for neuroscience research due to their nanoscale features, modifiable chemical functionalities, and tunable electrical properties. In this study, we developed a new cell culture system by immobilizing functionalized CNTs onto tissue culture polystyrene plates via covalent attachment, and studied the effects of this electroactive substrate on the neuronal differentiation of PC‐12 cells with a surface‐ entrapped PTEN inhibitor. Compared to glass coverslip control, the percentage of neuronal cells forming neurites under nerve growth factor (NGF) stimulation increased by a factor of 4.0, 7.8, and 10.0, when cultured on immobilized carboxylated CNT substrate, PEGylated CNT substrate, and PEGylated CNT substrate in the presence of 10 nM drug, respectively. The drug‐loaded, biofunctionalized CNT substrates down‐regulated the expression of PTEN and up‐regulated the Akt/ERK signaling pathway, thereby providing the mechanism for the improved neuronal differentiation of PC‐12 cells. These results highlight the promise of the electroactive and drug‐releasing CNT biomaterials for promoting neuronal culture and differentiation, and suggest their potential utility in future neural regeneration applications.
Antioxidant nanoceria‐decorated scaffolds (nanofiber, porous, films) for diabetic bone regeneration via adhesion‐mediated and TGF‐β1 stimulated MSC differentiation and endothelial function
1Research Professor, 2Professor, 3student
Restoring critical‐sized bone defects in patients with diabetes mellitus (DM) remains a challenge in clinical treatment because of high blood glucose levels, oxidative stress, and elevated reactive oxygen species (ROS) microenvironment in the bone defect region. Cerium oxide nanoparticles (C) have the ability to protect cells from oxidative damage by actively controlling the excessive cellular ROS and also by accelerating bone regeneration. In this study polycaprolactone electrospun nanofibers (P) were subsequently coated with clustered layer of cerium oxide nanoparticles (P@C) for scavenging ROS by multi‐catalase properties of C and also significantly improved the osteointegration under diabatic conditions. The Physico‐chemical characterization using AFM, XRD analysis, contact angle analysis, XPS analysis, and mechanical tests has confirmed the presence of homogeneously distributed C clustered nanoparticles on the surface of P nanofibers. Mesenchymal stem cells (MSC) recognized the nanoscale (tens of nm) topology of nCe‐scaffolds, presenting highly upregulated integrin sets and adhesion‐related molecules. The mechanistic pathway and interactions connecting P@C nanofibers with cell proliferation and differentiation was due to the upregulation of osteogenic potential via integrin‐mediated TGF‐β1 activation. In‐vivo implantation of P@C 3D porous scaffolds in the calvarium defect model showed that scaffolds could eliminate ROS production and also accelerate bone regeneration under diabetic conditions. We consider the currently‐exploited P@C scaffolds can be promising drug‐ and cell‐free therapeutic means to treat defective tissues like bone in diabetic conditions.
Strategies for regeneration and repair in the injured spinal cord
1Department of Neurosurgery, CHA University School of Medicine, CHA Bundang Medical Center, Seongnam, KOREA, 2Department of Biomedical Science, College of Life Sciences, CHA University, Seongnam, KOREA, 3School of Integrative Engineering, Chung‐Ang University, Seoul, KOREA, 4Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, KOREA
Traumatic spinal cord injury (SCI), in which the axon is physically damaged, is a serious condition that can cause permanent neurological dysfunctions including motor dysfunction and neuropathic pain. In this study, we designed a hyaluronic acid (HA)‐based hydrogel combined with decellularized brain matrix (DBM), polydeoxyribonucleotide (PDRN), TNF‐α/IFN‐γ primed mesenchymal stem cell‐derived extracellular vesicles (TI‐EV), and human embryonic stem cell‐derived neural progenitor cells (NPCs). This injectable formulation (DBM/PDRN/TI‐EV/NPC@Gel) could provide structural support within nerves, and we predicted that nerve regeneration would be possible due to the synergistic effects of the included substances. The results of this study demonstrated that the hydrogel promoted the proliferation and differentiation of NPCs in vitro. Functional motor recovery was also confirmed to be possible through the reduction of the inflammatory response and the formation of neurons and oligodendrocytes in in vivo animal tests. This novel bioactive injectable HA‐based hydrogel is an effective candidate for SCI regeneration.
Transplantation of human embryonic stem cells alleviates motor dysfunction in AAV2‐Htt171‐82Q transfected rat model of Huntington's disease
1Chungbuk univ, 2Chungbuk national univ, 3Chungbuk national univ hospital, 4Chungbuk nationl univ, 5Chungbuk National University
A calibrated forceps compression mechanical in vivo spinal cord injury model and behavioral assessment post injury
1Center of Tissue Engineering and Regenerative medicine, Faculty of Medicine, University Kebangsaan Malaysia, Jalan Yaacob Latif, 56000 Cheras, Kuala Lumpur, 2Department of Orthopaedics & Traumatology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, 3Institutes of Medical Science Technology, Universiti Kuala Lumpur, Kajang, 4Department of Surgery, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latiff, Bandar Tun Razak, Kuala Lumpur
Spinal cord injury (SCI) is a fundamental medical problem that can be associated with severe disability. Contusion SCI models are commonly used methods, however they need impactors. These impactors are extremely expensive and also not readily available. Compression SCI model especially forceps compression method are simple, inexpensive and manageable laboratory method.The objective of this study is to induce SCI by mechanical method and to observe sensory & locomotor assessments in injured & healthy groups. The mechanical injury was created using calibrated forceps method using Dumont # 5 forceps, producing compression for 15 sec at T12 vertebrae. Somatosensory evoked potentials (SEP) were also recorded at pre‐injury, post‐injury (within 30 min), 14 days & 28 days to observe any delay in latency and amplitude in conduction. The gait analysis of injured and healthy animals, was assessed for four weeks (28 days) using open field gait analysis apparatus, beside gait analysis other movement assessments such as running wheel, grid walk and inverted grip test at day 3, 7, 14, 21 & 28 days post injury was also scored for both groups by two blind observers. The animal completely lost lower limb movement reflecting efficacy and reproducibility of injury model. The injured group re‐gains some balance after 2 weeks; and didn't get complete movement until 4 weeks. The running wheels, grid walk and inverted grid test results also indicate slight improvement between 2‐4 weeks. Calibrated compression method can restrain hind limb motion producing paralytic rats, and it is a successful laboratory model to induce Spinal injury.
Heparin‐conjugated growth factors‐immobilized aligned electrospun nanofibers for nerve regeneration in tubular polycaprolactone/gelatin based nerve guidance conduits
1Department of Chemical Engineering, Faculty of Engineering, Graduate School, Kyushu University, 744 Motooka, Nishi‐ku, Fukuoka 819‐0395, Japan
Bioactive cues as well as morphological features of extracellular matrix (ECM) play significant role for tissue regeneration. Consequently, biomimicking ECM‐inspired features into tissue‐engineered scaffolds may help realize tissue‐like reparative functions. In spite of this, extensive ex vivo manipulations of growth factors (GFs) may compromise their bioactivity. Heparin‐functionalized biomaterials may simultaneously allow the sequestration of exogenous and endogenous GFs. Herein, we fabricated nerve conduits consisting of tubular polycaprolactone/gelatin (PCL/Gel) scaffolds and heparin‐installed GFs‐ immobilized aligned electrospun nanofibers. We systematically studied heparin and GFs immobilization on membranes as well as ascertained retention of GFs in vivo for up to 1‐wk in a sciatic nerve defect model in rats. Besides, a thorough analysis of material parameters was performed to gain an insight into the structural stability of conduits as well as evaluate the permeability of bovine serum albumin (BSA), a model protein in vitro. The bioactivity of nerve conduits was further assessed in short‐term for up to 1 week as well as for long‐term for up to 1 month in a sciatic nerve defect model in rats. Heparin and GFs were successfully immobilized onto membranes. Approximately, 0.1 mg of heparin was covalently conjugated to PCL/Gel nanofibers. Besides, basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) were electrostatically immobilized; the immobilization density of GFs was significantly higher in heparin‐conjugated membranes and increased in a concentration‐dependent manner. Nerve conduits were also populated by endogenous host cells. Conclusively, these nerve conduits may have broad implications for nerve regeneration as well as other bio‐related research disciplines.
Therapeutic potential of neural stem cells preconditioned with baicalein‐ enriched fraction (BEF) for ischemic stroke rat models
1School of Health Sciences, Health Campus, Universiti Sains Malaysia, Kelantan, Malaysia.
Ischemic stroke, triggered by the abrupt interruption of cerebrovascular blood flow, could lead to permanent neuronal cell death. Recently, multipotent neural stem cell (NSC) grafting has emerged as potential therapy to regenerate the damaged brain tissue. However, the hostile microenvironment in the ischemic brain region is challenging for the survival of transplanted cells. In this regards, NSC culture was optimized with baicalein‐enriched fraction (BEF) extracted from Oroxylum indicum, to enhance its survival rate and therapeutic potential. Ischemic stroke was induced in Sprague Dawley rats using endothelin‐1 (ET‐1) which constrict the middle cerebral artery (MCA), mimicking ischemic injury in human brain. In vitro expandable NSCs were preconditioned with BEF at optimum dosage of 3.125 μg/ml for 48 hours determined through MTT assay before the cells were transplanted into the ET‐1 induced ischemic stroke rat groups. Rat behaviours and stroke severity were observed and recorded for 14 days. Significant improvements in neurological behaviours occurred after the transplantation of BEF‐ preconditioned NSC compared to control and non‐preconditioned NSC transplantation groups. Through TTC staining, the non‐treated group showed brain infarct of 23.807 ± 2.60% while in the NSC‐treated and BEF‐preconditioned NSC treated groups, infraction volumes were 17.784 ± 2.33% and 11.535 ± 1.44%, respectively. BEF‐preconditioned NSC‐treated group induced angiogenesis and improved neuronal degradation, cell necrosis and inflammation. In conclusion, the present study has proved the potential of BEF extracted from O. indicum in contributing to the significant stroke recovery and brain tissue remodelling after the ischemic stroke injury.
Tri‐culture of intestinal epithelial cells, macrophage, and bacteria on a chip for culture model of inflammatory bowel disease
1Tokyo Institute of Technology, 2Shimadzu Corporation
Inflammatory bowel disease (IBD) is a chronic inflammatory disease caused by an over‐activation of the intestinal immune system against bacteria, however IBD development mechanism is still vague. It is difficult to co‐culture epithelial cells and bacteria because the rapid growth of bacteria induces cell‐death. Here, we aimed to establish a tri‐culture system of intestinal epithelial cells, immune cells, and bacteria using a microfluidic device having two channels to create an IBD model. C2BBe1, a colon cancer‐derived cell line, cells were cultured on a porous membrane in a microfluidic device. Tight junction formation of C2BBe1 cells was confirmed by increase of trans‐epithelial electrical resistance (TEER). Mouse macrophage‐like cell line: RAW264 was cultured on the lower layer of the device. TEER of C2BBe1 could be maintained before addition of E. coli, but was significantly decreased after the addition of E. coli into the RAW264 channel, suggesting that the tight junction of C2BBe1 was impaired. Expression levels of inflammatory cytokines such as mTNF‐α, mIL‐1β, and mIL‐6 were significantly increased in RAW264 cells after the addition of E. coli and those of hIL‐6 and hIL‐8 were increased in C2BBe1. Because several reports that TNF‐α, IL‐1, IL‐6, and IL‐8 were higher in the serum of the IBD patients correspond to our above results, our tri‐culture system can be expected to be useful for investigation of the molecular mechanism and the drug screening.
Engineering large and thick vascularized tissue constructs and its application in tissue engineering
1National Tsing‐Hua University
Nowadays, the main approaches to engineer vascularized tissues are to develop biomaterials combining with cell‐based therapy to achieve rapid vasculogenesis angiogenesis and anastomosis between engineered and host blood vessels. However, the thickness of engineered vascularized tissues in animals were still limted, because the hydrogel becomes less permeable with increasing thickness. To overcome this challenge, a diffusion‐based computational simulation was used to guide and optimize the geometry of hydrogel structures. Then, human derived blood vessel forming cells were encapsulated into prepolymer and patterned into different kinds of three‐dimensional (3D) hydrogel structures by photolithography micro‐patterning technique. After subcutaneous implantation into mice, the cell‐laden hexagonal structures can guide vasculogenesis and accelerated angiogenesis to form uniform distributed vascular networks in the large (diameter ≈2cm) and thick hydrogel (> 2 mm) within 7 days in mice. These vascularized soft tissues were subsequently used as the vascularized flap for the repair of volumetric muscle loss (VML) defects. After 1‐month, large portions of regenerated muscle with larger muscle fibers were well distributed at the site of injury in the group with high densities of perfused vascular networks. These findings suggest that pre‐engineered, perfused, vascularized soft tissue with well‐connected networks of capillaries prior to implantation accelerated muscle fiber repair through timely supply of sufficient blood and avoided invasion by host fibroblasts. This concept could serve as a platform technology for engineering various vascularized tissue for tissue engineering and regeneration.
Label‐free assessment of differentiation efficiency in induced pluripotent stem cell derived spinal cord progenitor cells via magnetic resonance relaxometry (MRR)
1School of Chemical and Biomedical Engineering, Nanyang Technological University, Interdisciplinary Graduate Programme, 2School of Chemical and Biomedical Engineering, Nanyang Technological University, 3Critical Analytics for Manufacturing of Personalized Medicine, Singapore‐MIT Alliance for Research and Technology, 4School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, 5Institute of Molecular and Cell Biology, A*STAR Research Entities
The advent of induced pluripotent stem cells (iPSC) has provided a promising solution to the replacement of damaged neurons, especially in spinal cord injuries. Despite its merits, iPSC and its differentiation into neurons is a highly variable process, prompting the need to reliably assess the degree of differentiation achieved in each batch of cells, and validate the quality and safety of the differentiated cells. iPSC phenotypes are usually detected through labelling cells with a fluorescent marker and analysing with a flow cytometer or immunofluorescence staining, which tends to perturb or destroy cells preventing them from further use. In this study, human iPSCs derived from Cord Lining Endothelial cells were differentiated into Spinal‐cord Progenitor Cells (SCPCs) through a 10‐day differentiation process. We performed a label‐free measurement of these cells at different timepoints of the differentiation using Magnetic Resonance Relaxometry (MRR), a rapid and label‐free technique to obtain critical cellular iron (Fe3+) content. MRR only requires 4μL samples of suspended cells (<50k) for measurements that takes up to 2 minutes without any chemical or biological sample preparation. We found that SCPCs have significantly different T2 relaxation time measurements as compared to iPSCs. Furthermore, SCPCs harvested at the end of the 10‐day differentiation process containing higher levels of residual pluripotent markers have lower T2 relaxation time measurements when compared to SCPCs with lower levels of these markers. Our technology will provide a quick label‐free method to determine critical quality attributes in differentiated iPSCs and significantly benefit cell therapy manufacturing.
Development of an in vitro microvasculature model for non‐penetrating trauma research
1Institute of Biomedical Engineering, University of Oxford, 2Department of Engineering Science, University of Oxford
Improvements in both survival and quality of life of major trauma patients have accelerated in the last decade; however, traumatic injury remains a leading cause of death and of devastating functional limitation. While microvascular injury following major trauma is central to many mechanisms for immediate and long‐term local and systemic pathology, suitable and ethically acceptable models for understanding mechanisms of injury are lacking. This research aims to assess and optimise hydrogel‐ based microtissues for use in non‐penetrating trauma research.
Microtissues were created from immortalised human dermal microvascular endothelial cells(HDMEC‐1) and a range of hydrogel compositions. Briefly, fibrinogen at different concentrations(1‐5%w/v) was prepared in PBS, serum‐free endothelial growth (medium EGM‐2) or EGM‐2 supplemented with VEGF. Cell viability and migration assays were considered when optimising hydrogel composition. Following the development of the microvascular networks, microtissues were subject to a non‐penetrating traumatic injury (blunt force/stretching/crush). Characterisation of the microvascular network was performed before/after injury to compare tissue integrity and establish relationships between key biomechanical inputs and the resulting injury.
EGM‐2+VEGF microtissues showed a higher cell viability and HDMEC‐1s in fibrin microtissues developed particularly well‐formed networks, with similar distribution between weight/volume concentrations. The microtissue damage highlights that some network segments are more likely to be damaged when sustaining a stretching injury; whilst there seems to be no difference following crush or blunt force trauma injuries.
This research provides an insight into tissue damage immediately after a traumatic injury–a highly‐ challenging feat to undertake in the clinical setting. Future work includes rheological characterisation and further development of the trauma‐loading device.
Interfacial cell migration‐based tattoo sticker‐like cell delivery platform
1Univ. postech, 2University of Cambridge
Cell sheet technology is a remarkable option to avoid the limitations of conventional tissue engineering techniques. Cell sheets are maintained intact cell‐cell junction and extracellular matrix proteins by a protease‐free cell harvesting and scaffold‐free cell delivery system. However, most cell sheet studies need artificial detachment triggering steps to harvest cell sheets. such as thermo‐, photo‐, magnetic‐, electro‐, or pH‐responsive, as well as ion‐induced cell sheet harvesting methods. In this study, we introduce a new unique and flexible cell delivery platform which can transfer both cell sheets and also single‐cell forms like tattoo stickers using self‐interfacial cell migration. This cell delivery method uses a highly biocompatible parylene thin‐film for the cell carrier, to culture and transfer the cell sheet. With the support of the parylene film, cell sheets can be simply designed in any shape and size, and easily transferred to the target surface while maintaining the original designs and even their own cytoskeletal structures. Different from other cell sheet harvesting methods, this cell delivery system is based on spontaneous interfacial cell migration without any external stimuli, then could minimize cell damage during the transferring process. In addition, the cell sheets could be easily stacked in heterotypic multilayered forms and applied to a wound site, confirming its potential application for wound patch transplantation. This easy‐to‐use cell delivery platform can contribute to the wider therapeutic application of cell sheet technology and a deeper understanding of cellular behaviors with interfacial migratory capability.
Micropore‐forming gelma bioinks for tissue bioprinting
1Harvard Medical School
Three‐dimensional (3D) bioprinting has emerged as a class of promising techniques in biomedical research for a wide range of related applications. It enables precise control over the composition, spatial distribution, and architecture of the bioprinted constructs, facilitating recapitulation of the delicate shapes and structures of target organs and tissues. In particular, the search for optimal bioink formulations has proven challenging as bioprinting materials must fulfill several, often contradictory requirements. However, the encapsulated cells can be restricted in spreading and proliferation by the dense biomaterial networks, which are mechanically strong and are usually required for maintaining good printing fidelity. To this end, the ability of precise control over the microporous structures within bioinks in a cytocompatible manner while retaining their manufacturability, is generally lacking, until we recently reported a method of formulating micropore‐forming bioinks based on gelatin methacryloyl (GelMA) and select porogen types. This talk will thus discuss our recently innovated micropore‐forming GelMA bioink toolbox, which simultaneously enables high‐fidelity advanced biomanufacturing as well as favorable cellular bioactivities. It is believed that this unique micropore‐forming GelMA bioink toolbox would pave new avenues for widespread applications in tissue biofabrication.
Biofabrication approaches enabled by covalent crosslinking of silk
1University of New South Wales, 2University of Otago Christchurch
Covalently crosslinked silk fibroin hydrogels have gained popularity over their physically crosslinked (via beta‐sheet formation) counterparts due to their elastomeric nature, transparency and ability to support cell encapsulation, as well as compatibility with modern biofabrication approaches. Covalent crosslinking is achieved via di‐tyrosine bond formation between tyrosines natively found in silk, without the need for any modification of the polymer chain. This reaction can be mediated through three broad strategies, including enzymatic‐, Fenton reaction‐, and photo‐initiated crosslinking approaches.
This talk will discuss the broad utility of ruthenium‐based photo‐crosslinking of silk for multiple biofabrication approaches and material formats, including hydrogels, microgels, granular hydrogels and lyogels. We demonstrate the formation of rapidly crosslinking hydrogels at concentrations as low as 1% wt/v silk with tunable compressive modulus (∼1‐100 kPa) and excellent optical properties (transmittance
>90%). These hydrogels can be fabricated into complex high‐resolution structures via sacrificial polymer templating or ice templating. Ice templating allowed formation of aligned pores and incorporation of complex structures such as channels, multiple material phases and drug encapsulation in large multi‐ centimetre scale constructs. Water‐in‐oil emulsion within a flow‐focusing microfluidic device allowed the formation of small (∼98 ± 18 um) or large (∼375 ± 28 um) silk microgels which when annealed together resulted in scaffolds with microporosity and supported excellent cell encapsulation, proliferation and tissue ingrowth in vivo relative to their bulk hydrogel counterparts.
This work offers new silk biomaterial platforms for biomedical applications and novel insights into di‐ tyrosine bond formation and the dynamic nature of silk crosslinking.
Advanced pathophysiological in vitro 3D models of the human heart using cardiac spheroids
1University of Technology Sydney, 2University of Newcastle, 3University of Sydney
Preclinical studies using currently available in vitro and in vivo models of the human heart fail at fully recapitulating the complex scenario typical of myocardial infarction (MI, or heart attack) and drug induced cardiotoxicity, limiting their translation from the bench to the bedside. Our laboratory has developed human cardiac spheroids (hCSs) as advanced in vitro models of the human heart. In an effort to establish advanced pathophysiological in vitro models to study the human heart tissue response to ischaemic/reperfusion (I/R) injury typical of MI and drug‐induced toxicity, we exposed hCSs to pathophysiological changes in oxygen (O2) levels, while others were treated with doxorubicin (DOX), respectively. Our analyses of cell toxicity ratios using calcein‐AM and ethidium homodimer staining, as well as our measurements of fractional shortening and contraction frequency in I/R‐ and DOX‐hCSs showed a statistically significant loss of cell viability and contractile function in both conditions compared to control (healthy) hCSs. Our 3D rendering analyses using IMARIS software of hCSs stained with cell‐specific and cell death‐markers demonstrated that changes in O2 levels are responsible for loss of viability in cardiac myocytes, whereas DOX is toxic in all cell types (cardiac myocytes, endothelial cells and fibroblasts). Changes in mRNA expression levels for genes regulating sarcomere structure, calcium transport, cell cycle, cardiac remodelling and signal transduction were measured in both I/R‐ and DOX‐hCSs. Current studies are evaluating novel potential therapeutics to hopefully prevent and treat myocardial injury in cardiovascular disease patients using in vitro I/R‐ and DOX‐hCSs.
3D bioprinting strategies for in vitro modeling of diseased skin
1Pusan National University
Skin bioprinting is an attractive biofabrication platform for structural recapitulation of native skin. As the technological maturation, many efforts of 3D bioprinted in vitro skin models have been made for preclinical cosmetic research, drug delivery, and pathophysiological mechanism. Nevertheless, engineering diseased human skin in vitro is poorly studied. In this study, we propose engineering in vitro disease models via 3D bioprinting strategies for diabetic skin and metastatic melanoma. In this diabetic artificial skin, slow re‐epithelialization, a typical feature of diabetic skin, was observed. In addition, when the diabetic fat tissue layer containing blood vessels was added, insulin resistance, adipocyte hypertrophy, pro‐inflammatory response, and vascular dysfunction, which are commonly observed in diabetes, were confirmed. In metastatic melanoma, the main focus was to recapitulate tissue‐level metastatic microenvironment with precisely controlled cancer and vascular interactions via in‐bath spheroid and vascular bioprinting processes. As per the change of the distance between melanoma and vascular channel, metastatic properties were varying, which indicates the possibility as a personalized cancer in vitro platform. Taken all together, we anticipate it to be a way to replace animal models that have been conventionally used to observe skin diseases. It is significant that its applicability as a disease model for new drug development and unfound pathophysiological study has been proven.
3D printed PEEK/silicon nitride scaffolds with triply periodic minimal surface structure for spinal implants
1ETH Zurich, 2SINTX Technologies, Inc.
Spine‐related disorders, such as intervertebral disc degeneration and herniation, are a global healthcare concern. To restore the function of spine, the use of implants has become a standard solution. Therefore, it is of great interest to develop biomaterials and structures with a high osteogenic capacity, exhibiting comparable mechanical properties and dynamic response to the host bone. In this study, polyether ether ketone/silicon nitride (PEEK/SiN) scaffolds were fabricated by 3D printing. The PEEK/SiN scaffolds have a triply periodic minimal surface (TPMS) structure, which has the advantages of providing a large surface area and uniform stress distribution under load bearing. We evaluated the mechanical properties and dynamic response of PEEK/SiN scaffolds with different porosities by mechanical testing and finite element analysis. The PEEK/SiN TPMS scaffold with 30% porosity showed an elastic modulus (734 ± 64 MPa) and compressive strength (34.56 ± 1.91 MPa) that matched those of trabecular bone. Additionally, the PEEK/SiN scaffold showed good damping properties, as evidenced by dynamic loading tests. The biological properties of the PEEK/SiN scaffolds were evaluated in vitro using a non‐composite PEEK scaffold as a control. The results showed that the PEEK/SiN scaffold stimulated the proliferation and differentiation of mouse pre‐osteoblast cells (MC3T3‐E1) and resulted in a better expression of relevant osteogenic genes. On the other hand, we found that the PEEK/SiN scaffold showed better antibacterial properties than the PEEK scaffold. Overall, the results of mechanical and biological tests suggest that PEEK/SiN TPMS scaffolds are good candidates for spinal implants and more generally for bone tissue engineering applications.
Evaluation of poly vinyl alcohol based membranes as shape memory polymers
1Chang Gung University
Shape‐memory polymers (SMPS) can be used in many fields such as drug delivery, tissue engineering, bone, aerospace engineering, medical device, sensor, energy applications etc. SMPs are different categorized as per their respective external stimuli such as heat, light, pH, electricity, magnetic field and water/moisture. Among these, water‐induced SMPs have received immense attention for biomedical applications because in the human body, there are many functions that happened due to fluid of the body; therefore, water can act as an alternative fluid in the human body. Hence, water induced shape‐memory properties is the better and safer option for biomedical applications.
Poly vinyl alcohol (PVA) has unique properties, such as water solubility, multiple OH groups for further decoration, FDA‐approved biocompatibility, and low toxicity, the use of PVA as a biomaterial has attracted great attention in biomedical applications. PVA has excellent and easy film‐forming properties and can be mixed with synthetic and natural polymers due to its water‐soluble, biodegradable, non‐ carcinogenic, and biocompatible characteristics through inter and intra molecular hydrogen bonding. Due to the presence of abundant hydroxyl groups in PVA, it is easy to modify or crosslink. Moreover, the modification process may lead to changes in the physical parameters of membranes, such as crystallinity, mechanical strength, pore diameter, and ionic mobility in the swollen state. Series studies of PVA based membranes has been evaluated recently in our lab and these results will be presented in this report.
Fabrication of MOF gel‐immobilized polymer substrates as a cell scaffold for mammary grant epithelial cells
1Tohoku University
Metal organic frameworks (MOFs) are crystalline porous materials composed of metals and organic ligands. This study attempted to fabricate a cell scaffold that mimics an extracellular matrix structure by using an ordered structure of MOFs. MOFs of gamma‐cyclodextrin (CD) were immobilized onto the surface of a polymer substrate by combining surface‐graft polymerization of glycidyl methacrylate and crosslinked to form gels with water‐insolubility. A cell adhesion peptide containing IKVAV sequence was introduced into the ordered structure of CD‐MOF gel, and cell adhesion was evaluated using mammary grant epithelial cells. Quantitative PCR analysis revealed that beta‐casein gene expression was upregulated for epithelial cells only on the surface of the cell adhesion peptide‐introduced CD‐MOF gels. In contrast, without CD‐MOF gels, no upregulation was observed for epithelial cells, even on the surface of the cell adhesion peptide. These results suggest that combining cell adhesion peptides with CD‐MOF gels allows us to maintain epithelial cells in culture.
Adhesive nanofibrous membrane for photobiomodulated wound healing
1Pusan National University
Wound healing is the dynamic tissue regeneration process to replace the devitalized and missing tissue layers. With the development of photomedicine techniques in wound healing, safe and noninvasive photobiomodulation therapy is receiving intensive attention. In this talk, we introduce the adhesive hyaluronic acid–based gelatin nanofibrous membranes integrated with multiple light‐emitting diode (LED) arrays as a skin‐attachable patch for wound management in photobiomodulation. The nanofibrous wound dressing is expected to mimic the three‐dimensional structure of the extracellular matrix, and its adhesiveness allows the wound sites and the flexible LED patch to be tightly coupled together. The series of results on the experiments demonstrate that our medical device accelerates the initial wound healing process by the synergetic effects of the wound dressing and LED irradiation. Our proposed technology holds great potential in progressing the improved wound healing management and other possible biomedical applications.
Evaluation of poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate) [p(3hb‐co‐ 4hb)] based biocomposite for translational biomedical applications
1University College London, 2Universiti Sains Malaysia, 3Dankook University
Optimisations of newly formulated biomaterials hold significant impacts in ensuring dynamic progress and highly output quality of current research. This study aims to investigate production of poly(3‐ hydroxybutyrate‐co‐4‐hydroxybutyrate) [P(3HB‐co‐4HB)] copolymer via shake flask fermentation and scale up in bioreactor. Optimisation of P(3HB‐co‐4HB)'s composition and purity was further functionalised with 45S5 bioactive glass and graphene. P(3HB‐co‐4HB) copolymer, from Cupriavidus sp. USM1020, was harvested via shake flask cultivation and bioreactor inoculation system. 45S5 bioactive glass was prepared using sol‐gel technique meanwhile graphene monolayers were fabricated using liquid exfoliation of graphite. P(3HB‐co‐4HB) biocomposite was prepared via facile blending in chloroform followed by ultrasonication with bioactive glass and graphene, before cast via solvent technique. Homogeneity was assessed via SEM, FT‐IR and DSC. Disc diffusion antibacterial assay was tested against Escherichia coli and Staphylococcus aureus. Biocompatibility was studied via live/dead, MTS, AB, LDH‐release and inflammatory cytokine expression against human dermal fibroblasts (HDF). In vivo studies were performed via subcutaneous implantation. Difference in 4HB composition via both fermentations shown different yields and properties affecting the miscibility and further functionalisation of biocomposite. Incorporation of bioactive glass and graphene in biocomposite seen higher water contact angle measurements, improved mechanical profiles and antibacterial activities. Results shown enhanced cells activity with increasing concentration of bioactive glass and graphene, and demonstrated good biocompatibility and interaction with native tissues via animal study. Thus, this study demonstrated vital optimisations parameters of P(3HB‐co‐4HB)‐bioactive glass‐graphene, which opens up its endpoint applications considerably as versatile wound dressing and non‐load bearing scaffold in biomedical engineering field.
Mussel‐inspired mechano‐activatable adhesive microcapusles as a strain‐ regulated on‐demand therapeutics delivery system
1Kyungpook National Unviersity
Bacterial infection of a wound is a major complication that can significantly delay proper healing and even necessitate surgical debridement and other complications. Conventional non‐woven fabric dressings, including gauzes, bandages and cotton wools, often fail in treating wound infections in a timely manner due to their passive release mechanism of antibiotics. Here, we propose adhesive mechanically‐activated microcapsules (MAMCs) capable of strongly adhering to a fibrous matrix to achieve a self‐regulated release of antibiotics upon uniaxial stretching of a non‐woven fabric dressings. To achieve this, a uniform population of polydopamine (PDA)‐coated MAMCs (PDA‐MAMCs) are prepared using a microfluidics technique and subsequent oxidative dopamine polymerization. The PDA‐MAMC allows for robust mechano‐activation within the fibrous network through high retention and effective transmission of mechanical force under stretching. By validating the potential of a PDA‐MAMC‐laden gauze to release antibiotics in a tensile strain‐dependent manner, we demonstrate that PDA‐MAMCs can be successfully incorporated into a woven material and create a smart wound dressing for control of bacterial infections. This new mechano‐activatable delivery approaches will open up a new avenue for a stretch‐triggered, on‐ demand release of therapeutic cargos in skin‐mountable or wearable biomedical devices.
Sound induced morphogenesis: A new contactless bioprinting's technology for tissue regeneration
1AO Research Institute
Morphogenesis, a complex process, ubiquitous in developmental biology and tissue regeneration, is based on self‐patterning of cells. Spatial patterns of cells, organoids, or inorganic particles can be forced on demand using acoustic standing waves, such as the Faraday waves. This technology allows tuning of parameters (sound frequency, amplitude, chamber shape) under contactless, fast and mild culture conditions, for morphologically relevant tissue generation. We call this method Sound Induced Morphogenesis (SIM). First, we introduce the use of SIM to achieve tight control over patterning of endothelial cells and mesenchymal stem cells densities within a hydrogel, with the endpoint formation of vascular structures. This sound‐induced cell density patterning and subsequent vasculogenesis requires less cells than the conventional cell culture. Finally, we give an overview on the Sound Guided Tissue Regeneration in Musculoskeletal area, aiming to explore SIM as a holistic approach, from a size and time perspective, to control tissue organization toward clinical functionality. We advocate for the use of SIM for rapid, mild, and reproducible morphogenesis induction and further explorations in the regenerative medicine and cell therapy fields.
Effect of polydeoxyribonucleotide and polynucleotide on rotator cuff healing and fatty degeneration
1Chuncheon Sacred Heart Hospital, Hallym University Medical College
Polydeoxyribonucleotide (PDRN) is a tissue regeneration activator that is composed of a mixture of nucleotides and activates adenosine A2A receptors, stimulating vascular endothelial growth factor (VEGF) expression and the activity of fibroblasts. In addition, polynucleotides (PNs) are polymeric molecules that undergo enzymatic cleavage and progressively release both water molecules and smaller oligonucleotides with a longer effect. In diabetic ulcer of rat model, PDRN showed an effect of wound healing. Recently, PDRN has been used for healing of tendon, ligament, cartilage, or bone. PN had been investigated about the effect of tissue healing with a longer duration.
Rotator cuff tears are not uncommon, especially for older patients. Regardless of surgical techniques or devices, recent reports have shown that retears rate of repaired cuffs is considerably high. Moreover, in DM or hypercholesterolemia, the healing of repaired cuffs was compromised according to the literatures. Therefore, the biologic materials can be used for enhancing cuff regeneration. Several studies showed that PDRN might improve rotator cuff healing. Also, a study reported that PDRN and PN showed might improve tendon healing and decrease fatty degeneration in a rat cuff repaired model. Our unpublished studies showed that PDRN and PN had a similar effect on rotator cuff healing and fatty degeneration in diabetic and hypercholesterolemic rat model, also. In addition, PDRN combined with platelet rich plasma (PRP) did not show a satisfactory effect although PN with PRP did a good effect in another unpublished study, which probably resulted from a negative feedback between PDRN and PRP.
Novel bottom‐up tissue engineering strategy enabled by 3D‐printed microscaffolds
1TU Wien
Current approaches in tissue engineering (TE) can be roughly categorized into scaffold‐based and scaffold‐free. In this contribution a proof‐of‐concept of a third TE strategy, combining the advantages of these seemingly opposing approaches while circumventing their drawbacks, is presented. This novel strategy is based on self‐assembly of tissue units (TUs) consisting of highly porous microscaffolds containing cell spheroids. Realization of such microscaffolds, which are only about 300 micrometers in diameter and resemble a buckyball structure, is enabled by laser‐based high‐resolution 3D printing. It is demonstrated that human adipose‐derived mesenchymal stem cells (hASCs) rapidly form spheroids directly within such 3D printed microscaffolds. The presence of the microscaffolds does not have a negative effect of fusion of neighboring spheroids. Furthermore, compared to scaffold‐free spheroids, TUs maintain their shape and roundness much better over 4 weeks of culture, while also reducing the compaction of the spheroids. The resulting TUs showed high viability and preserved their chondrogenic and osteogenic potential. The successful differentiation towards the osteogenic lineage was verified by calcium deposition quantification fluorescent calcein green staining, while the successful chondrogenic differentiation was verified by quantification of sulfated glycosaminoglycans and total protein amount. The formation of larger tissue constructs is possible, using these differentiated TUs as “building blocks”. Our results indicate that the microscaffolds carrying high density of cells are promising building blocks for cartilage and bone TE facilitated by bottom‐up assembly.
Bone matrix derived bone‐tendon enthesis for repair of irreparable rotator cuff tears
1University College London
Rotator cuff tear is a common musculoskeletal disorder associated with damage to the tendon which connects bone and muscle. Surgery to repair a torn rotator cuff involves re‐attaching the tendon to the bone with sutures. In this procedure, naturally derived or synthetic “tendon” is used to establish the reattachment of tendon to the humerus. Despite the growing clinical use of synthetic scaffold devices for the repair of rotator cuff tears, the main limitations of these medical products are unable to mimic the tendon‐bone junction microenvironment and are unable to form a stable fixation of the tendon.
The junction between tendon and bone is a progressively evolved “multi‐tissue” structure: tendon‐ fibrocartilage ‐ mineralized fibrocartilage ‐ bone. These tissues have progressively increased Young's moduli, which distribute the forces generated in the muscles onto the bone. The authors postulate that a bone matrix derived may provide biological enhancement and tension‐free mechanical augmentation for stable rotator cuff repairs.
In this study, we developed a novel bone matrix processing method and developed a naturally derived bone‐tendon enthesis. The novel enthesis comprises a fibrocartilaginous section (for tendon attachment) naturally attached to decellularised bone section (for insertion into the bone). The in vitro evaluation has demonstrated the developed enthesis can provide tension‐free attachment of the tendon to the bone with an appropriate physiological environment to support cells ingrowth and guide cell differentiation into chondroblasts in the superficial regions, stimulate ossification in the immediate distal regions, and direct formation of bone at the distal region.
Neuropeptide calcitonin gene‐related peptide engineered to bind the extracellular matrix restores diabetic wound healing via immunoregulation
1European Molecular Biology Laboratory Australia, Australian Regenerative Medicine Institute, Monash University, Australia, 2Laboratory of Host Defense, World Premier Institute Immunology Frontier Research Center, Osaka University, Osaka, Japan
The immune system is a key regulator of tissue repair and regeneration. In addition, emerging evidence suggests that neuropeptides released by the nervous system are key regulators of immune activities. Thus, interactions between these two systems may control tissue healing. Nociceptors constitute a significant subset of peripheral sensory neurons innervating the skin and muscle. Using mouse models, we found that calcitonin gene‐related peptide (CGRP), a neuropeptide expressed by nociceptors, plays a positive immunomodulatory role during skin and muscle healing. Thus, delivering CGRP into damaged tissues could be an attractive approach to promote tissue healing, if it has a controlled spatio‐temporal release system. To address this, we engineered CGRP to allow it to remain localised upon delivery in vivo through binding to extracellular matrix (ECM) components. To evaluate the therapeutic effectiveness of ECM‐binding CGRP, we used a diabetic mouse model displaying neuropathy complications where skin and muscle healing is known to be delayed, accompanied by dysfunctional immune responses. Mice received full‐thickness skin wounds or volumetric muscle loss injuries. Then, ECM‐binding CGRP was topically applied on skin wounds or delivered via a fibrin hydrogel in the muscle. We demonstrated that ECM‐binding CGRP accelerated skin wound closure and increased muscle regeneration, compared to wild‐type CGRP or saline control. Notably, the ECM‐binding CGRP re‐established a pro‐healing microenvironment by reducing pro‐inflammatory immune cell accumulation and upregulating anti‐ inflammatory mediators. This suggested that our engineered CGRP improved tissue healing by mimicking neuro‐immune interactions, thus highlighting its potential for treating chronic wounds where the immune activation is dysregulated.
Dysregulated energy production during scaffold‐guided bone regeneration In type 2 diabetes
1Berlin Institute of Health at Charite‐universitatmedizin Berlin, 2Berlin Institute of Health ‐ Metabolomics Platform, Berlin, Germany
New strategies and animal models for craniomaxillofacial tissue regeneration
1Tufts University School of Dental Medicine, Boston MA, 2Wake Forest Institute for Regenerative Medicine, Winston‐Salem, North Carolina, 3University of Texas Health Science Center at Houston, Houston TX
Craniomaxillofacial (CMF) defects affecting jaws and teeth currently affect more than 15M individuals in the US alone. Craniofacial birth defects, including cleft lip/palate which occur in 1/700 live births, often require multiple sequential jawbone and tooth repair procedures over time in order to achieve proper form and function, particularly in growing children. The need for oral, head and neck cancer resections and the widespread occurrence of accidental and battlefield injuries to the CMF complex all add to the significant and highly complex surgical interventions needed to restore proper form and function to jaws and dentition. As such, the need for more effective Tissue Engineering and Regenerative Medicine (TERM) therapies for CMF jaw and tooth regeneration are a significant health concern. Here we present potential new approaches and therapies that may be used to overcome current limitations in CMF tissue repair and provide better patient outcomes. These studies are anticipated to transform the fields of Tissue Engineering and Regenerative Medicine and Dentistry and provide new hope for patients experiencing a variety of debilitating CMF defects. These studies were supported by NIH/NIDCR/NIBIB R01 DE026731 and AFIRM2 W81‐XWH‐14‐2‐0004.
Stem cell factor & cKIT as novel therapeutic targets for vascular eye diseases
1Chung‐Ang University, 2Chung‐Ang University, College of Pharmacy
Aberrant neovascularization (NV) is a leading cause of blindness in several eye diseases, including age‐ related macular degeneration (AMD). The identification of key regulators of pathological ocular NV has been a subject of extensive research and great therapeutic interest. Recently, we found that the previously unrecognized role of stem cell factor (SCF) and its receptor, cKIT, in the pathological ocular NV process. At hypoxia, a crucial driver of NV, SCF/cKIT signaling is highly enhanced in endothelial cells, which significantly contributes to the pathological NV in the retina and choroid via regulating beta‐catenin signaling pathway and endothelial glycolysis. Blockade of SCF/cKIT signaling using cKIT mutant mice substantially suppressed the pathological NV in the eye, which suggests that anti‐SCF/cKIT therapy would be a novel pharmacological strategy for treating vascular eye diseases. We then developed a fully human monoclonal antibody targeting cKIT (NN2101). In a murine model of neovascular AMD, intravitreal injection of NN2101 substantially inhibited the choroidal neovascularization (CNV), with efficacy comparable to aflibercept, a vascular endothelial growth factor inhibitor. A combined intravitreal injection of NN2101 and aflibercept resulted in an additive therapeutic effect on CNV. NN2101 neither caused ocular toxicity nor interfered with the early retinal vascular development in mice. Ocular pharmacokinetic analysis in rabbits indicated that NN2101 had a pharmacokinetic profile suitable for intravitreal injection. These findings provide the first evidence of the potential use of the anti‐cKIT blocking antibody, NN2101, as an alternative or additive therapeutic for the treatment of neovascular AMD.
Regeneration of airway mucosa: Scaffolds to epithelial cells
1Ewha Womans University, College of Medicine
Trachea and bronchus connect the larynx and the lungs. These upper airways provide the structural support which prevent airway from collapsing and provide immune defense. Histologically, the cartilage of the upper airway supports the structure and respiratory epithelia prevent infection by excretory function and ciliary movement.
We previously reported the ready‐made, acellular patch‐type tracheal prosthesis. This prosthesis was fabricated using 3D printing technique with medical grade polyurethane. Animal study showed optimistic result. The limitation of this scaffold is that full‐functional recovery of the respiratory epithelium delayed until 2 months after the transplantation, since regeneration of the respiratory mucosa depends only on the growth of the surrounding tissues.
For the functional recovery of the epithelium, we developed acellular respiratory mucosal matrix (ARMM) using decellularization method. Unlike the whole trachea decellularization, mucosal decellularization needs only small amount of decellularization agent and time. ARMM produced by decellularization protocol demonstrated low antigenicity and cytotoxicity. In vivo transplantation reveal that this scaffold has proper biocompatibility. When transplanted in tracheal mucosal defect animal model, crust forming and stenosis were prevented by implanting this ARMM.
For the instant cellular regeneration of the respiratory epithelium, we are developing the differentiation protocol for functional airway epithelial cells using tonsil‐derived mesenchymal stem cell. Using the stepwise application of the agents based on the embryology and air‐liquid interface culture system, we developed respiratory epithelium mimicking cells.
Combination treatment of stem cells and cartilage acellular matrix injection for osteoarthritis
1Kangstem Biotech, 2Seoul National University
Osteoarthritis (OA) is a general joint disease. Cartilage damage is associated with a decreased density of extracellular matrix and chondrocytes in the cartilage tissue. Mesenchymal stem cells (MSCs) differentiate into adipocytes, osteocytes and chondrocytes, and are an excellent source of cell therapy. Cartilage‐derived extracellular matrix (ECM) promotes chondrogenesis of MSCs. However, the role of MSCs stimulated by ECM is not well known in OA. Our previous study demonstrated that human umbilical cord blood‐derived MSCs (hUCB‐MSCs) combined with cartilage acellular matrix injection (CAM Inj.) represent potential therapeutic agents for structural improvement and anti‐inflammatory effects in a rabbit model of OA. Additionally, this study indicated that therapeutic potential of UCB‐ MSCs with CAM is mediated via BMP6 in OA. Based on a previous study, we evaluated the safety and efficacy of hUCB‐MSCs combined with CAM Inj. in an anterior cruciate ligament transection (ACLT) with medial meniscectomy (MMx) in a goat model. Lameness and radiographic parameters were assessed 6 months after administration, and macroscopic and histological evaluations of the goat articular cartilage were performed 6 months after intervention. As a result, the K&L score and gross findings showed significant improvement by treatment of hUCB‐MSCs combined with CAM Inj.
In conclusion, treatment with a combination of hUCB‐MSCs and CAM Inj. reduced OA symptoms and induced effective cartilage tissue repair in a goat model. We suggest the combination of hUCB‐MSCs and CAM Inj. as an alternative therapy for OA.
Bioactive microcapsules for cultivation of stem cells
1Mayo Clinic
Encapsulation technology is an important platform for scaling up stem cell cultures. Microcapsules may be used as stem cell carriers to protect cells from shear stress of stirring and to deliver inductive signals driving stem cell differentiation. This presentation will describe recent work in our lab aimed at encapsulation, maintenance and differentiation of pluripotent stem cells. Specifically, we will focus on heparin‐containing bioactive microcapsules that may be loaded with growth factors in sequentially manner to mimic traditional multi‐step stem cell differentiation protocols. Hepatic differentiation of encapsulated stem cells will be discussed.
Machine learning‐based predictive model for printability and shape fidelity in biofabrication
1Rapid Manufacturing Platform, GeM Laboratory, CNRS UMR 6183, Centrale Nantes, Nantes, France, 2PIMM Laboratory, CNRS UMR 8006, Arts et Metiers Institute of Technology, Paris, France
Today, hierarchically organized living constructs are developed using additive manufacturing techniques. This emerging technology is named three‐dimensional bioprinting.
These biofabricated organized structures are conceived thanks to the use of hydrogels. One of the most critical challenges in bioprinting is the development of bioinks that are suitable for additive manufacturing. These hydrogels are subjected to different physicochemical constraints such as temperature, speed, and pressure during the bioprinting procedure. The bioprinted structure's fidelity can significantly be affected by these constraints.
To support our assumption, we have collected data through experiments to understand the more significant parameters in the process. This data was used for conducting a forward machine learning approach. Indeed, an inverse model was developed that allows specifying the machine's operating data according to the dimensions of the desired structure.
To obtain an accurate construct, we present a new standard of digital biofabrication, developing a predictive model using artificial intelligence for microextrusion bioprinting. Considering these previously mentioned physicochemical variables, our model allows us to predict the best parameters to establish in microextrusion bioprinting.
A biphasic 3D osteo‐vascular on a chip for recapitulating the bone marrow environment
1Korea National University of Transportation
Recent developmental engineering efforts aim to recapitulate endochondral ossification (EO), an important step in bone formation. However, this process involves condensing mesenchymal stem cells (MSCs) into cartilage templates, which require long‐term culture and are difficult to scale. Here, a biomimetic scaffold is developed that enables fast and self‐sustaining EO without the occurrence of initial hypertrophic cartilage. The design comprises a porous chondroitin sulfate cryogel decorated with white site calcium phosphate nanoparticles and a soft hydrogel occupying the porous space. This complex system allows human endothelial colony‐forming cells (ECFCs) and MSCs to quickly assemble into the osteovascular niche. Moreover, in such a system, an environment is created in which hematopoietic stem cells (HSCs) can be cultured. Through bone and blood vessel regeneration, the expression of SCF protein level is increased around it, thereby recruiting hematopoietic stem cells through blood vessels to the supporting area, creating the bone marrow‐like environment. We propose a novel biotechnology approach to finally implement these supports in a Lab on‐chip environment to reconstruct bone marrow niches in the context of in vitro models.
Toward an edible engineered meat‐like tissue from assembled bovine cell fibers fabricated by tendon‐gel integrated bioprinting
1Joint Research Laboratory (TOPPAN) for Advanced Cell Regulatory Chemistry, Graduate School of Engineering, Osaka University, 2Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 3Osaka University
Cultured meat is a current hot topic but is still at an early stage where reproducing structured steak is challenging. Since actual meat is indeed made of muscles surrounded by fat tissue and blood capillaries and these tissues are aligned fibers connected to the tendon for the actions of contraction and relaxation, bioprinting appeared suitable. For this purpose, tendon‐gel integrated bioprinting was developed with supporting bath printing technology where three types of fibers (muscle, fat and endothelial cells) were reproduced from isolated bovine cells. Since the fat content is the key point to reach the actual taste and juiciness of the meat, further studies were then done to determine the suitable fatty acids mixture and their concentrations for the adipogenesis of the bovine adipose‐derived stem cells (bADSC) used in the fat fibers. To ensure the scale‐up of this model and the edibility of the final meat product, other improvements focused on the bovine cell isolation method to get then the maximum possible cell expansion along with maintained differentiation potential. As antibiotics cannot be used in food applications, a food‐compatible peracetic acid based microbiocide solution was used to wash the tissues before cell isolation and culture in antibiotics‐free conditions. bADSC were then found with a good differentiation at least until passage 12 (2.3‐3.2 times more lipid accumulation than undifferentiated condition). All in all, this technology appeared promising for the fabrication of custom desired types of steak‐like cultured meats.
Reference: Kang, D. H., Louis F. et al., Nature communications (2021):12(1),1.
Strategies for the controlled release of therapeutics
1Vidyasirimedhi Institute of Science and Technology
We present herein several strategies for controlling the release of therapeutics upon trigger with a stimulus relevant to biomedical applications. Drugs were conjugated to polymers in the side chain or in the main chain of polymers and linked via cleavable chemical groups. A particularly good control over the release of the drug could be achieved by encapsulating a polyprodrug in nanocapsules. Upon degradation of the polyprodrug by hydrolysis in mild acidic conditions, an anti‐inflammatory diffused through the semi‐permeable wall of the nanocapsules. KCs in the liver demonstrates the potential for this nanocarrier system to be applied in future studies for the treatment of chronic or acute intrahepatic inflammatory diseases. The results emphasized the potential of this type of material for the treatment or chronic or acute intrahepatic inflammatory diseases.
Pathogen mimicking polysaccharide nanocapsule for cancer immunotherapy
1Inha University
Microbial pathogens provoke “danger” signals at the site of infection and confer cell‐mediated protective immunity by inducing proinflammatory cytokines and recruiting and activating T lymphocytes at the site of infection. In particular, pattern recognition receptors (PRRs) expressed on innate immune cells recognize the conserved molecular structure of the microbial cell walls, termed pathogen‐associated molecular pattern (PAMP), to initiate and orchestrate the host response to infection. Provision of PAMP signals into local tumor may elicit an antitumor immune response by reawaking local immune cells otherwise immunosuppressive or tolerant, and thus to commence protective immune response against tumor cells.
We utilized fungal mannan, a well‐known PAMP derived from the microbial cell wall, for developing a new class of subunit microbial nano‐vaccine for cancer immunotherapy. We engineered mannan into a hollow nanocapsule (Mann‐NC) to construct a synthetic nano‐PAMP. Mann‐NC demonstrates unique nanoscale geometry that allows displaying desirable mannose arrangement on the surface with deformable physical properties for enhanced biodistribution and immunological performances. In a study, we utilized Mann‐NC as an mRNA carrier for mRNA‐based cancer vaccine development.
On another hand, Th17 immunity plays a key role in infection, autoimmune disease, and cancer. However, it remains largely unknown how to pharmacologically induce antitumor Th17 cells in vivo and exploit Th17 immunity as the basis for cancer immunotherapy. Mannan has been shown to trigger Dectin‐ 2 and Toll‐like receptor (TLR)‐4 activation in DCs and Th17 immunity, thus we report here that Mann‐ NC displays mannose in a multivalent manner as fungi can elicit potent antitumor Th17 response.
Controlled local photothermal therapy using nano‐functionalized stent
1Asan Medical Center
Placement of self‐expandable metallic stent (SEMS) is currently the most common therapeutic strategy for treating malignant and selected benign non‐vascular strictures. However, the use of SEMSs is greatly limited by tumor or hyperplasia tissue embedment that occurs in the uncovered portion of the SEMSs, which can lead to new stricture formation with recurrent symptom. Controlled local heat treatments have been introduced for cancer therapy and managing of tissue hyperplasia. Thermal therapy using various nanoparticles such as different shaped gold nanoparticles (AuNPs) and magnetic nanoparticles has been studied extensively for inducing controlled heat treatments with high efficiency and accuracy. In here, the AuNPs‐coated SEMS for controlled local photothermal therapy (PTT) was synthesized a facile two‐step process. Surface of control SEMS was first modified using polydopamine (PDA), which helps AuNPs adhere to the surface of the nitinol stent, and then AuNPs attached to the PDA‐coated surface of the SEMS. The temperature of the AuNPs‐coated SEMS was increased in proportion to the near infrared (NIR) intensity. The PT properties can be easily controlled by adjusting NIR irradiation power. Controlled local PTT using AuNPs‐coated SEMS successfully treated granulation tissue formation after stent placement in the rat esophageal and gastric outlet models. AuNPs‐coated stent effectively suppressed tumor growth after stent placement by heat‐induced tumor necrosis in an orthotopic, obstructive rectal cancer mouse model. Our results indicate that this novel strategy of localized heat treatment could be a promising option for prolonged stent patency with decreased tumor volume and suppression of stent‐ induced tissue hyperplasia.
Biomimetic peptides for sustained drug release from silk fibroin hydrogel
1Biomedical Engineering Program, Faculty of Engineering, Chulalongkorn University, 2Chulalongkorn University, 3Department of Biochemistry, Faculty of Science, Chulalongkorn University
Sustained release of bioactive proteins from such as growth factors from hydrogel can be beneficial for tissue engineering and regenerative medicine. In this work, we demonstrated a novel strategy to sustain a model protein namely green fluorescent protein (GFP) from silk fibroin (SF) hydrogel. We used genetic engineering to conjugate SF mimicking peptides that had varied lengths ((GAGAGS)n; n = 0‐6) to GFP. The resulting (GAGAGS)n‐GFP with a greater n led to a slower release of GFP from the hydrogel while it did not affect the gelation time. FTIR revealed that the GFP might interfere with the beta‐sheet formation, but the (GAGAGS)n helped restore the beta‐sheet fraction in the hydrogel. The longer (GAGAGS)n also increased the mechanical stiffness and the strength of the SF hydrogel. This work highlights the potential of genetic engineering in conjugating (GAGAGS)n to a protein that will be used for sustained‐release application.
Three‐dimensional microphysiological system‐inspired scalable vascularized tissue constructs for regenerative medicine
1Department of Mechanical Engineering, Seoul National University, 2School of Chemical and Biological Engineering, Seoul National University, 3KIST
Microphysiological systems (MPSs), based on microfabrication technologies and cell culture, can faithfully recapitulate the complex physiology of various tissues. However, three‐dimensional tissues formed using MPS have limitations in size and accessibility; their use in regenerative medicine is, therefore, still challenging. Here, we designed an MPS‐inspired scale‐up vascularized engineered tissue construct that can be used in regenerative medicine. We sandwiched endothelial cell‐laden hydrogels between two through‐hole membranes. The microhole array in the through‐hole membranes enabled the molecular transport across the hydrogel layer, allowing the long‐term cell culture. Furthermore, the time‐ controlled delamination of through‐hole membranes enabled the harvesting of cell‐cultured hydrogel constructs without damaging the capillary network. Importantly, when the tissue constructs were implanted in a mouse ischemic model, they protected against necrosis and promoted functional recovery to a greater extent than did implanted cells, hydrogels, and simple gel‐cell mixtures.
Ion‐assisted plasma polymerization: Surface engineering of biomimetic interfaces
1University of Newcastle
The sub‐optimal biocompatibility of bare metal surfaces, used as bone implants, often leads to adverse reactions such as infection, requiring additional medical interventions. The integration of metallic implants with local host tissues can be strongly improved by an ion‐assisted plasma polymerized (IPP) coating functionalized with biomimetic molecules. The stability of the IPP layer in body fluids is indispensable, and the coating must resist failure. Here we report a novel approach using a combination of plasma immersion ion implantation and plasma polymerization for the fabrication of highly robust polymeric coatings on titanium surfaces. The chemical and mechanical stability of the coatings in simulated body fluid (SBF) was examined by incubation of samples in Tyrode's solution at 37°C for durations of up to 2 months. The IPP coating resisted failure, and no delamination, cracking, or buckling was observed after scratching and subsequent incubation in SBF solution. X‐ray photoelectron spectroscopy (XPS) results revealed that the excellent interface adhesion is linked to the formation of metallic carbide and carbonate bonds, induced by ion implantation, at the early stages of film growth. Such atomic interfacial mixing also resulted in the formation of a continuous smooth film near the substrate as suggested by atomic force microscopy (AFM) and time of flight secondary ion mass spectroscopy (ToF‐SIMS) data. Our findings demonstrate that multifunctional protein layers, peptide molecules, or silver nanoparticles can be covalently immobilized on such radical‐rich interfaces for improved osteoblast activity and enhanced antimicrobial properties.
The importance of CMC in your R&D; How to integrate commercial thinking into your discovery
1Amplifybio
Historically, the field of cell therapy has operated under the mantra of "The Process is the Product". This mantra and associated operating cadence came from and continue contributing to the complexity of personalized medicines like autologous cell therapy. Having progressed from the early days of exploratory studies and rescue trials where the primary clinical objective was "do no harm", we now have access to state‐of‐the‐art, highly quantitative, and qualitative tools to return to the original mantra of pharmaceutical science, "The Product is the Product."
This talk will detail what, where, when, and how of advanced therapies, especially when identifying the right partners at the right time for your advanced therapy drug development process. We will look at some of the newer tools and capabilities that are available to researchers and how to start thinking about commercialization from day one.
Regain of the function by the cell therapy: New wave of regenerative medicine
1Cellatoz Therapeutics, Inc.
Today we are focusing on the peripheral neuropathy caused by various diseases including diabetes, chronic liver or kidney disease, lymphoma, multiple myeloma, CMT (Charcot‐Marie‐Tooth), Guillain‐ Barre syndrome, amyloidosis, rheumatoid arthritis, lupus, etc. However, apart from relieving pain with painkillers, few treatments are available and even those treatments are neither fundamental but only delay or reduce symptoms.
Cellatoz is utilizing novel cells called Neuronal Regeneration Promoting Cells for the treatment of peripheral neuropathy starting with CMT diseases. To develop effective cell therapies, we need to know the characteristics of cells well enough to control. Defining your cells (product) well enough can assure you the efficacy of your cells (products).
Many people claimed that the process is the product in cell therapy area since the process is critical to make your cells (product) reproducibly. Nowadays the development of the process become a big issue as COG (Cost of Goods) is the center of attentions. However, we have to be careful to change your process without knowing your products well. Defining your cells is the first step of process development.
With well‐characterised and sure‐efficacious Cellatoz' NRPC, pre‐clinical animal models were examined. The mutant animals cannot be reversed their symptoms by nature or immune modulation (common stem cells' MOA). NRPC showed the change of the behaviors and symptoms of mutant animals suggesting that NRPC can be a life changer of patients.
The first‐in human studies would tell us that Cellatoz could make new wave for CTP version 2.0 +.
AffyXell: New modality for restoring immune balance
1AFX R&D Center, AffyXell Therapeutics Co., Ltd., 2AffyXell Therapeutics Co., Ltd., 3Strategy & Planning Team, AffyXell Therapeutics Co., Ltd.
Past 20 years, mesenchymal stem cells (MSCs) have proven their safety and effectiveness through various clinical trials for rare and intractable diseases, including immune disorders, but there are still obstacles to overcome in order to increase their usability as a therapeutic agent.
Recently, cell therapy is having shifted a new paradigm to genetic‐engineered cell therapy through the successful development of CAR‐T. As such MSCs also, in order to overcome immune disorders, it is necessary to develop novel therapeutic assets through cutting‐edge technology and manufacturing innovation.
AffyXell is a novel MSC‐based therapeutic platform that improves the immunomodulatory function of MSC based on the platform convergence of DW‐MSC and Affimer®.
The DW‐MSC, pluripotent stem cell‐derived MSC, developed by Daewoong Pharmaceutical is a next‐ generation MSC platform that overcomes the limitations of mass production of adult tissue‐derived MSCs. In addition, DW‐MSC is also suitable for genetic‐modified MSC‐based cell therapy through mass production properties.
The Affimer® platform developed by Avacta Life Science (UK) is a non‐IgG scaffold protein designed to have antibody‐like target binding properties. And Affimer is based on Stefin A derived from the human body with specific target binding affinity in the nanomolar range. Therefore, it has the advantage of good expression through genes introduced in cell‐based on their small size.
Today, we intend to introduce a novel therapeutic modality for regulating immune homeostasis that can induce immune tolerance to overcome immune disorders using AffyXell, and perspective for a future development direction for cell & gene therapy using MSCs.
Stem cell therapy for intractable neurological disorders
1Central Research Center, Corestem Inc.
Recent studies show developments of therapeutic agents based on mesenchymal stem cells for intractable neurological disorders. Mesenchymal stem cells (MSCs) are stromal cells capable of self‐renewal and multiple lineage differentiation. Extracellular vesicles and cytokines secreted from MSCs are widely known to have the effects of anti‐inflammation, neuro‐protection, neuro‐regeneration, and thereby are considered as therapeutic agents with a complex mechanism of action for intractable neurological disorders.
Corestem Inc. is a biotechnology company established in 2003, specializing in personalized stem cell therapy research and development for intractable neurological disorders. Lenzumestrocel (Neuronata‐ R®), targeting amyotrophic lateral sclerosis (ALS), was conditionally approved by the Ministry of Food and Drug Safety (MFDS) in 2015. Its phase 3 clinical trial, approved in Korea and the U.S. in 2019, is currently in progress. Additionally, commercial clinical trials for multiple system atrophy (MSA) and cerebellar ataxia (CA) are in progress with allogeneic MSCs, and those for neuromyelitis optica spectrum disorder (NMOSD) are planned.
NMOSD is an inflammatory demyelinating disease of the central nervous system that causes optic neuritis or transverse myelitis. Although anti‐inflammatory agents and monoclonal antibodies have been developed to delay the progression or to prevent its recurrence, there are no effective agents for restoring and regenerating the neurons. Corestem has demonstrated the anti‐inflammatory and neuro‐protective effects of MSC‐based therapeutic strategies in NMOSD disease models, both in vitro and in vivo, and the safety has been proved by toxicity tests.
Corestem continues to strive to create the future and provide hope for patients with intractable neurological disorders.
Korean fund for regenerative medicine (KFRM): Present and future
1Korean Fund for Regenerative Medicine
Korean Fund for Regenerative Medicine (KFRM) is a government‐funded agency created in 2021 to promote public health and advance scientific progress by supporting research in all fields of regenerative medicine. KFRM is an inter‐governmental organization operating under the Ministry of Science and ICT and the Ministry of Health and Welfare. KFRM supports promising research projects that aim to produce regenerative cures. We fund a wide range of regenerative research including cell therapy, (ex vivo) gene therapy, tissue engineering therapy and convergence of the above regenerative therapies stated in the Advanced Regenerative Bio‐Act. The Fund is committed to expanding knowledge by strengthening the linkage between projects in different development stages from exploratory and translational research to clinical trials through early commercialization, technology transfer, and treatment for rare and incurable diseases. KFRM's vision is to maximize the value of regenerative medicine technology by fostering collaboration among research groups and actively engaging with government organizations such as KFDA, KNIH, K‐Bio, KISTA, etc. The Fund also directly reaches out to researchers through a program called Science Ambassador (SA) whose job is to hear and discuss issues our researchers frequently encounter. Its members are composed of KFRM staffs and experts from intellectual property, non‐clinical studies, and CMC (chemistry, manufacturing, and controls). Altogether, KFRM incorporates our hands‐on experience and professional expertise to create and amend our request for proposals (RFPs). This talk will briefly introduce KFRM, its goals and current status of our ongoing projects.
Understanding the regenerative response induced by biomaterials systems: Insight into the role of glycosylation
1CÚRAM, University of Galway
Biomaterials are no longer considered innate structures and using functionalisation and biofabrication strategies to modulate the desired response whether it is a host or implant is currently an important focus in current research paradigms. Fundamentally, a thorough understanding of the host response will enable us to design appropriate strategies. The input from the host response needs to be weighed in depending on the host disease condition. Our current inputs have been through a thorough understanding of glyco‐ proteomics‐based tools which we are developing in our laboratory. In addition, biomaterials themselves provide immense therapeutic benefits which need to be accounted for in the design paradigm. Using functionalisation strategies such as enzymatic and hyperbranched linking systems, we have been able to link biomolecules to different structural moieties. The programmed assembly of biomolecules into higher‐ order self‐organized systems is central to innumerable biological processes and the development of the next generation of scaffolds. Recent design efforts have utilized a glycobiology and developmental biology approach toward both understanding and engineering supramolecular protein and sugar assemblies.
Engineering musculoskeletal soft tissues: Material and mechanobiologic considerations
1University of Pennsylvania
Treatment strategies to address musculoskeletal (MSK) dense connective tissue pathology are limited in part due to the low number of endogenous cells, the density of the matrix, and the limited vascularity. A number of pro‐regenerative implants have emerged as commercial products to treat injured dense tissues. However, current strategies still do not reflect the native structural, biochemical, and mechanical characteristics of the tissues, which potentially causes problems of inappropriate tissue formation and scaffold instability. To further development in this area and to overcome limitations with current strategies, we have developed biomimetic multiphasic biomaterial systems with tunable chemo‐ mechanical properties for the dense MSK connective tissue repair and regeneration. These new biomimetic systems allow us to program biochemical and biomechanical properties of tissue‐engineered scaffolds, which can recapitulate anisotropic MSK native tissue microenvironments. In addition, the cells including their progenitors in the tissues continuously sense alterations in the stiffness and biochemical components of their ECM, and are subjected to various mechanical forces during development, homeostasis, and degeneration to regulate their phenotype. Thus, elucidating the mechanism by which cells respond to their physical environment is important for understanding the physiologic, pathologic, and repair responses of the MSK tissues. In this seminar, we will also discuss our recent studies on how 3D genome architecture changes with the dense MSK tissue development or degeneration, and how chemo‐mechanical cues regulate these processes using super‐resolution stochastic optical reconstruction microscopy (STORM) imaging.
Confinement as a strategy for inducing stem cell differentiation
1National University of Singapore
Self‐induced cellular confinement has been shown to play a role in a wide variety of biological processes, including cancer invasion and metastasis, immune cell navigation, and mechanosensitive gene expression and localization. However, the ability of adult stem cells to enter confinement has been less studied, despite the fact that stem cells are capable of finely tuned mechanotransduction, must migrate from their home niche to their regenerative niche, and have been shown to integrate past stimuli in a form of 'mechanical memory'. Here, we characterize interstitial spaces in cleared tissue, providing a physiological basis for the design of biomimetic microchannel devices. Next, we show that adult stem cells are capable of entering and permeating PDMS‐based microchannels as narrow as 3 μm. Patterns of microchannel permeation as a function of width are similar to those found in cancer cells, despite the fact that these stem cells are more well‐spread and exhibit higher cell diameters. Both narrow and wide confinements were shown to induce an upregulation of the osteogenic differentiation marker CBFA1. Interestingly, narrow confinements led to enhanced CBFA1 expression compared to wide channels, suggesting that the level of confinement imposed upon a stem cell via its extracellular environment ultimately plays a role in differentiation. This relationship is time dependent, as short confinements did not induce the same level of osteogenic differentiation. Future work will discern if the migratory journey a stem cell undergoes during development and regeneration, and the confinement it experiences along the way, drive tissue‐specific stem cell differentiation.
Directed assembly of keratin based scaffolds for tissue engineering
1School of Materials Science and Engineering, Nanyang Technological University, Singapore, 2Environmental Chemistry and Materials Centre, Nanyang Environment and Water Research Institution, Singapore, 3Center for Nanotechnology and Nanotoxicology, Harvard T.H. Chan School of Public Health, Harvard University, United States
Keratins from various sources have emerged as a viable source of biomaterial. Coupled with the pursuit of sustainable approaches, interest in keratins has intensified. In particular, human hair keratins (HHK) extracted from abundant hair waste have been progressively studied as templates to support tissue regeneration. While potential in terms of bioactivity and cell/tissue compatibility have been demonstrated, keratins are particularly interesting because of their propensity to self‐assemble and the abundance of functional groups for crosslinking and conjugation. The versatility of processing HHK into a wide range of formats is well documented in the literature including coatings, fibers, films, gels and sponges. More recently, we have developed a cryogelation technique to fabricate 3D HHK scaffolds with tunable physical properties and microarchitecture. Under optimal freeze‐thaw conditions, cryogelation was found to improve the storage and compression moduli of HHK hydrogels. Strain‐stiffening behavior was also observed when HHK‐dopamine composite constructs were produced using a similar cryogelation strategy. Separately, we have shown that conditions can be optimized to induce self‐assembly of keratins in vitro to form intermediate filament‐like networks which can serve as coatings for cell culture. This ability to form organized structures provides the possibility to fabricate resilient films and fibers through simple methods such as casting and freeze‐drying. Thiolate interaction with cations can also be exploited to produce novel gradient hydrogels through a single‐step fabrication process. In this presentation, various HHK templates we have developed will be described. Overall, HHK provide an alternative biomaterial platform with new functionalities for exploitation in multiple applications.
Harnessing the bio‐responsive adhesion of immuno‐bioGlue for cancer immunotherapy
1Ewha Womans University
Despite the great promise of immune checkpoint blockade (ICB) therapy for cancer treatment, the currently available options for ICB treatment pose major clinical challenges, including the risk of severe systemic auto‐ immune responses. Here, we developed a novel localized delivery platform, immuno‐ bioglue (imuGlue), which is inspired by the intrinsic underwater adhesion properties of marine mussels and can allow the optimal retention of anti‐PD‐L1 drugs at tumor sites and the on‐demand release of drugs in response to the tumor microenvironment. Using a triple‐negative breast cancer and melanoma models, we found that imuGlue could significantly enhance anti‐tumor efficacy by eliciting a robust T cell‐mediated immune response while reducing systemic toxicity by preventing the rapid diffusion of anti‐PD‐L1 drugs into the systemic circulation and other tissues. It was also demonstrated that imuGlue could be successfully utilized for combination therapy with other immunomodulatory drugs to enhance the anti‐tumor efficacy of ICB‐based immunotherapy, demonstrating its versatility as a new treatment option for cancer immunotherapy.
Reproducing kinematic load in vitro for chondrogenesis studies
1AO Research Institute Davos
Articulating cartilage experiences a multitude of biophysical cues. Due to its primary function in distributing load with near frictionless articulation, it is clear that a major stimulus for cartilage homeostasis and regeneration is the mechanical load it experiences on a daily basis. While these effects are considered when performing in vivo studies, in vitro studies are still largely performed under static conditions. Therefore an increasing complexity of in vitro culture models is required, with the ultimate aim to recreate the articulating joint as accurately as possible.
We have for many years utilized a complex multiaxial load bioreactor capable of applying tightly regulated compression and shear loading protocols. Using this bioreactor, we have been able to demonstrate the mechanical induction of human bone marrow stromal cell (BMSC) chondrogenesis in the absence of exogenous growth factors. Building on previous bioreactor studies that demonstrated the mechanical activation of endogenous TGFβ, and subsequent chondrogenesis of human bone marrow derived MSCs, we have been further increasing the complexity of in vitro models. For example, the addition of high molecular weight hyaluronic acid, a component of synovial fluid, culture medium leads to reduced hypertrophy and increased glycosaminoglycan deposition.
The ultimate aim of all of these endeavors is to identify promising materials and therapies during in vitro/ ex vivo studies, therefore reducing the numbers or candidates that are finally tested using in vivo studies. This 3R approach can improve the opportunities for success while leading to more ethically acceptable product development pathways.
Microfracture with collagen scaffold for cartilage repair and osteoarthritis
1The Catholic University of Korea, 2Institute of Medical Sciences, Faculty of Health and Social Sciences, Canterbury Christ Church University
The autologous collagen‐induced chondrogenesis technique is described, and the results of a 6‐year follow‐up clinical study using this technique are presented.
30 patients with International Cartilage Repair Society (ICRS) Grade III/IVa symptomatic chondral defects of the knee treated with enhanced microdrilling using atelocollagen were prospectively examined in this clinical series. The median age of the patients was 39.0 years (range 19–61 years). Patients were followed up to 72 months. Clinical evaluation was performed using functional knee scores and radiologically. Both quantitative and qualitative assessments were performed.
Statistically significant and clinically relevant improvement was observed in 2 years and was sustained for the 6 years of the study observation. At 6 years, the mean Lysholm score was 79.7 (SD 6.8) compared to 52.6 (SD 10.7) pre‐operatively (p < 0.05). The symptomatic Knee Injury and Osteoarthritis Outcome Score (KOOS) improved from 68.3 (SD 11.4) to 90.2 (SD 4.3) (p < 0.05). The subjective International Knee Documentation Committee (IKDC) also showed improvement from 39.1 (SD 4.1) to 81.6 (SD 7.8) (p < 0.05). The calculated T2* relaxation times were 26.0 (SD 4.2) seconds and 30.3 (SD 6.2) seconds for the repair tissue and native cartilage, respectively. The average magnetic resonance observation of cartilage repair tissue (MOCART) score was 78.5 (SD 9.6) for all lesions.
The enhanced microdrilling using atelocollagen is an enhancement of the traditional microfracture method using an off‐the‐shelf product. When used to treat moderate to severe chondral lesions, this enhancement produces hyaline‐like cartilage with a corresponding improvement in symptoms.
BMAC, ACI, cartilife for cartilage repair and osteoarthritis
1Yonsei University College of Medicine
Osteoarthritis is one of the common joint diseases, which is characterized by the loss of joint functions including articular cartilage degenerations. It is known that articular cartilage has a very limited healing capacity when it is damaged. Arthritis causes degenerative changes of cartilage, ultimately causing the irreversible destruction of the joint. In particular, cartilage lesions with moderate to large size in physically active and young patients can cause rapid joint destruction and early arthritis. Accordingly, cartilage lesion that has not yet progressed to severe arthritis requires restoration treatment.
There are various treatment methods of recovering cartilage lesion. Microfracture is the most common treatment method that has been used for a long time. Clinical outcomes have been reported to be relatively good as a long‐term follow‐up result. However, many studies have reported that microfracture has poor clinical results over time and large lesions also have poor clinical results.
Cell‐based therapies are emerging as promising treatments for knee osteoarthritis and cartilage lesions. In 1987, Brittberg first succeeded in harvesting cartilage from patients, separating and culturing cartilage cells, and then transplanting them into cartilage lesions. He reported the results in The New England Journal of Medicine in 1994. Autologous chondrocyte implantation (ACI) has the advantage that regenerated cartilage is hyaline like cartilage that is similar to joint cartilage. However, ACI has disadvantages of two‐stage surgery and a relatively very high cost. In addition to ACI, many kinds of cell‐ based therapies have been attempted to treat cartilage lesions.
Allogeneic umbilical cord blood–derived mesenchymal stem cells implantation for large cartilage defects in older patients
1Chung‐Ang University, 2Samsung medical center, Sungkyunkwan university
Currently, there is no optimal method for cartilage restoration for large full‐thickness cartilage defects in elderly patients. This study was to determine whether implantation of allogeneic umbilical cord blood‐ derived MSCs (UCB‐MSCs) with hyaluronate (HA) results in reliable cartilage restoration in patients with large full‐thickness cartilage defects, and whether any clinical improvements can be maintained up to 5‐years. A randomized controlled phase 3 clinical trial was conducted for 48 weeks, and the populations were followed by an observational extended follow‐up study of 5 years. Cartilage defect was treated with UCB‐MSCs‐HA implantation or with microfracture. Primary outcome was proportion of subjects improved by ≥1 ICRS grade at 48 weeks. Secondary outcomes included histologic assessment, VAS pain, WOMAC, IKDC scores, and adverse events.
Among 114 randomized participants (55.9 years), 89 completed the clinical trial. 73 were enrolled in the 5‐year follow‐up study. Mean defect size was 4.9 (UCB‐MSCs‐HA) and 4.0 cm2 (microfracture). At 48 weeks, improvement by ≥1 ICRS grade was in 97.7% (UCB‐MSCs‐HA) versus 71.7% (microfracture) (p = 0.001), and the overall histological assessment was superior with UCB‐MSCs‐HA (p = 0.036). No between‐group differences in adverse events were observed. Improvement of pain, WOMAC, and IKDC score were similar between the groups at 48 weeks. However, the clinical results were significantly better in the UCB‐MSCs‐HA group at 3‐year and 5‐year follow‐up (p < 0.05). UCB‐MSCs‐HA implantation showed improved cartilage grade at 48 weeks and provided more durable improvement of pain and function in patients with large cartilage defects.
The application of pulsed electro‐magnetic field for cartilage regeneration
1National University of Singapore, 2National University Health System
Mesenchymal stem cells (MSCs) could participate in cartilage repair by direct differentiation to chondrogenic cells, or through the secretion of a plethoria of trophic factors, exerting paracrine function to the surrounding cells. MSCs are “environmentally responsive” to local micro‐environmental cues and biophysical perturbations, including pulse electromagnetic field (PEMF) exposure. In this study, using an in‐house designed PEMF delivery system producing precise magnetic field of low intensity and frequency, we demonstrate the augmentation of MSC chondrogenesis by PEMF exposure. PEMF inductive effect are exquisitely dependent on the pulsing intensity, duration, dosage as well as the MSC culture platform (scaffold free pellet culture, hydrogel or fibrous scaffold culture). Contrary to conventional practice of administering prolonged and repetitive exposures to PEMFs, optimal chondrogenic outcome is achieved in response to brief (10 minutes), low intensity (<2 mT) exposure at 15 Hz, administered only once at the onset of chondrogenic induction. By contrast, repeated exposures diminished chondrogenic outcome. Further, PEMF‐induced MSC secretome was capable of enhancing the migration of chondrocytes and MSCs as well as mitigating cellular inflammation and apoptosis. In vivo study using an osteochondral rabbit defect model, we provided evidence that brief exposure to low amplitude PEMFs enhanced the ability of MSCs‐based cartilage repair. Collectively, our results indicate that PEMF stimulation could have broad clinical and practical ramifications for the enhancement and re‐ establishment of cartilage regeneration.
Preclinical evaluation of a novel osteochondral scaffold in sheep joint
1University College London, 2University of Portsmouth
Pollen‐based materials innovation for sustainable technologies
1Nanyang Technological University
Throughout history, new materials have been the foundation of disruptive technologies. From bronze, paper, and ceramics to steel, polymers, and semiconductors, each material enabled far‐reaching advances and defined the era. Seventy years ago, the synthesis of pure semiconductors as single crystals led to a complete transformation of the electronics industry and sweeping changes in communications, computing, and transportation. Today, inspired by the United Nation's Sustainable Development Goals (SDGs) – a blueprint to achieve a better and more sustainable future for all – another new class of materials is emerging—one with both the potential to alleviate environmental burden, provide radically new functions, and to challenge our notion of what constitutes a “material.” These materials, inspired and co‐opted from biology, combine (1) hybrid‐composite design, combining disparate building blocks; (2) compartmentalized architecture, encapsulating desirable biomolecules while excluding others; and (3) hierarchical organization. Together, they enable unique and remarkable combinations of properties, including adaptability, plasticity, multi‐functionality, and environmental responsiveness – far beyond those achieved by monolithic materials of the synthetic world. An extraordinary example is pollen, a discrete mesoscale compartment, which encapsulates, protects, and transports male genetic material in flowering plants enabling the biological imperative of reproduction. In this talk, I will introduce our ongoing efforts to explore the materials science of pollen and to transform pollen into a valuable commodity to produce pollen‐based materials innovation as a sustainable solution to numerous outstanding environmental challenges. Key examples that will be covered include digital printing of shape‐morphing materials, recyclable and reusable paper, and oil‐absorbing sponges.
Metformin alters the mechanical behavior of common carotid arteries in type 2 diabetic mice
1Sunchon National University
Type 2 diabetes is the most popular type of diabetes and is associated to the increased risk for other organs including heart and blood vessels. Metformin is a most common medication to treat type 2 diabetes. To understand the mechanics of the arterial wall in the type 2 diabetes, we investigated the mechanical behavior of left common carotid arteries (LCCs) with and without metformin treatment in Leptin receptor‐deficient (db/db) male mice. After confirming high blood glucose levels at 7 weeks of age, they were fed with compounds of normal diet and 0.05% metformin for 6 weeks. Age matched m+/db mice and db/db mice fed with normal diet were used for comparison. To investigate the nonlinear viscoelastic mechanical behavior, the outer diameter (OD) of LCCs and axial force were recorded for analysis.
The OD of untreated db/db LCCs are significantly larger than the control near physiological pressure. However, the metformin treated db/db LCCs show the decreased ODs, which are close to the normal. The circumferential stretch and stress of the untreated db/db LCCs are greater than the control, but metformin treatment results in a less increase of the stretch and stress
The increased stretch and stress of db/db LCCs cause more compliant in stress‐stretch curve than the control, but it becomes similar to the control with metformin treatment. It suggests that metformin probably prevents the abnormal change of the arteries in type 2 diabetes. Further studies include histological investigation and mathematical modeling to understand the alteration of the arterial mechanics.
Skin tissue engineering using decellularized scaffolds
1Indian Veterinary Research Institute, 2Banaras Hindu University, 3Sardar Vallabhbhai Patel University of Agriculture and Technology, 4Narendra Deva University of Agriculture and Technology
Wound healing is a complex and dynamic process of restoring cellular structures and tissue layers. One of the major applications of tissue‐engineered skin substitutes for wound healing is to promote the healing of cutaneous wounds. In this respect, many important clinical milestones have been reached in the past decades. However, currently available skin substitutes for wound healing often suffer from a range of problems including wound contraction, scar formation, and poor integration with host tissue. Engineering skin substitutes by tissue engineering approaches have relied upon the creation of three‐dimensional scaffolds as extracellular matrix (ECM) analogs to guide cell adhesion, growth, and differentiation to form skin‐functional and structural tissue. The three‐dimensional scaffolds can cover wounds and give a physical barrier against external infection as a wound dressing and provide support for dermal fibroblasts and the overlying keratinocytes for skin tissue engineering. In the present study, skin from rats and rabbits was used for decellularization. The skin was harvested from rats and rabbits which were sacrificed for other reasons. These skins were then subjected to different combinations of physical and chemical decellularization techniques for decellularization. These decellularized scaffolds underwent physical characterization to ascertain their physical properties which were then compared with the natural skin. These decellularized scaffolds were then used to treat experimentally created cutaneous wounds on the dorsum of rats. The study concluded that the decellularized skin scaffolds accelerated the wound healing process and had complete early healing.
Enhancing effect of placental extract on regeneration of crush injured facial nerve
1Chonnam National University, 2Department of Veterinary Anatomy, College of Veterinary Medicine and BK21 FOUR Program, Chonnam National University, 9 Gwangju, 3BK21 FOUR Education Research Group for Age‐Associated Disorder Control Technology, Department of Integrative Biological 6 Science, Chosun University, Gwangju, 4K21 FOUR Education Research Group for Age‐Associated Disorder Control Technology, Department of Integrative Biological 6 Science, Chosun University, Gwangju
The aim of the present study was to investigate the effects of topical placental extract on recovery after a crush injury to the rat facial nerve, by functional, electrophysiological, and morphological evaluation. The viability was significantly increased in RSC96 Schwann cells treated with PE. The PE improve the migration of RSC96 cells by immunoblot test. The qPCR shows that PE increased neurotropic gene expression compared to control. The recovery of the vibrissa fibrillation in the placenta group enhanced compared to the control group. Threshold of electrically stimulated action potential. PE group statistically reduced threshold compared to the PBS group. Histopathological findings showed that crushed facial nerves treated with PE exhibited larger axons as compared with non‐treated control group. Surrounding myelin sheaths were also more distinct and thicker in PE treated groups as compared with control. From these results, PE may be considered as a topical therapeutic agent for traumatic facial nerve paralysis. No images/photos/figures/ tables.
Edible plant‐based scaffold for cultured meat
1Nanyang Technological University, School of Materials Science and Engineering
Lab cultured meat has garnered great interest over the last decade with the realization that livestock production will no longer be able to sustain the protein needs of the human population soon and the detrimental effects it has on global environment. The reliance on gelatin as a scaffold material for cultured meat is heavy as it is an edible natural polymer with chemical similarities to the extracellular matrix in native tissues. However, gelatin is also sensitive to enzymatic degradation and have poor mechanical properties on its own. To overcome these drawbacks and to further reduce the reliance on livestock production, there is value in exploring plant‐based scaffolds. Plant‐based polymers could comprise of cellulose, carbohydrates, alginates, plant proteins or plant extracts. Not only are they a sustainable source of food, but they are also highly tunable and processible materials. Pectin is polysaccharide material which has been employed in tissue engineering as it has a simple gelling mechanism, and can be easily adapted to various structures such as hydrogels, films and nanoparticles. While the benefits of using plant‐based materials are vast, one major barrier is the lack of cell‐adhesion sites. Our lab has established that pectin coating is able to support cell adhesion and can be further explored to be incorporated as a scaffold for cultured meat.
High antioxidation‐capacity stem cells enhance cartilage regeneration
1seoul national university hospital
The key functions of stem cells (SCs) are critically regulated by their cellular redox state. Glutathione is the most abundant non‐protein thiol that functions as an antioxidant and redox regulator. However, investigation of the relationship between glutathione‐mediated redox capacity and SC activity has been hampered by the lack of probes. We show that cyanoacrylamide‐based coumarin derivatives are suitable ratiometric probes for real‐time monitoring of glutathione levels in live SCs. We have confirmed that FreSHtracer is an effective tool for visualization, measurement, and real‐time monitoring of GSH levels in intact live MSCs. In addition, GSHhigh stem cells purified with FreSHTracer were shown to increase the maintenance of stem cells. GSHhigh stem cells can be increase the stem cell function and improve therapeutic efficiency in the treatment of cartilage defects. The results of this study are thought to maximize the efficacy of stem cell therapeutics that induce cartilage regeneration through high‐functional stem cell culture and isolation using FreSHtracer.
Confirming the ability of mesenchymal stem cells to promote fracture healing in a rat model of long bone shaft fracture and assessing the optimal cell concentration with the maximum therapeutic effect
1Kyung Hee University, 2Kyung Hee University Hospital at Gangdong
Ninety‐six adult male Wistar rats (8 weeks old) were used to establish a femur shaft fracture rat model. They were divided into four groups: the normal saline‐injected group and groups administered with different concentrations of MSCs (2.5 × 106, 5.0 × 106, or 10.0 × 106 MSCs were directly injected into the fracture site [u1]). The group injected with normal saline was used as the control group to confirm the effect of MSC administration on bone union. At two and six weeks post‐fracture, the rats were sacrificed to evaluate fracture healing. New bone formation (bone volume [BV] and percentage bone volume [PBV]) was evaluated using micro‐computed tomography (CT). Histological analysis was performed using hematoxylin and eosin staining to evaluate the fracture healing score of femur specimens. In addition, the mRNA expression of factors related to AD‐MSC migration (monocyte chemoattractant protein‐1 [MCP‐1] and transforming growth factor‐beta 1 [TGF‐β1]) and angiogenesis (vascular endothelial growth factor [VEGF]) was evaluated using real‐time quantitative polymerase chain reaction (RT‐qPCR). The protein expression of cytokines (bone morphogenetic protein‐2 [BMP‐2] and transforming growth factor‐beta 1 [TGF‐β1]) associated with osteogenesis were quantitatively evaluated via western blot analysis.
The direct injection of various concentrations of MSCs enhanced fracture healing in a rat model of long bone shaft fracture. Furthermore, among the various concentrations tested, the administration of 5.0 × 106 MSCs was the optimal concentration to maximize the effect on fracture healing.
Development of iPSC‐derived neuronal model for studying mitochondrial defects in leigh's syndrome
1Centre for Tissue Engineering and Regenerative Medicine (CTERM), Universiti Kebangsaan Malaysia (UKM), 2Institute for Medical Research (IMR), National Institutes for Health (NIH), Ministry of Health (MOH), Malaysia, 3Department of Biomedical Engineering, Faculty of Engineering, Universiti Malaya, Malaysia, 4Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia (USM)
Leigh's Syndrome (LS) is progressive neurodegenerative disorder linked to mitochondrial dysfunction. Pathogenesis of LS is not fully understood due to limitations of current disease models. Development of induced pluripotent stem cells (iPSCs) has opened the possibility of elucidating disease mechanisms. Here, we aimed to generate iPSC‐derived neural stem cells (NSC) from fibroblasts obtained from a LS patient carrying ND6‐mutation and healthy individuals and analyse the mitochondrial function in these NSCs.
We reprogrammed fibroblasts from healthy individuals and an LS patient by lentiviral transduction. iPSC clones were expanded and characterized by immunostaining and real‐time quantitative polymerase chain reaction (qRT‐PCT) for pluripotency and genetic stability. iPSCs were differentiated into NSCs and neurons by defined medium. iPSC‐derived NSCs were analysed by high content image analysis for analysis of mitochondrial membrane potential.
iPSCs from LS patients (LS‐iPSCs) exhibited characteristic morphology and highly expressed pluripotency markers.LS‐ iPSCs showed no chromosome aberrations and formed embryoid bodies that spontaneously differentiated and expressed markers of all three germ layers. LS‐iPSCs successfully differentiated into NSCs expressing SOX2 and Nestin and subsequently mature into neurons expressing BIII‐tubulin and MAP2. Reduction in mitochondrial membrane potential was seen in LS‐derived NSCs with no significant differences in mitochondrial mass compared to control iPSCs.
We successfully obtained iPSCs, NSCs and neural cells from fibroblasts of an LS patient without genetic aberration. These LS‐iPSC‐derived NSCs and Control‐iPSC‐derived NSCs may further assist the understanding of the LS pathogenesis and develop the new strategies for clinical diagnosis and therapeutic drug targets.
Generation of human tonsil epithelial organoids as an ex vivo model for SARS‐CoV‐2 infection
1Infectious Diseases Therapeutic Research Center, Korea Research Institute of Chemical Technology (KRICT), Daejeon, Republic of Korea, 2Department of Microbiology, CHA University School of Medicine, Seongnam, Republic of Korea., 3a Department of Microbiology, CHA University School of Medicine, Seongnam, Republic of Korea. b CHA Organoid Research Center, CHA University, Seongnam, Republic of Korea. c R&D Institute, Organoidsciences Ltd., Seongnam, Republic of Korea, 4Department of Otorhinolaryngology‐Head and Neck Surgery, the Research Institute, Konkuk University School of Medicine, Seoul, Republic of Korea, 5Department of Biomedical informatics, CHA University School of Medicine, CHA University, Seongnam, Republic of Korea, 6Infectious Diseases Therapeutic Research Center, Korea Research Institute of Chemical Technology (KRICT), Daejeon, Republic of Korea Graduate School of New Drug Discovery and Development, Chungnam National, 7a Department of Microbiology, CHA University School of Medicine, Seongnam, Republic of Korea. b CHA Organoid Research Center, CHA University, Seongnam, Republic of Korea., 8Department of Microbiology, CHA University School of Medicine, Seongnam, Republic of Korea. b CHA Organoid Research Center, CHA University, Seongnam, Republic of Korea.
The palatine tonsils (hereinafter referred to as “tonsils”) serve as a reservoir for viral infections and play roles in the immune system's first line of defense. The aims of this study were to establish tonsil epithelial cell–derived organoids and examine their feasibility as an ex vivo model for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection. The tonsil organoids successfully recapitulated the key characteristics of the tonsil epithelium, including cellular composition, histologic properties, and biomarker distribution. Notably, the basal layer cells of the organoids express molecules essential for SARS‐CoV‐2 entry, such as angiotensin‐converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and furin, being susceptible to the viral infection. Changes in the gene expression profile in tonsil organoids revealed that 395 genes associated with oncostatin M signaling and lipid metabolism were highly upregulated within 72 h after SARS‐CoV‐2 infection. Notably, remdesivir suppressed the viral RNA copy number in organoid culture supernatants and intracellular viral protein levels in a dose‐ dependent manner. Here, we suggest that tonsil epithelial organoids could provide a preclinical and translational research platform for investigating SARS‐CoV‐2 infectivity and transmissibility or for evaluating antiviral candidates.
Adipose stem cell transplantation using viscous liquid phase protein‐ based adhesive coacervate for cartilage reconstruction
1Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 37673, Korea, 2Research institute of Convergence Life Science, Dongguk University, Goyangsi, Gyeonggido, korea
The articular cartilage is the smooth and rigid tissue that covers the end of bones to allow normal glide of joint. However, damaged cartilage can trigger pathogenesis of osteoarthritis (OA) by occurrence of friction between adjacent bones. Because cartilage has limited intrinsic healing capacity, application of stem cells for grant of regenerative function is required. To secure the regenerative effect of transplanted stem cells, high retention, viability, and proliferation are required. Thus, the scaffolds made of many types of materials such as metals, polymers, fibrin, and collagen have been developed as attempts to transplant stem cells into the cartilage environment. However, most of the previous methods of implanting scaffolds into cartilage are invasive due to stiffness of scaffolds and complicated processes for fixation of scaffolds. In the present work, bioengineered mussel adhesive protein (MAP) was applied to secure high viability and maintenance of implanted stem cells on cartilage defect site. Especially, we propose a stem cell‐ encapsulated injectable high viscos liquid phase system for filling cartilage defect using bioengineered MAP. The MAP was processed into complex coacervate by using high viscos hyaluronic acid (HA) for functional underwater adhesion. The MAP‐HA coacervate might satisfy the requirements for cartilage reconstruction through feasible injection from its shear thinning ability and effective encapsulation ability of adipose stem cells (ASCs). In this regard, we developed biocompatible adhesive which enables implanted ASCs being stably fixed to the lesion site of defected cartilage, and thus the implanted cells could survive lengthily in defect site, acting on cartilage regeneration.
Opportunities and challengues of light‐based bioprinting technologies
1Maastricht University
Light based bioprinting has been actively investigated as one of the enabling technologies for the impending era of personalized medicine. These technologies, which use light for polymerization of biomaterials, have made significant progress in the quality, resolution and generation of precise complex tissue structures. Numerous fields have been disrupted by its introduction, such as tissue engineering and regenerative medicine. This is often attributed to the technology's inherent versatility, unique ability to fabricate intricate designs, and cost‐effectiveness compared to traditional manufacturing processes. Here will be explained commonly used light‐based bioprinting technologies, including, Laser‐based SLA, Mask‐based SLA, two‐photon bioprinting (TTP), laser induced forward transfer (LIFT), laser‐direct bioprinting (LDP). In particular we will focus on recent results on multimaterial digital light projection (DLP) bioprinting and volumetric 3D printing approaches; and their application to tissue engineering and regenerative medicine. An illustration the key factors for light‐based bioprinting, including the cell‐ polymer‐light interactions will be considered.
Cell encapsulation in gelatin bioinks impairs 3D resolution and the capacity of volumetric DLP bioprinting
1New Jersey Institute of Technology, 2Maastricht University
3D digital light processing (DLP) bioprinting is an additive manufacturing technology based on a series of 2D light projections, and it has been recently applied in tissue engineering and regenerative medicine. Cell‐laden hydrogels are commonly used as DLP bioinks which can be cured via light‐assisted crosslinking to form the desired 3D shape. Gelatin methacryloyl (GelMA) bioinks have been extensively used for DLP bioprinting. The quality of the process depends on the resolution or the smallest feature size in DLP bioprinted constructs. We studied how fibroblast cell distribution may impact the resolution in DLP bioprinted GelMA (5% to 10% w/v). Our results show that planar and out‐of‐plane resolutions were impaired by the change of the cell density. The team then studied the role of cell density in volumetric DLP bioprinting (which is subjected to three perpendicular projections). The feature size increased by 25% when the cell density increased from 100 K cells/ml to 20 M cells/ml. The 3D light scattering was also increased by nearly five times. The present results may provide some criteria by which 3D bioprinting parameters can be optimized for (volumetric) DLP bioprinting and GelMA‐based bioink formulations. This can be extended to other natural hydrogels in biofabrication.
Silk fibroin as a bioink for cardiovascular applications
1University of Technology Sydney, 2University of New South Wales, 3Garvan Institute of Medical Research
3D bioprinting has emerged as a potential optimal approach to biofabricate viable and functional cardiac tissues to promote regeneration in patients. Our laboratory has developed a novel approach to engineer vascularized bioprinted cardiac tissues using bioinks containing alginate (Alg) and gelatin (Gel) biomaterials, with optimal biocompatibility, printability and durability to promote cardiac regeneration in vitro. Given its tunable properties and low immunogenic response in the human body, we have investigated the potential use of silk fibroin (SF) as biomaterial for cardiac bioink formulations. SF is a natural protein extracted from the Bombyx mori silkworm thread and as aqueous solution was mixed with Alg and Gel to obtain hybrid hydrogels. SF‐based hybrid hydrogels were evaluated for their morphology, mechanical properties, printability, durability, cardiac cells viability and expression of the endothelial cells marker CD31. Our preliminary results showed that the addition of SF to Alg/Gel hydrogels does not affect the printability and durability of bioprinted hydrogels up to 28 days in cell culture medium. However, increasing concentrations of SF to Alg/Gel hydrogels change the morphology of the hydrogels and they resulted with higher viscosity and less elasticity. Current studies are investigating the biocompatibility of SF‐based hybrid hydrogels using cardiac cells and they showed that cells growth kinetics are regulated by the addition of SF to Alg/Gel hydrogels. Furthermore, cardiac endothelial cells expressed CD31 in Alg/Gel hydrogels containing higher concentrations of SF. Future studies will evaluate the in vitro testing for cardiac cell contraction and vascular network formation in Alg/Gel hydrogels.
Whole human extracellularmatrix (ECM) derived from neonatal dermal fibroblast and bone marrow MSC culture achieved complete burn wound regeneration
1ROKIT HEALTHCARE, Inc
WHO reports over 11 million people suffer from sever burn wound not only in traumatized body condition but also in post injury psychological trauma. Currently, there is no one time treatment that prevent further infection and promotes scarless skin regeneration at the same time. We derived human whole dermal ECM from neonatal dermal fibroblast culture and prepared in lyophilized powder form for burn wound treatment. In porcine model, 4cm x 4cm burn wounds (between 2nd and 3rd degree burn) were created. ECM powder dissolved in saline was sprayed directly on the derbidemented wounds. Re‐ epithelialization was achieved in 14 days and 35 days follow up showed fast skin regeneration pattern without contraction compared to control group. At day 35, ECM treated group showed complete re‐ epithelialization with hair without scar while control group showed scar formation due to contraction. Furthermore, proteomic analysis showed ECM applied group had minimal amount of collagen type I content similar to that of normal undamaged skin. In histological analysis, rich collagen type III, which is main collagen type in dermis, was found in newly formed dermis of ECM treated group. However, collagen type I was main collagen type in control group. We also confirmed the reformed basement membrane. Moreover, in immunohistological analysis, CTGF (connective tissue growth factor associated with fibrotic tissue formation) level was 4 folds less than scar formed control group. We confirmed that whole human ECM from matching tissue type cell provided a regeneration niche that is a minimal requirement for dermal regeneration.
Enthesis regeneration in anterior cruciate ligament reconstruction
1Biomedical Engineering, National University of Singapore, 2Orthopaedic Surgery, National University of Singapore
The fibrocartilaginous type enthesis interfacial tissue provides the strength to secure ligament to bone. In surgical reconstruction of ACL with tendon graft, optimal healing of the tendon graft within the bone tunnel is not adequately achieved. Therefore, the proposed solution involves the use of a silk fibroin (SF)‐based sheath, loaded with nanoparticles of low crystallinity hydroxyapatite (nHA), and sleeved onto tendon autografts to complement their use and promote enthesis formation. The sheath was sutured onto both ends of the graft and pulled through both femoral and tibial bone tunnels in a porcine ACL reconstruction model. All animal experiments were approved by the respective IACUC. The enhancement in osteointegration of tendon autograft was evident within the femoral and tibial ends of the graft from as early as 1‐month post reconstruction. Continuous host integration and bone remodeling were observed through the 9 months period, with significant bone tunnel narrowing in the test groups observed by the end of the study. Mechanical study showed that the sheaths enhanced graft tensile strengths. The SF‐ based sheath serves as delivery platform for cellular and bioactive components. Progenitor cells attracted from the host into the porous sheath could have reconstitute the native cellular environment of the enthesis by differentiating into chondrocytes and osteoblasts. Consequently, there was enhanced graft‐to‐ host integration progressively over the 9 months implantation period, which resulted in overall mechanical properties closer to that of the native bone‐ACL‐bone construct.
Osteochondral scaffold innovation for repair of large osteochondral defects: From bench to bedside
1University College London
The repair of osteochondral defects is one of the major clinical challenges in orthopedics. Well‐ established osteochondral tissue engineering methods have shown promising results for the early treatment of small defects. However, less success has been achieved for the regeneration of large defects, which is mainly due to the mechanical environment of the joint and the heterogeneous nature of the tissue.
In this study, we developed a multi‐layered osteochondral scaffold to match the heterogeneous nature of osteochondral tissue by harnessing additive manufacturing technologies and combining the established art laser sintering and material extrusion techniques. The developed scaffold is based on a titanium and polylactic acid matrix‐reinforced collagen “sandwich” composite system. The microstructure and mechanical properties of the scaffold were examined and its safety and efficacy in the repair of large osteochondral defects were tested in an ovine condyle model.
The in vivo evaluation revealed extensive and significantly higher bone in‐growth in the multi‐layered scaffold compared with the collagen‐HAp scaffold, and the achieved stable mechanical fixation provided strong support to the healing of the overlying cartilage. The histological examination showed that the regenerated cartilage in the multi‐layer scaffold group was superior to that formed in the control group. Chondrogenic genes such as aggrecan and collagen‐II were upregulated in the scaffold and were higher than those in the control group. The findings showed the safety and efficacy of the cell‐free “translation‐ ready” osteochondral scaffold, which has the potential to be used in a one‐step surgical procedure for the treatment of large osteochondral defects.
Human synovial joint‐mimicking organ‐on‐a‐chip systems for disease modeling and drug testing
1The Chinese University of Hong Kong
Disorders of the synovial joint, such as osteoarthritis and rheumatoid arthritis, are well‐recognized but unsolved medical problems. The significant unmet medical need for joint disease treatment is primarily caused by the lack of an appropriate model that can faithfully recapitulate the pathophysiology of the native joint. Organ‐on‐a‐chip (OoC) platforms for modeling human synovial joints have undergone rapid development in the past decade. While the concept of multi‐component joint OoC for modeling joint diseases has been proposed, the feasibility has yet to be validated. This presentation first provides an overview of the development of joint OoCs with the key milestones highlighted. Next, a miniaturized, joint‐mimicking OoC, named miniJoint, is reported. The components of the miniJoint include human mesenchymal stem cell‐derived osteochondral tissue (bone and cartilage), synovial‐like fibrous tissue (SFT), and adipose tissue. These tissues could maintain their respective tissue phenotypes for at least four weeks. Furthermore, “synovitis” induced by insulting the SFT with the proinflammatory cytokine interleukin‐1β caused pathological changes both locally and in other tissues, as shown in RNA‐seq and Multiplex assay results, indicating active crosstalk between all miniJoint tissues. In addition, the established disease model was further employed to evaluate the efficacy of a “systemically administered” non‐steroidal anti‐inflammatory drug (used in all medium streams) and four “intra‐articularly injected” drugs (added to the “simulated synovial fluid” only). The results support the physiological relevance and potential of the miniJoint in mechanistic studies and preclinical drug testing. Finally, the challenges and future perspectives on the development of joint OoCs are presented.
Probing lateral integration of engineered cartilage spheroids in healthy and diseased microenvironments – a 3D in‐vitro tissue fusion model
1University of Otago, 2Univerisity of Otago
Tissue engineered (TE) cartilage constructs often display limited lateral integration with host cartilage tissue, restricting clinical translatability. This study therefore aims to investigate lateral tissue‐tissue interactions between cartilage microtissues and engineered implants in healthy and diseased microenvironments.
Healthy cartilage microtissues were fabricated by centrifuging human articular chondrocytes (hACs) [1,2]. Exposure to inflammatory cytokines (TNF‐α/IL‐1β) resulted in osteoarthritic (OA) microtissues. TE cartilage spheroids were created by encapsulating clinically relevant human mesenchymal stromal cells (hMSCs) and/or hACs in biomaterials (allylated gelatin;GelAGE or native vitreous humor; VH) [3]. Bio‐assembly of tissue spheroids adjacently in extrusion‐based 3D‐printed polycaprolactone (PCL) cages allowed characterisation of tissue formation (GAG, DNA, IHC: Safranin‐O/Col I/II) and cellular migration at the tissue‐tissue [interface 5].
Fusion between healthy microtissues displayed impaired migration and fibroblastic extracellular matrix (ECM) formation at the tissue interface (collagen II/I ratio:0.5), recapitulating in‐vivo [observations 4]. Introducing hAC‐laden photo‐crosslinked GelAGE hydrogel spheroids improved migration, however, significant collagen‐type‐1 deposition was observed. Cell‐instructive VH supported hMSC‐VH‐spheroid self‐assembly and stimulated migration of both hACs and the encapsulated hMSCs towards the tissue‐ tissue interface, inducing hyaline cartilage tissue formation (collagen II/I ratio:1.25, p < 0.05). Successful fusion between both healthy and OA microtissues was observed, confirming additional clinical relevance of the 3D model.
HMSC‐VH‐spheroids supported cellular migration and healthy tissue formation at tissue‐tissue interfaces, while GelAGE‐based spheroids provided insufficient biological cues to support hyaline cartilage ECM deposition. Ultimately, this 3D fusion model allows novel TE constructs to be systematically screened and optimised in high‐throughput, in both healthy and diseased microenvironments, for lateral tissue‐ tissue interaction prior to (pre)clinical studies.
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[2]‐Schon.B;et‐al.‐Ann.Biomed.Eng.45(1).2017; [3]‐Lindberg.G;et‐al.‐Acta.Biom.85.2019;
[4]‐Khan.I.M;et‐al.‐European.cells.and.materials.16.2008. [5]‐Lindberg.G;et‐al.‐Adv.Science.8(22).2021
The role of microRNA‐1 in cartilage repair and osteoarthritis
1University of Technology Sydney, 2Peking University People's Hospital, 3Tianjin Hospital
Osteoarthritis (OA) is an incurable joint disease characterised by significant pain and tissue degradation. Epigenetics have been increasingly studied as important mechanisms contributing to OA pathogenesis. In particular, microRNAs are found to have critical roles in controlling inflammatory pathways and matrix regulation in articular cartilage. In this study, we investigate for the first time the role of microRNA‐1 (miR‐1) in the pathogenesis of OA, previously shown to contribute to normal cartilage development, but its role in OA has not been elucidated.
From its involvement in other diseases, we hypothesised that miR‐1 may attenuate OA pathogenesis and cartilage degradation by regulating Wnt/β‐catenin signalling in chondrocytes. By comparing gene expression in chondrocytes from human donors with and without OA, we found that microRNA‐1 is under‐expressed in OA chondrocytes. Using SW1353 cells as a human chondrocyte cell model, we found that over‐ and under‐expression of miR‐1 respectively reduced and enhanced gene expression for catabolic enzymes associated with cartilage degradation, including MMP9, MMP13 and ADAMTS5. We further determined through luciferase reporter assay that miR‐1 can bind to FZD7 as a molecular target, which inhibits downstream Wnt/β‐catenin signalling in chondrocytes by increasing the levels of phosphorylated (inactivated) β‐catenin. This subsequently reduces gene expression in chondrocytes associated with catabolic enzyme activity, manifesting as reduced cartilage degradation. Increased miR‐1 expression may therefore attenuate OA pathogenesis.
This study provides the first evidence of a functional link between miR‐1 and Wnt/β‐catenin signalling in chondrocytes, uncovering a new mechanism that may be used towards developing new treatment targets in OA.
Biomimicking scaffolds for neural tissue regeneration
1Nanyang Technological University
The extracellular matrix plays critical roles in dictating cell fate and tissue regeneration. Biophysical signals, such as matrix architecture and compliance affect cellular response. In combination with biochemical signals from drugs, growth factors, nucleic acids and/or cells, synergistic effects on directing endogenous cell phenotypic changes and tissue regrowth are often seen. Here, we will present our approaches towards designing bio‐mimicking constructs to direct cell fate. We will also share our findings of combining the appropriate biophysical and biochemical signals into tissue‐engineered scaffolds to promote nerve regeneration and remyelination in the central nervous system.
Piezoelectric peptide‐based injectable hydrogels for bone tissue engineering
1Soft Matter Physics and Biophysics Section, KU Leuven, Leuven, Belgium, 2Prometheus Division of Skeletal Tissue Engineering, KU Leuven, Belgium; Skeletal Biology and Engineering Research Center, KU Leuven, Belgium; Biomechanics Section, KU Leuven, Belgium, 3Prometheus Division of Skeletal Tissue Engineering, KU Leuven, Belgium; Skeletal Biology and Engineering Research Center, KU Leuven, Belgium; Biomechanics Section, KU Leuven, Belgium; GIGA In Silico Medicine, University of Liege, Liege, Belgium , 4Department of Physics and Astronomy, KU Leuven, Leuven, Belgium, 5Biomechanics Section, KU Leuven, Belgium
Bone undergoes piezoelectric stimulation under mechanical loading (e.g., stresses, strains, interstitial fluid flows) due to the non‐centrosymmetric crystal organization of the collagen network. Piezoelectricity is a phenomenon when asymmetric shifts in ions create electrical forces in response to mechanical loading. In bone, the piezoelectricity stimulates cells by micro voltage charges recruiting macromolecules and ions in the extracellular matrix (ECM). Thus, piezoelectric materials are gaining attention in bone regeneration strategies with the aim of mimicking bone piezoelectricity. Peptide self‐assembly is a novel concept in hydrogel‐based biomanufacturing strategies for tissue engineering and regenerative medicine. Among them, diphenylalanine (FF), and derivatives (e.g., Fmoc‐FF, Boc‐FF) are promising for osteogenic differentiation with tunable piezoelectric and mechanical properties.
In this study, GelMA was formulated with Fmoc‐FF to form injectable piezoelectric hydrogels. GelMA/Fmoc‐FF hydrogels were stable at 37 oC, providing the possibility to handle the piezoelectric hydrogel at physiological temperature. The hydrogelation process depends on dual crosslinking; (i) ionic crosslinking and self‐assembly of Fmoc‐FF and (ii) covalent crosslinking with ultraviolet light. Surface characterization was performed using atomic force microscopy (AFM), scanning electron microscopy (SEM) and Fourier‐transform infrared spectroscopy (FTIR). Piezoelectricity of the hydrogels was measured with piezoresponse force microscopy (PFM) and in‐house piezoelectric measurement device which applies controlled force. Cell viability and differentiation experiments were done using hTERT‐ human Bone Marrow Mesenchymal Stem Cells (hTERT‐hBMMSCs) which confirmed high cell viability. Taken together, we conclude that GelMA/Fmoc‐FF blends are versatile injectable hydrogels for bone tissue engineering.
Synthetic, tuneable and dynamic organoid matrices using novel self‐ assembling peptide hydrogels
1University of New South Wales
Organoid culture has traditionally relied on natural hydrogels like Matrigel as extracellular matrix mimics. Synthetic peptide hydrogels have emerged as promising alternatives as they are fully defined, tunable, and enable the decoupling of biophysical properties of the microenvironment from cell behaviour and fate. Certain peptide motifs can self‐assemble into nanofibers that form entangled networks reminiscent of native tissue matrices. However, identifying new self‐assembling peptides from sequence or experimentation alone remains highly challenging.
Here, we present our combined computational and experimental approach to identify novel peptide sequences that form dynamic hydrogels via a tryptophan‐zipper (trpzip) motif. High performance computing was used to run coarse‐grain molecular dynamics simulations of hundreds of trpzip peptides within a physiologically solvated simulation box. Simulations and subsequent experimental studies confirmed the successful gelation ability of new trpzip peptide sequences. The viscoelastic properties of our peptide hydrogel were characterised using rheology, where we observed the self‐healing of our hydrogel to stiffnesses of 10 kPa within 10 minutes. Human fetal fibroblasts were cultured in the hydrogel, where we observed high cell viability in both 2D and 3D settings. Due to the tryptophan‐rich peptides, our hydrogel demonstrates strong antimicrobial resistance against both gram‐positive and gram‐ negative bacteria. Human small intestinal organoids and foregut lung organoids were encapsulated in our hydrogel and their viability, morphological changes and gene expression profiles assessed using confocal fluorescence microscopy and RT‐qPCR. Due to its dynamic and stress‐relaxing behaviour, we anticipate our peptide hydrogels will be versatile matrices for promoting organoid growth and morphogenesis in vitro.
Impact of alginate and monolayer culture on intervertebral disc progenitor
1The University of Hong Kong
Alginate hydrogel is widely used as a bioactive scaffold for cartilage and intervertebral disc engineering. Alginate encapsulation can promote extracellular matrix (ECM) production and limit cellular de‐ differentiation. Intervertebral disc is a semi‐cartilaginous spinal joint tissue, and its degeneration leads to spinal disability and pain. Accumulating evidence indicates a heterogeneous nucleus pulposus (NP) cell population and decreased disc progenitors in disc degeneration. Disc progenitors are thought to have a therapeutic capacity for disc repair. We investigated the impact of alginate culture on disc progenitors. We found that alginate cultured NP cells demonstrated a reduced colony formation capacity, implying alginate inhibited progenitor formation. Transcriptome analysis suggested that alginate encapsulated NP cells have upregulated ECM production, receptor ligand interaction and lipid transportation. Enriched genes include membrane components with functions in cell adhesion and ECM organization, as well as ERK1/2 signaling which is essential to osmolarity control and apoptosis of NP cells. Pathway analysis indicated activation of signaling related to calcium and rheumatoid arthritis. In contrast, analysis of monolayer‐cultured NP cells showed enriched genes involved in cell cycle, p53 activation, and cellular senescence. Notably, genes involved in oocyte meiosis and maturation were largely upregulated, implying a regulation of progenitor activity. Altogether, our findings suggest that alginate culture tends to maintain the differentiation state and heterogeneity of human NP cells, whereas monolayer culture may in contrast lead to the expansion of proliferative cells, such as disc progenitors.
Culture and differentiation of human pluripotent stem cells on mixed oligopeptide‐grafted hydrogels
1National Central University
Human pluripotent stem cells (hPSCs) have unlimited proliferation and pluripotency to differentiation into the cells derived from three germ layers. Xeno‐containing Matrigel are typically used to culture and differentiation of hPSCs where Matrigels contain collagen IV, laminin‐111, etc. Typically, single oligopeptide‐grafted hydrogels have been used for hPSC culture and differentiation. Considering multiple components contained in Matrigel, we designed mixed peptide‐grafted hydrogels, which may improve hPSC proliferation and differentiation using different binding site of hPSCs. We prepared vitronectin‐ derived peptide (GCGGKGGPQVTRGDVFTMP) and laminin β4‐derived peptide (KKGCGGKGGPMQKMRGDVFSP)‐grafted poly(vinylalcohol‐co‐itaconic) hydrogels using N‐ hydroxysuccinimide (NHS)/1‐ethyl‐3‐(‐3‐dimethylaminopropyl) carbodiimide (EDC) chemistry where the elasticity of the hydrogels was controlled at 25.3 kPa. Human PSCs could proliferate on the mixed peptide‐grafted hydrogels compared to single peptide‐grafted hydrogels efficiently. Hydrogels prepared with low concentration of mixed peptides (200:200 μg/mL) could support hPSC adhesion and pluripotency. Furthermore, positive amino acid (K, lysine) insertion on the first sequence of the peptide contributed to enhancement of surface grafting density of peptide‐grafted hydrogels. This is explained that the amino group on the side chain of the first position of peptide (
Adoptive T‐cell therapy using marrow‐infiltrating lymphocytes for multiple myeloma
1Chonnam National University Hwasun Hospital
Adoptive immunotherapy is a promising treatment approach for multiple myeloma (MM). In this study, we generated expanded MILs from MM patients based on anti‐CD3/CD28 beads. Anti‐CD3/CD28 beads were used to expand MILs in the presence of IL‐2, IL7 and IL15. Expansion rate, proportions of effector cells such as CD8, CD4 T cells, NK cells, memory T cells and functions of expanded MILs were determined over two weeks of culture. The study demonstrated that co‐culturing of MIL with anti‐ CD3/CD28 beads in the presence of IL2, IL7, and IL15 resulted in remarkable expansion of MIL over 14 days culture period. In addition, expanded MILs showed increased proportions of CD8+ T cells, central memory T cells. Interestingly, eMILs showed a higher proportion of central memory T cells (>80%) while the proportions of myeloid‐derived suppressor cells (MDSCs) and regulatory T cells (Tregs) are very low at days 14 or 21 of culture. Compared with expanded PBLs, eMILs demonstrated increased cytotoxicity towards target MM cells, particularly CD138+ primary MM cells from autologous patients. Increased activity of eMILs was further confirmed by CD107a degranulation, Incucyte cytotoxicity assay, and IFN‐γ production. However, the expression of inhibitory receptor (TIM3) is increased after MIL expansion (∼40%) that may be a limitation of eMIL, therefore we are currently investigating the combination of eMIL plus a checkpoint blockade against MM. Therefore, MILs are a distinctive set of T cells that have been shaped by the unique BM microenvironment and may play a future role as a novel immunotherapy for MM.
Development and in vivo evaluation of anti‐CD19 chimeric antigen receptor (CAR)‐γδ t cells
1Asan Medical Center, 2Asan Institute for Life Sciences
Background: The chimeric antigen receptor (CAR) T‐cell therapy is intended to artificially enhance the function of T cells against specific tumor antigens. Gamma delta T (γδ T) cells are a distinct subset of T cells exhibiting both adaptive and innate‐like behaviors. This study was to develop and evaluate in vivo efficacy of CAR‐γδ T cells.
Methods: Lentiviral vector to express CD19‐CAR was transduced into γδ T cells. NSG mice were intraperitoneally injected with CFSE‐labeled SU cells and γδ T cells transduced with CD19‐CAR. Peritoneal lavage was analyzed by flow cytometry.
Results: Vδ1 γδ T cells were expanded 10‐fold in the presence of IL‐2+IL‐15, while Vδ2 γδ T significantly proliferated 114 in the presence of IL‐2+ZOL+IL‐15. γδ T cells were confirmed to express the activating (NKG2D, CD16, DNAM‐1), cell death (TRAIL, FasL), and immune checkpoint (PD‐1, BTLA) molecules. Except for PD‐1, Vδ2 γδ T cells expressed the markers listed above approximately 1‐2 fold more, compared with Vδ1 γδ T cells. In vitro, CAR‐γδ T cells exhibited about 2‐5% higher cytotoxicity than that of γδ T cells without CAR. In vivo, mice injected with ex vivo expanded CAR‐ γδ T cells showed 11 times lower levels of SU cells than mice injected with SU only.
Conclusion: This study shows γδ T cell expansion to be engineered with CAR under different conditions. As a result, γδ T cells transduced with CAR exhibited advanced tumor cell killing ability in vitro and in vivo without compromising the expression of multiple receptors.
Impaired T cell function in microenvironment of multiple myeloma – implications for immunotherapy
1Ajou University School of Medicine
While immune checkpoint inhibitors have achieved significant clinical success in many types of solid tumors, recent clinical trials of immune checkpoint blockade performed in patients with multiple myeloma failed to demonstrate significant anti‐tumor efficacy. To enhance the clinical efficacy of immune checkpoint blockade in multiple myeloma, elaborate characterization of tumor antigen‐specific T cells is an essential prerequisite.
We investigated the immunophenotypic and functional characteristics of tumor antigen‐specific T cells in patients with multiple myeloma. In addition, using direct ex vivo techniques, we tried to examine how to manipulate the immunosuppressive microenvironment to maximize anti‐myeloma responses of the tumor‐ specific T cells. Although PD‐1/PD‐L1 axis acts as a major component of immunosuppressive milieu in multiple myeloma, the efficacy of PD‐1 blockades in multiple myeloma might be hampered by complicated microenvironment consisting of T cell‐intrinsic and ‐extrinsic factors.
TGF‐β produced by clonal plasma cells and bone marrow stromal cells is critical component consisting of immunosuppressive microenvironment of multiple myeloma. We found that combination of TGF‐β signaling blockade with anti‐PD‐1 significantly increased the frequencies of IFN‐γ‐ and/or TNF‐α‐ producing CD8+ T cells in response to ex vivo TCR stimulation, compared to a single PD‐1 or a single TGF‐β blockade. Likewise, myeloma antigen‐specific proliferation of CD8+ T cells was significantly enhanced with addition of TGF‐β signaling blockade. These results provide an ex vivo evidence of incorporating TGF‐ β signaling blockade to immune checkpoint inhibition to enhance anti‐tumor T cell responses in multiple myeloma for future clinical trials.
Natural killer cell activation receptor overexpression strategy through NKG2D mRNA‐loaded polyplex
1Dongguk Univ., 2Inha Univ.
One of the most promising cancer treatments is adoptive cell transfer using natural killer (NK) cells. However, the treatment of solid cancer is limited due to pathophysiological characteristics such as intrinsic negative regulatory mechanisms in tumor microenvironments. To improve receptor/ligand dependent cellular interaction and following anticancer efficacy, NK cells should be genetically modified for enhanced cancer cell specificity. Particularly, in vitro transcribed (IVT) mRNA can be translated directly from the cytoplasm without reaching the cell nucleus, allowing the desired protein to be expressed faster than DNA delivery. Therefore, we developed genetically engineered NK cells with overexpressed NKG2D surface receptors via polyplex‐mediated mRNA delivery. For polyplex fabrication, cationic polymer polyaspartamide 2‐cyclohexyl diethylenetriamine (PAsp(CHE/DET)) containing (1) a DET moiety for endosomal escape and (2) CHE moiety for stabilizing particle formation. PAsp(CHE/DET) polyplex did not show toxicity to cells in varying polymer's amine‐to‐ mRNA's phosphate (N:P) ratio, and the delivery of the model Fluc‐mRNA using polyplex exhibited higher luminescence intensity as compared to that in free mRNA transfection. Polyplex‐mediated NKG2D mRNA delivery effective upregulation of this activation receptor onto NK cell surfaces. Co‐ culture with cancer cells expressing MICA/B (representative ligand of NKG2D) and genetically engineered NK cells resulted in higher cancer cell death, indicating facilitated anticancer efficacy of transfected NK cells. These results confirmed (1) the successful transfection of NK cells via mRNA delivery using PAsp(CHE/DET)‐based polyplex, and (2) enhanced in vitro cytotoxicity to cancer cells. Therefore, our mRNA delivery platform is suitable and functional genetic engineering tool for immune cell modulation.
T‐cell‐derived nanovesicles for cancer immunotherapy
1Seoul National University
Despite of successful treatment of acute lymphoblastic leukemia (ALL) by chimeric antigen receptor (CAR)‐T therapy, solid tumor treatment is not well achieved by current cancer immunotherapy. A major hurdle of solid tumor treatment is T cell exhaustion in immunosuppressive tumor microenvironment (TME), which is mainly comprised of tumor programmed death‐ligand 1 (PD‐L1) and transforming growth factor‐beta (TGF‐β). Herein, we developed T cell derived nanovesicle (TCNV)s, which are produced by the serial extrusion of cytotoxic T cells through nanopore membranes. These TCNVs inhibit T‐cell exhaustion and simultaneously exhibit antitumoral activity, and these antitumor mechanisms are stably maintained in the immunosuppressive TME. TCNVs, which have programmed cell death protein 1 (PD‐1) and TGF‐β receptor on their surface, block PD‐L1 on cancer cells and scavenge TGF‐β in the immunosuppressive TME, thereby preventing cytotoxic‐T‐cell exhaustion. In addition, TCNVs directly kill cancer cells via granzyme B delivery. Attacking tumor by both indirect and direct mechanisms, TCNVs successfully suppressed tumor growth in syngeneic solid tumor bearing mice. Taken together, TCNV offers an effective cancer immunotherapy strategy to overcome the tumor's immunosuppressive mechanisms.
Reduced bone resorption by polyphosphoesters having a high mineral affinity
1Kansai University
Biodegradable polyphosphoesters (PPEs) are of increasing interest due to their promising biomedical applications. Polyphosphodiesters (PPDEs), formed by polyphosphotriester (PPTE) dealkylation, have mineral affinity and therefore are promising polymer drug candidates for bone disease treatment. In the current study, the effects of PPDEs on both osteoblastic and osteoclastic differentiation were investigated with cell culture experiments to clarify biological functions of PPDEs. Moreover, in vivo experiments were also performed to clarify biodistribution of PPDEs and their reduction efficacy of bone resorption. Two types of PPDEs, e.g., poly(ethylene sodium ohosphate) (PEP·Na) and poly(propylene sodium phosphate) (PPP·Na) have been synthesized. When osteoblastic‐like cells (MC3T3‐E1) were cultured with PPDEs, the gene expression of differentiation markers and mineralization were effectively enhanced with the polymer uptake quantity. Interestingly, intracellular distribution of PEP·Na and PPP·Na was difference and hydrophilic PEP·Na spread homogeneously in cytoplasm. The effects of PPDEs on osteoclastic differentiation was also investigated. The size of osteoclasts formed in the media containing PPDEs was significant small compared with that in the ordinal media. Bone distribution of PEP·Na was more remarkable compared with that of PPP·Na. Furthermore, the efficacy of PEP·Na for reducing bone resorption in OVX mouse was also observed.
Effect of mechanical dose on characteristics of cartilaginous matrix production
1Department of Medical Engineering, Graduate School, Kyung Hee University, 26, Kyungheedae‐ro, Dongdaemun‐gu, Seoul, 02447, South Korea., 2Medical Science Research Institute, Kyung Hee University Medical Center/ Impedance Imaging Research Center, Kyung Hee University, 26, Kyungheedae‐ro, Dongdaemun‐gu, Seoul, 02447, South Korea., 3Kyung Hee University, 4Department of Biomedical Engineering, School of Medicine, Kyung Hee University/ Medical Device Research Center, Medical Science Research Institute, Kyung Hee University Medical Center, 26, Kyungheedar‐ro, Dongdaemun‐gu, Seoul, 02447, South Korea
Meniscal cartilages exert protective effect on articular cartilage by supporting weight load in patellar joint. However, upon damage, therapeutic option for meniscus regeneration is very limited. Tissue engineering approach to reconstruct meniscus tissue resembling its natural tissue features could present therapeutic possibility. For this purpose, scaffold‐free tissue building blocks constructed with in vitro‐ expanded meniscal chondrocytes were assembled to form macro‐scale tissue to subjected to mechanical stimulus. The tissue constructs subjected to chondrogenic differentiation without tensile stimulus resulted in abundant cartilaginous matrix production without any trace of tissue alignment. However, tensile stimulation exerted negative effect on chondrogenic differentiation resulting in severely attenuated glycosaminoglycans production. To promote abundant cartilaginous matrix production with uniaxial tissue alignment, optimal duration of tensile stimulus was determined by screening tensile dose. Unlike previous report showing irreversible influence induced by long term mechanical stimulus in mesenchymal stem cells, meniscal chondrocytes showed reversible mechanotransduction status even after long term exposure. When the tissue constructs were subjected to tensile‐stimulation for first 7 days during 3 weeks of chondrogenesis, cartilaginous matrix was produced to the comparable level to normal control with potentiated type I collagen production. Based on nuclei angle, uniaxial tissue alignment was observed in the optimized mechanical dose group in the majority of the cells even after the tension release. For tissues of mechanical loading, tissue microstructure plays important role for the tissue functionality. These results indicate that optimized tensile stimulus can modulate the characteristics of matrix production, and lead to the successful reconstruction of fibrocartilage.
Elastic photo‐cross‐linking hybrid hydrogel with high compressive modulus for bone tissue regeneration
1Korea Institute of Materials Science (KIMS)
Polymer‐based hydrogel has been investigated in the biomedical application field due to its high water content, easily tunable mechanical strength, and biodegradation. However, relative weak mechanical properties have been aware of critical limitations as scaffolds and substitute for bone tissue regeneration. To overcome the limitations, we tried to combine α‐TCP (alpha‐tricalcium phosphate) inside polymer material to increase the mechanical properties that solidified ceramic part inside the polymer hydrogel due to their hydrolysis. The compressive modulus of the hybrid hydrogel was dramatically improved after solidification despite maintaining its elastic properties like a typical hydrogel. This hybrid hydrogel might be able to utilize for bone tissue engineering as the scaffold and 3D printing materials.
Guided bone regeneration using 3D‐printed resorbable PCL/β‐TCP membrane
1Jeonbuk National University College of Dentistry
The aim of this study was to evaluate the clinical and radiographic outcomes of osseous changes following the application of guided bone regeneration technique using a 3D‐printed resorbable polycaprolactone/ β‐tricalcium phosphate (PCL/β‐TCP) membrane in comparison with a collagen membrane on peri‐implant dehiscence defects.
For a total of 10 patients, alveolar bone augmentation was accomplished with either pre‐shaped 3D‐ printed PCL/β‐TCP membranes as a test group or collagen membranes as a control group in conjunction with bovine‐derived xenograft. The cone‐beam computed tomography (CBCT) scans were performed at two points, immediately after implant placement (baseline) and at re‐entry surgery after 4‐5 months of healing. The horizontal thickness (HT0, HT2, HT4), vertical thickness (VT), and VT at 45° angle (45‐ VT) of the augmented hard tissue were measured to compare alveolar bone changes through CBCT images as follows: HW0 at the level of the implant shoulder, HW2 and HW4 at respective depths of 2mm and 4mm apical to the implant shoulder, VT and 45‐VT were measured upward from the implant shoulder.
The intergroup CBCT comparison between the test and control group showed a statistically significant difference (p < 0.05) in mean horizontal and vertical bone dimensions observed at HW0, VT, and 45‐VT. A significantly higher amount of bone regeneration was observed in the 3D‐printed PCL/β‐TCP membrane group.
Within the limitations of the present study, peri‐implant dehiscence defects could be effectively treated with a pre‐shaped 3D‐printed resorbable PCL/β‐TCP membrane in terms of stability and improved bone regeneration at both horizontal and vertical augmented regions.
Subchondral bone remodelling spatially linked to the overlying cartilage degeneration in osteoarthritis progression
1University College London
Early diagnosis of osteoarthritis (OA), before to the onset of irreversible changes is crucial for understanding the disease process and identifying potential disease‐modifying treatments from the earliest stage. However, the spatial relationships between cartilage lesion severity (CLS) and microstructural changes in the subchondral plate and trabecular bone remain elusive.
In this study, we collected femoral heads from hip arthroplasty for primary osteoarthritis (OA group) and femoral neck fracture (non‐OA controls). The specimens were assessed for cartilage lesions using Outerbridge classification and entire femoral heads were micro‐CT scanned and analysed. Principal component analysis (PCA) was employed to assess differences between OA and non‐OA samples, and the spatial relationship between CLS and subchondral bone changes.
The mapping of the trabecular bone microstructure in OA patients with low CLS revealed trabecular organisation resembling non‐OA patients, whereas clear differences were identifiable in subchondral plate architecture. It was reported that the greater articular cartilage deterioration in OA was regionally‐linked with lower BV/TV, TMD and thickness, and greater BS/BV and porosity in the subchondral plate; and with thinner, less separated trabeculae with greater BMD and BS/BV in the trabecular bone. Our findings suggest that impairment of subchondral bone microstructure in early stage of OA is more readily discernible in the cortical plate and that morphological characterisation of the femoral head bone microstructure may allow for earlier OA diagnosis and monitoring of progression.
Functional scaffolds and biomimetic matrices for 3D culture of mesenchymal stem cells
1National Institute for Materials Science
Porous scaffolds and biomimetic matrices play an important role in controlling stem cell functions for tissue engineering applications. We have developed some functional scaffolds of biodegradable polymers and extracellular matrices. Porous scaffolds of collagen and gelatin have been prepared by using ice particulates and PLGA sponges as templates. The porous scaffolds showed well controlled and interconnected pore structures to facilitate cell penetration and distribution. Human bone marrow‐derived mesenchymal stem cells (hMSCs) were cultured in the porous scaffolds. The interconnected porous structures facilitated cell‐cell interaction and promoted chondrogenic differentiation. Composite scaffolds of synthetic polymers and natural polymers have been prepared by hybridizing these two types of polymers. The composite scaffolds combined the advantages of both types of polymers, showing high mechanical strength and excellent cell interaction. The composite scaffolds have been used for tissue engineering of skin, cartilage and bone. Biomimetic extracellular matrices have been prepared from cultured cells. Their composition could be controlled by cell type and phenotype. Influence of the biomimetic matrices on differentiation of hMSCs was dependent on the matrix composition. Photothermal composite scaffolds have been prepared by hybridizing biodegradable polymers with photothermal convention nanoparticles. Influence of the photothermal composite scaffolds on adipogenic differentiation of hMSCs was investigated. Hybridization with photothermal nanoparticles did not affect the promotive effect on adipogenic differentiation. Meanwhile, the photothermal composite scaffolds showed photothermal ablation capacity to breast cancer cells under near infrared laser irradiation. Therefore, the photothermal composite scaffolds might be used for breast reconstruction after surgical resection of breast cancer.
Biomaterials grafted with several designed peptides for human pluripotent stem cell culture and differentiation
1National Central University
Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are attractive sources of cells for regenerative medicine. One of the difficulties of applying human pluripotent stem cells (hPSCs; hESCs and hiPSCs) in the clinic is that hPSCs cannot be cultured on conventional tissue culture polystyrene dishes. In particular, extracellular matrix (ECM) protein‐derived peptide‐grafted cell culture biomaterials are attractive because completely synthesized surfaces can be prepared for the peptide‐ grafted biomaterials. In this study, we developed poly(vinyl alcohol‐co‐itaconic acid) (PV) hydrogels grafted with laminin‐derived peptides that had different joint segments and several specific designs, including dual chain motifs. PV hydrogels grafted with a peptide derived from laminin‐β4 (PMQKMRGDVFSP) chain containing a joint segment, dual chain motif and cationic amino acid insertion could attach hPSCs where the PV hydrogels were adjusted to have an optimal elasticity for hPSC cultivation. The PV hydrogels grafted with a laminin‐β4 peptide having a joint segment, dual chain motif and cationic amino acid promoted high expansion folds in long‐term culture (over 10 passages) with low differentiation rates and could differentiate into cardiomyocytes with high efficiency, whereas hPSCs attached poorly on PV hydrogels grafted with laminin‐α5 (PASYRGDSC) peptides that had joint segments with and without a cationic amino acid or on PV hydrogels grafted with laminin‐β4 peptides containing the joint segment only. The inclusion of a cationic amino acid in the laminin‐β4 peptide was critical for hPSC attachment on PV hydrogels, which contributed to the zeta potential shifting to higher values (3–4 mV enhancement).
Alginate‐chitosan microencapsulated cells for improving hematopoietic stem cell's maintenance and expansion
1Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, 2Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 3Department of Materials Engineering Science, Graduate School of Engineering Science, Osaka University, 4Department of Obstetric and Gynecology, Faculty of Medicine, Universitas Indonesia ‐ Dr. Cipto Mangunkusumo General Hospital, 5Department of Biotechnology, Faculty of Engineering, Osaka University
Protocols for isolation, characterization, and transplantation of hematopoietic stem cells (HSCs) have been well established. However, the difficulty in finding human leucocyte antigens (HLA)‐matched donors and the scarcity of HSCs are still the major obstacles to allogeneic transplantation. In this study, we developed a double‐layered microcapsule to deliver paracrine factors from non‐matched or low‐ matched HSCs to other cells. The umbilical cord blood‐derived hematopoietic progenitor cells, identified as CD34+ cells, were entrapped in alginate polymer and further protected by chitosan coating. The microcapsules showed no toxicity for surrounding CD34+ cells. When CD34+ cells‐loaded microcapsules were co‐cultured with bare CD34+ cells that have been collected from unrelated donors, the microcapsules affected surrounding cells and increased the percentage of the CD34+ cell population. This study is the first to report the potency of alginate‐chitosan microcapsules containing non‐HLA‐matched cells for improving proliferation and progenitor maintenance of CD34+ cells.
Engineering of hybrid spheroids of mesenchymal stem cells and drug depots for immunomodulating effect in islet xenotransplantation
1Department of Precision Medicine, School of Medicine, Sungkyunkwan University Suwon, 16419, 2Department of Pharmaceutical Science, College of Pharmacy, Keimyung University, Daegu 42601
Immunomodulation is an essential consideration for cell replacement procedures. Unfortunately, lifelong exposure to non‐specific systemic immunosuppression results in immunodeficiency and has toxic effects on non‐immune cells. Here, we engineered hybrid spheroids of mesenchymal stem cells (MSCs) with rapamycin‐releasing poly(lactic‐co‐glycolic acid) microparticles (RAP‐MPs) to prevent immune‐rejection of islet xenografts in diabetic C57BL/6 mice. Hybrid spheroids were rapidly formed by incubating cell‐ particle mixture in methylcellulose solution while maintaining high cell viability. RAP‐MPs were uniformly distributed in hybrid spheroids and sustainably released RAP for ∼3 weeks. Locoregional transplantation of hybrid spheroids containing low doses of RAP‐MPs (200‐4000 ng RAP/recipient) significantly prolonged islet survival times and promoted the generation of regional regulatory T cells. Enhanced Programmed Death‐Ligand 1 (PD‐L1) expression by MSCs was found to be responsible for the immunomodulatory performance of hybrid spheroids. Our results suggest that these hybrid spheroids offer a promising platform for the efficient use of MSCs in the transplantation field.
Recent update of modeling of human kidney diseases using hPSCs‐ derived kidney organoids
1Cell Death Disease Research Center, College of Medicine, The Catholic University of Korea/Division of Nephrology, Department of Internal Medicine, The Catholic University of Korea
The establishment of differentiation protocols of kidney organoids from human pluripotent stem cells (hPSCs) provides potentials in applications of kidney organoids in regenerative medicine. Modeling of kidney diseases and investigating new therapeutic option are attractive applications. Although much progress still remains to be made in the development of kidney organoids, recent advances in clustered regularly interspaced short palindromic repeat (CRISPR)‐CRISPR‐associated system 9 (Cas9) genome editing have contributed to the application of kidney organoids in genetic kidney disease. However, kidney organoids derived from hPSCs have some pitfalls for the applications of kidney organoids to kidney disease modelling. To overcome the pitfalls and improve the clinical applications for disease modeling using human kidney diseases, many efforts have performed research in this field. In this session, I will review the recent advances and share our experiences in the modeling of human kidney diseases using hPSCs‐derived kidney organoids.
Modeling heart disease using hiPSC‐derived cardiac organoids
1NEXEL Co., Ltd.
Human induced pluripotent stem cells (hiPSCs)‐derived cardiomyocytes (CMs) propose a promising source for heart disease modeling and drug screening. Recently, developing organoid technology enables to study how hiPSC‐derived cardiomyocytes interact together, and the organoid system mimics the tissue environment and behavior of the cardiac cells in the heart. Although the cardiac organoids (COs) derived from hiPSCs exhibited mature phenotypes and functions, the cellular compositions in human heart should be established in the organoids for efficient modeling heart disease. Here, we generated a self‐organizing cardiac organoid (COs) from hiPSCs with cardiomyocytes, fibroblasts, and endothelial cells (multi‐ cellular COs) that recapitulates cellular compositions of the human heart. Under hypoxic condition, the multi‐cellular COs resulted the increase of myocardial infarction (MI) markers with defective functions. Furthermore, cardiac fibrosis also could be induced in the multi‐cellular COs under pro‐fibrotic conditions. This result could be an important implication for the application of in vivo‐like 3D heart and disease modeling, and these hiPSC‐derived MI model might provide a promising alternative to an animal experimental model for studying cardiac disease and drug screening system for finding therapeutic targets.
Acknowledgement: This study was supported by grants from the National Research Foundation funded by the Ministry of Science and ICT of Korea (No. 2019M3A9H1103718 and 2022M3A9H1014160).
Alveolar organoids from human pluripotent stem cells: Research and applications
1Department of Internal Medicine, School of Medicine, Kangwon National University
Recently, the development of three‐dimensional alveolar organoid (AO) system using human pluripotent stem cells (hPSCs) has attracted great attention compared to conventional monolayer alveolar epithelial cell cultures for studying early alveoli development, modeling pulmonary diseases, screening for novel drugs and evaluation of toxic materials. Although AO technology is consistently and rapidly being developed, its limitations still remain. One shortcoming of the AO is the lack of crucial immune cell components, such as macrophages and neutrophils, that play a key role in the pathological development. This deficiency limits our capacity to model various diseases and subsequent drug screening approaches using AO. Furthermore, detailed understanding of the development of pulmonary disorders and the biological mechanisms how environmental toxic materials affects the lungs are remain incompletely understood, which attributed to lack of in vitro human models that recapitulate the architecture and function of alveolar tissue. In this lecture, we discuss the progress and potential applications in AO technology and hPSC‐derived macrophages that are particularly relevant to recapitulate pulmonary fibrosis. We also discuss the potential use of in vitro AO platform for evaluating the adverse effects of environmental toxins including particulate matter and heavy metals (This research was supported by the KFRM grant funded by the Korea government, 22A0304L1‐01).
Development of
in vitro
mechanical spinal cord injury model in differentiated NSC‐34 cells
1Center of Tissue Engineering and Regenerative medicine, Faculty of Medicine, University Kebangsaan Malaysia, Jalan Yaacob Latif, 56000 Cheras, Kuala Lumpur, 2Department of Orthopaedics & Traumatology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur 56000, 3Institutes of Medical Science Technology, Universiti Kuala Lumpur Malaysia, Kajang 43000, 4Department of Surgery, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latiff, Bandar Tun Razak, Kuala Lumpur 56000
Spinal cord regeneration is considered as an ultimate achievement in fields of neuroscience. Scientists are inventing newer strategies to regenerate damaged neuronal tissue following spinal cord injuries. The in vitro model to induce spinal cord injuries using mice or rat neuronal cells can mimic certain key aspects of SCI in humans. The objective of this study was to develop mechanical SCI model in vitro using mechanical scratch. At first NSC‐34 cell lines, were differentiated into motor neurons, using retinoic acid (RA) and Prostaglandin E2 (PGE2) for 5‐7 days. Subsequently, the mechanical injury was created using yellow pipette tips, with two parallel vertical and two horizontal scratches, followed by treatment with a proprietary product for 3 days. The neuroregenerative ability of the proprietary product was estimated using: neurite outgrowth using time lapse assay (3 days), viability assay, immunocytochemistry (ICC) and AKT‐PI3K regenerative pathway analysis by western blotting. The result obtained showed that NSC‐ 34 cells lines can differentiate into mature motor neuron by RA (7 days) and PGE2 (5 days). Mechanical scratch injury model damaged the neuronal cells at the scratch area, breaking motor neurons. The time lapse assay showed treated cells have significant higher neurite outgrowth as compared to untreated cells. The AKT‐PI3K regenerative pathway analysis and ICC showed more expression of regenerative markers (p‐AKT, p‐GSK, ATF‐3, GAP43, p‐53 & elF2β) at concentrations 1000 & 1200 μg/mL then untreated cells. In vitro SCI can be created using mechanical scratch method in the neuronal cells to study the neurodegenerative diseases.
The developmental metabolism of brain organoids
1Seoul National University, 2Seoul National University College of Medicine
Brain organoid has the application for Bio‐Health field, from basic science to regenerative medicine. We focused on metabolic synthesis during brain development using brain organoids.
Human induced pluripotent stem cells (hiPSCs) were used source of Brain organoids that has induced with neuronal morphogens including ROCK inhibitor, bFGF, and extracellular matrix (ECM). Neural epithelium has matured Brain organoids with different composition of media and ECM for the evaluation of nutrient and scaffold effect. From this growth, Brain organoid has not difference than control group that means neuronal nutrient were supported brain organoid development.
And brain organoids were developed from Glioblastoma multiforme (GBM) patient derived hiPSCs and compared with normal hiPSCs. After over a month, this brain organoid size was revealed with normal group that seems like growth retardation, whereas neuronal markers were detected such as SOX2, Tuj1, PAX6 in the immunohistochemical analysis.
In addition, GBM cell line and 3D spheroids were analysis between the other culture condition. From this result, metabolic related pathway genes were expressed as like Fatty acid transporter (FABP3), Triglyceride metabolism (LIPE), and Mitochondrial fatty acid oxidation disorders (CTP2).
In conclusion, we provide evidence that Brain organoid culture condition has available for various application and related with metabolic synthesis.
Cell therapy: From fibroblasts to CAR‐T/NK and organoids beyond stem cells
1Seoul National University
Cell therapy has been established from bone marrow transplantation since 1963, however, actually, cell therapy has been recognized in stem cells for isolation of mesenchymal stem cells from bone marrow (1970). There is a breakthrough of stem cell area in 2006. Prof. Shinya Yamanaka reported a generation of induced pluripotent stem cells using Oct‐4, Sox‐2, Klf, c‐myc, which cells showed human embryonic stem cells characterisctics and properties. Mesenchymal stem cells (MSCs) are an important type of cell that are highly recognized for their safety and efficacy as a cell therapy agent. In order to obtain MSCs, primary tissues (adipose tissue, bone marrow, and umbilical cord blood) must be used; however, these tissues, especially umbilical cord blood, are difficult to obtain due to various reasons, such as the low birth rate trend. We have worked and developed stem cell therapies using human umbilical cord bloods for targeting immune‐related diseases such as Atopic dermatitis, Rheumatoid arthritis and so on.
Recently, cell therapy was intensively developed for cancer treatment, for examples, Car‐T cell thepray for leukemias. It also extensively expanding to solid cancer for target. Now, iPSC‐derived CAR‐T cells and CAR‐NK cells has been also extensively developed for the concept of Off‐the‐self beyond allogenic. A recently developed human pluripotent stem cell (hPSC)‐derived Brain and skin organoid model has opened up new avenues for investigating organ development, disease, and regeneration.
The newest R&D trend in Organoidsciences' technologies
1ORGANOIDSCIENCES, LTD, 2ORGANOIDSCIENCES, LTD
ORGANOISCIENCES is one of the leading biotech companies developing an innovative organoid technology with the aim of contributing to human health by developing medical technologies, which can solve problems of organ damage and shortage based on advanced science.
Organoids, miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro‐anatomy. They include Multiple organ‐specific cell types, grouped together and spatially organized like an organ and capable of recapitulating some specific functions of an organ.
Therefore organoids are promising strategies in the field of regenerative medicine and transplantation with high levels of regenerative capacities. We have successfully developed a platform called “ATORM (Adult Tissue derived Organoid based Regenerative Medicine)” with oncoming first in human clinical trial pipelines and its GMP process for organoid therapeutics. We would like to present the current limitations for clinical and industrial application of organoids and other innovative platforms based on organoid.
Allogenic pre‐osteoblast cells (CF‐M801) to treat osteonecrosis of the femoral head
1CEFO Co., Ltd.
Osteonecrosis of the femoral head (ONFH) is a disease in which bone necrosis is caused by poor blood circulation in the femur. We developed CF‐M801, allogenic pre‐osteoblast cell therapy, which is from umbilical cord mesenchymal stem cells (MSC) by optimizing the stiffness of hydrogels in three dimensions. CF‐M801 is distinguished from their undifferentiated MSC and from their differentiated osteocytes and has the dual function of osteogenesis and angiogenesis, necessary for bone regeneration. We confirmed the efficacy of CF‐M801 using the bone defect models of goat and mouse and the safety of toxicity, biodistribution, and tumorigenicity. We determined potency markers of CF‐M801 reflecting their functions as osteogenicity, osteoinductivity, osteoconductivity, and angiogenicity. The competitiveness of CF‐M801 is that the final drug product is supplied in frozen status and in more than 10,000 doses from a single donor. We carry on phase 1 clinical trial in which we planned to confirm the toxicity (DLT) and investigational efficacy of CF‐M801 in ONFH patients (AROC stage 1 & 2) in three doses.
Role of recombinant human hyaluronan and proteoglycan link protein 1 on dry eye disease
1HAPLN SCIENCE
Dry eye disease (DED) has been closely associated with disruption of tear film homeostasis as a chronic and progressive multifactorial disease occurring to the ocular surface. The global prevalence of DED is greatly growing.
The endogenous HAPLN1 proteins in human localize in extracellular matrix (ECM) and play crucial roles in regulation of ECM homeostasis as a link protein. Additionally, HAPLN1 are involved in a variety of biological processes such as regeneration, inflammation, ROS, and aging. The depletion of HAPLN1 has been reported to link with the age‐related diseases in human. Here, we present a recombinant human hyaluronan and proteoglycan link protein 1 (rhHAPLN1) as a novel potential therapeutics against DED. We found the treatment of rhHAPLN1 promotes wound healing, reduces production of reactive oxygen species (ROS), suppresses inflammation responses, and delays cellular senescence in human primary corneal epithelial cells exposed to hyperosmotic stress environment which represents in vitro DED model. Moreover, rhHAPLN1 upregulates gene expression of Mucin5AC and enhances Mucin5AC secretion in primary conjunctiva epithelial cells.
In vivo efficacy experiments, we found rhHAPLN1 repairs damaged corneal surface and increases tear volume production in scopolamine‐induced DED mouse model. In the histopathological analysis, we identified the treatment of rhHAPLN1 inhibits corneal epithelial cell detachment, increases goblet cell number in Fornix of ocular tissue, and represses inflammatory responses in lacrimal gland. We confirmed the efficacy of rhHAPLN1 in the benzalkonium chloride (BAC)‐induced DED rabbit model. Taken together, we consider rhHAPLN1 as a therapeutic agent to be able to cure DED through multi‐functional mechanisms.
An effective therapy for critical limb ischemia using endothelial progenitor cells from human umbilical cord blood
1YOUTH BIO GLOBAL
The ischemic vascular diseases such as cardiac vascular disease, cerebrovascular disease, and peripheral vascular disease, etc are categorized as one of the highest mortality rate with over 40% in the world. Endothelial Progenitor Cells(EPCs) have been proven to be prominently effective for the aged and injured blood vessels and ischemic tissue regeneration, etc. YOUTH BIO GLOBAL has a cutting‐ edge technology with a new type of culture media not using animal materials and we say xeno‐free media. And this new technology has the advanced culture method that enhances the clinical therapeutic effects using completely safe Endothelial Progenitor (Cells EPCs). The culture environment has a considerable influence on the characteristics of the primary cells that are cultured in the exposed conditions. Although the endothelial cells do not proliferate during prolonged culturing process, xeno‐free cultured EPCs (X‐EPCs) outstandingly represent not only high CD34 surface expressions but also the maintenance of stemness. When compared with different types of EPCs by other researchers, YBG's X‐ EPC noticeably represented similar or enhanced properties of tube‐structure formation, LDL metabolism, secretion of angiogenic factors, and also SDF‐1α dependent migration. In addition, it showed up the rapid restoration of revascularization in the ischemic area by constructing critical limb ischemia mice model and then Intramuscular injection of XEPC will be conducted.
In conclusion, YBG's novel culture media and method strategy clearly helps enhance the regenerative function of EPC selected in xeno‐free condition and represents both angiogenesis and vasculogenesis. Thereby, it provides a positively accessible clinical use of critical limb ischemic diseases indication.
Xcell therapeutics: groundwork for reliable and reproducible regenerative medicine solutions
1Xcell Therapeutics
Cell culture media is a very crucial component in regenerative medicine. As the science, technology, and commercial applications of regenerative medicine advances and matures, the need for safe, reliable, and reproducible media solutions becomes ever so essential. We here at Xcell Therapeutics focus on addressing those needs, striving to provide animal‐free, chemically‐defined, and optimized media solutions. During this presentation, we will breifly touch upon the growing trends in cell culture media for regenerative medicine, highlighting some of our efforts and solutions in overcoming the hurldes in the industry.
Introduction of CARM
1Council for Advanced Regenerative Medicine (CARM), Inha University College of Medicine
Korea has a favorable environment for commercialization of regenerative medicine (RM). With strong initiatives of Korea government, Korea has 18 cell and gene therapies approved in the market including 4 products using mesenchymal stem cells (MSCs). Council for Advanced Regenerative Medicine (CARM) was established in May 2016 with the support of the Ministry of Health and Welfare (MoHW), Korea. The goals of CARM were to improve regulatory framework for RM products, activate the domestic RM industry by encouraging partnership between venture companies and pharma companies, and facilitate international collaboration with overseas partners. CARM has more than 100 companies including venture companies developing cell and gene therapies, pharma companies, contract research organizations (CROs), contract manufacturing organizations (CMOs), those in the enabling technology, venture capitals and related associations. For its activities, CARM has three committees of policy committee dealing with policies and regulations on RM, international cooperation committee in charge of domestic and international partnering and industry partnering committee to facilitate communication between RM companies in Korea. Currently, CARM is working closely with many RM stakeholders and operating many partnering events and forums to deal with key agendas in RM and foster RM industry in Korea. For international collaboration, CARM has signed memorandum of understandings (MOUs) with Alliance for Regenerative Medicine (ARM) in the US, Forum for Innovative Regenerative Medicine (FIRM), Japan, AusBiotech, Australia and International Society for Cell and Gene Therapies (ISCT). The presentation will introduce CARM history and current activities. Please visit CARM website for more details (www.carm.or.kr).
Surface functionalization and coating of titanium alloys
1Politecnico di Torino
Several molecules of natural origin (derived from plants or animals) are of great interest for surface functionalization (grafting of a molecular layer) or coating (nano or micrometric continuous films) of titanium alloys. They can be peptides, polypeptides, proteins, vitamins, oils, organic compounds, or natural polymers. Functionalization or coating of titanium must be coupled to a proper surface chemistry and topography according to the type of biomolecule to be grafted and the specific application or purposes. The modified titanium surfaces acquire biomimetic or antibacterial functionalities and/or ability to guide the tissue growth. Positive outcome on inflammatory or anticancer properties can be also induced. New characterization protocols (biological, chemical, physical, mechanical tests and analyses) are needed for characterizing the modified surfaces and the post‐processing steps (packaging, sterilization) must be adapted.
An overview of the strategies and the benefits from grafting or coating titanium with nisin, vitamin E, tocopherol phosphate, mentha essential oil, chitin derivative, polyphenols will be presented.
Matrix ligand/protein conjugated hydrogels for tissue engineering applications
1POSTECH
Tissue engineering is an interdisciplinary area aimed at maintaining, rebuilding, and promoting the normal function of organs and tissues using biomaterials and live cells. To understand the interface between biomaterials and cells where cells respond to subtle changes of microenvironmental cues to eventually control cell phenotypes and fate decisions, cell adhesion onto the surface of biomaterials is critical. However, engineering cell culture microenvironments to recapitulate the 3D dynamic and complex nature of tissues at the biomaterial‐cell interface is a major challenge. To tackle the engineering issues, hydrogel‐based biomaterials could be smartly constructed as cell culture platforms to improve tissues' function, structure, and maturity comparable to native tissues in the body. Here I will present several hydrogel‐based model systems where cells could adhere and then be guided through multiple biophysical, mechanical, and biochemical signals to program (e.g., stem cell differentiation) and reprogram (de‐differentiation of cancer cells). These studies demonstrate the importance of the precise synchronization of microenvironment parameters existing in engineered biomaterials during cellular decision‐making for disease modeling and regenerative therapies.
Cell/tissue adhesive, antibacterial hydrogels based on fungal‐derived carboxymethyl chitosan and tannic acid via drug‐free approach for wound healing applications
1School of Chemical Engineering and Research Institute of cell culture, Yeungnam University, Gyeongsan‐38541, Republic of Korea
In the present work, we developed antibacterial, tissue adhesive hydrogels composed of fungal derived carboxymethyl chitosan (FCMCS) and tannic acid (TA) within polyacrylamide (PAM) network. The hydrogels have good swelling, mechanical and compressive properties. The antibacterial experiments revealed against E. coli, and S. aureus indicated effective killing capacity. Furthermore, the hydrogels are biocompatible towards skin fibroblasts and keratinocyte cells. Owing to the presence of TA, the hydrogels showed excellent antioxidant properties. Therefore, drug free antibacterial, and tissue adhesive properties developed within hydrogels have potential for wound dressing for infected wounds.
Controllable and switchable drug delivery platform by carbon‐based 3D hydrogel sponges
1Korea Carbon Industry Promotion Agency, 2Dankook University
Smart hydrogels that are responsive to various external (e.g. electrical and/or thermal) stimulation have become increasingly popular in recent years for simple, rapid, and precise drug delivery that can be controlled, turned on or off with external stimuli. For such a switchable drug delivery material, highly homogeneous dispersion and distribution of the hydrophobic, electrically conductive nanomaterials throughout a hydrophilic three‐dimensional (3D) hydrogel network remains a challenge and is essential for achieving well‐connected electrical and thermal conducting paths. Herein we developed electrical and thermal stimulus‐responsive 3D hydrogels based on i) carbon nanotubes (CNT) as the core unit and an electrical/thermal conductor, ii) chitosan (Chit) as the shell unit and a hydrophilic dispersant, and iii) poly(NIPAAm‐co‐BBVIm) (pNIBBIm) as the drug carrier and a temperature‐responsive copolymer. By formulating the CNT‐core and Chit‐shell units and constructing a CNT sponge framework, uniform distribution and 3D connectivity of the CNTs were improved. The 3D hydrogel based on the CNT sponge, namely the 3D framed CNT‐Chit/pNIBBIm hydrogel, delivered approximately 37% of a drug, ketoprofen used for treatment of musculoskeletal pain, during about 30% shrinkage after electrical and thermal switches on/off and exhibited the best potential for future use in a smart transdermal drug delivery system. These physicochemical, mechanical, electrical, thermal, and biocompatible characteristics of this nanocarbon‐based 3D framed hydrogel led to remarkable electrical and thermal stimulus‐responsive properties capable of developing an excellent controllable and switchable drug delivery platform for tissue and biomedical engineering and medicine applications.
Polypeptide‐fueled transient volume phase transition of a hydrogel
1Division of Applied Chemistry, Graduate School of Engineering, Osaka University
The metabolic cycle, comprising fuel intake from and waste excretion to the external environment combined with internal cascade anabolic and catabolic pathways, is essential for life forms operating under out‐of‐equilibrium conditions. The dissipative nature of living systems contributes to their dynamic and unique properties, including the(re)construction, maintenance, and growth of diverse biological components. Recently, chemically fueled artificial materials that perform work under out‐of‐equilibrium conditions have attracted significant attention.[1] However, a polymer hydrogel that exhibits the transient structural change fueled by a biomacromolecule has not been reported. We developed an metabolic cycle‐ inspired hydrogel (MC gel) that exhibits biomacromolecule‐fueled transient volume phase transition (TVPT) under the dissipative condition. This acrylamide‐based hydrogel has the affinity and digestive capacity for a fuelα‐poly‐L‐lysine by incorporating acrylic acid and trypsin. The TVPT is driven by the transient change in the thermodynamic state with the kinetic imbalance between fast cross‐linking and slow de‐cross‐linking processes. The TVPT of the gel was exploited to realize the transient release of the payload. This study forms the basis of a strategy for engineering hydrogels that afford the dynamic regulation of biological processes via the target biomacromolecule‐fueled TVPT. [2]
[1] K. Das, L. Gabrielli, L. J. Prins, Angew. Chem. Int. Ed.
[2] M. Nakamoto, S. Kitano, M. Matsusaki, Angew. Chem. Int. Ed.
Uterus‐specific extracellular scaffolds and stem cells promote tissue regeneration after extensive injury in rodents
1Laboratory for Transplantation and Regenerative Medicine, Department of obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Kvinnokliniken, Blu00e5 stru00e5ket 6, 413 45, Gu00f6teborg, Sweden, 2Laboratory for Transplantation and Regenerative Medicine, Department of obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, 3IVI Foundation Edificio Biopolo, instituto de Investigaciu00f3n Sanitaria la Fe Avenida Fernando Abril Martorell, 106 Torre A, Planta 1a 46026, Valencia
Recellularization with mesenchymal stem cells contribute to an immunotherapeutic microenvironment in vivo after decellularized rat uterus tissue transplantation
Artificial oviduct system for preimplantation embryo based on the mechanical mimic condition
1Department of Biomedical Sciences, CHA University, 2CHA University, 3CHA Fertility Center Seoul Station
In vivo condition is not a plastic dish but soft tissue. But we culture embryos on plastic dishes. Therefore, optimal soft tissue like the oviduct's mimic condition develops for the optimal culture of the embryo. Mechanical properties are fundamental properties of the cells and tissues of living organisms. In this study, we amine that the development of the oviduct environment mimics the optimal culture dish for optimal embryo culture.
We developed an oviduct tube mimicking polyacrylamide hydrogel as 2kPa, 4kPa, and 10kPa dish. Then we collected embryos and cultured them in a time‐lapse system incubator with a different hydrogel culture dish. And we evaluated embryo development used for each check and recorded images. We check‐up development ratios and cleavage time point of the embryo during time‐lapse culture. Then we classified the quality of the embryo depending on the cleavage speed and blastocyst ratios.
10kPa culture dish shows fast development and high ratios of blastocyst formation compared with the control dish. Also, hatched‐out ratios of blastocyst significantly increase on the 10kPa culture dish compared with control. Then the blastocyst on the hydrogel culture dish reveals significantly higher Oct4, Sox, and Nanog as fetal formation‐related gene expression ratios than the control group.
In conclusion, an optimal hydrogel base mechanical culture dish could be applied to improve the clinical outcome of in vitro fertilization patients to high pregnancy and healthy live birth.
Differentiation into follicle‐like cells using ovarian spheroid
1Seoul National University Hospital
The ovarian follicles are one the final frontier for fertility preservation. Several studies tried to produce artificial follicles using stem cells, however, due to the complex structure and hormonal regulation, the trial is still a struggle. In this study, we tried to produce ovarian spheroid derived from ovarian somatic cells and differentiated into follicle‐like cells using a 2D or 3D in vitro environment.
Ovaries of 8 weeks old female C57BL6 mice were isolated and dissolve into single cells. The isolated cells were further cultured for 7 days and dispersed. The cells were placed on a spheroid formation dish and the formation of the spheroid was assessed. The formed spheroid was induced to differentiate in resuspension and replated for further differentiation. The expression of specific genes and secretion of E2 was evaluated.
The ovarian cells formed spheroid successfully in different 3D culture environments (40% vs 22%). And differentiation efficiency into ovarian cells was lower with PSC‐derived hEBs. Furthermore, the expressions of specific markers were significantly varied in ovarian spheroid.
In this study, we established in vitro formation of ovarian spheroid applicable as the progenitor of further differentiation into ovarian cells and proved their differentiation potentials (2019R1A2C1010163 and 2022R1A2B5B01002541).
Application of experimental in vitro model to evaluate uterine receptivity for embryo
1Korea University
The uterus consists of many cells and extracellular matrix molecules, which comprise the stratification of all uterine layers, endometrium, myometrium, and perimetrium. Furthermore, the specific part of this organ, such as the salpinx, endometrium, and cervix, should represent the respective functional act to keep the competence as the central organ of the reproductive system. Most of all, it is notable that the systemic endocrinologic network regulates the role of the uterus. Our system using endometrial cell organoids showed features mimicking the endometrial implantation period. This finding suggested that the endometrial cell culture could offer a useful in vitro model to evaluate the hormonal and pharmacological effect on uterine receptivity for the embryo. Further study should be necessary for their experimental and clinical application.
Biomaterials design of tissue engineering to enhance natural‐hearing potentials for regenerative medicine
1Laboratory of Biomaterials, Institute for Life and Medical Sciences, Kyoto University
A new therapeutic trial based on the natural‐healing potential of body itself to induce tissues regeneration and repairing, has been recently expected. To realize this regenerative therapy, there are two approaches of cell transplantation and tissue engineering. Tissue engineering is a biomaterial technology or methodology to artificially create a local environment which enables cells to enhance their proliferation and differentiation for tissue regeneration. If a cell scaffold or a key bio‐active is supplied to the right place at the right time or concentration, the body system initiates to physiologically function, resulting in the natural induction of cell‐based tissue regeneration.
The biological functions of bio‐active molecules are promoted by combining with drug delivery system (DDS) technology. Biodegradable hydrogels enabled the controlled release of various growth factors and chemokines to realize the cell healing potential‐based regeneration and repairing of various tissues through the in vivo recruitment and activation of cells. This release and/or cell scaffold technologies can be combined with cell transplantation to significantly enhance the therapeutic efficacy in tissue regeneration. The biomaterials technology of regenerative medicine is also applicable to the basic research of stem cells biology. Further development of stem cells biology will contribute to an enhanced therapeutic efficacy of cell‐based tissue regeneration.
In this paper, several applications of DDS and cell scaffold technologies to the tissue regeneration therapy as well as the basic research of stem cells are introduced to emphasize the necessity and significance of tissue engineering technologies in regenerative medicine.
A human osteoarthritis cartilage explant model for assessing the effectiveness of antibody‐conjugated cell micromass targeted delivery ease
1Universiti Kebangsaan Malaysia, 2Nitta Gelatin, 3Kyoto University
This work reports the characterisation of an osteochondral explant from a patient with osteoarthritis. In addition, this model was used to investigate the phenotypic persistence of antibody‐conjugated human bone marrow‐derived mesenchymal stem cells cultured on gelatin microspheres (Ab‐BMSCs‐GM). The model's sensitivity to external stimulation in an environment conducive to OA was explored.
Extracted joint tissues from total knee replacement procedures were dissected into many miniatures, standardised osteochondral explants. Before co‐culturing with Ab‐BMSCs‐GM, the chondrogenic differentiation and hypoxia responsiveness of the cell micromass and explant cultures were exhaustively validated at the molecular level. In addition, the efficacy of targeted delivery of the Ab‐BMSCs‐GM to an osteochondral defect explant was demonstrated.
Antibody conjugated MSCs micromass (Ab‐BMSCs‐GM) to a chondrocyte phenotype is effective under hypoxic conditions and a 3D culture environment, as evidenced by an increase in cartilage‐related biomarkers and biosynthesis of a glycosaminoglycan‐positive matrix. Evaluation of the osteochondral defect explant demonstrated that the Ab‐BMSCs‐GM could adhere to the osteochondral surface after 48 hours. In addition, OA explant culture cells maintained their chondrogenic and OA‐specific features for four weeks. In addition, it was discovered that the engineered cartilage could respond to biomimetic environmental circumstances (oxygen tension). In conclusion, we established a human osteoarthritis (OA) osteochondral explants culture and evaluated the feasibility and potential of employing this model to evaluate emerging cartilage therapies in vitro.
Osmoprocessor for enabling highly sensitive biomarker detection via lateral flow assays
1Department of Bioengineering, University of Washington, 2Department of Mechanical Engineering, University of Washington
Lateral flow assays are low‐cost devices suitable for point‐of‐care testing, particularly in low‐resource settings. However, some of the lateral flow assays exhibit limited diagnostic utility because the assays can only sample <100μL specimen and the biomarker concentration is significantly lower than the assay detection limit, which compromise the sensitivity. To address the challenge, we have developed the osmoprocessor that statically and spontaneously concentrated biomarkers via osmosis. The specimen in the device interfaces with the aqueous polymer solution via a dialysis membrane. The polymer solution induces an osmotic pressure difference that extracts water from the specimen, while the membrane retains the biomarkers. The evaluation demonstrated that osmosis induced by various water‐soluble polymers efficiently extracted water, ca. 15 mL/hr. The water transport kinetics can be adjusted by varying polymer molecular weights and mass concentrations. The osmoprocessor concentrated the specimens to improve the lateral flow assays' detection limits for the model analytes—human chorionic gonadotropin and SARS‐CoV‐2 nucleocapsid protein. The device processed a 10 mL specimen into a 100μL concentrated sample. Then, the lateral flow assays detected the corresponding biomarkers in the concentrated specimens. The test band intensities of the assays with the concentrated specimens were very similar to the reference assays with 100‐fold concentrations. The mass spectrometry analysis estimated the SARS‐ CoV‐2 nucleocapsid protein concentration increased ca. 200‐fold after the osmosis. With its simplicity and flexibility, this device demonstrates a great potential to be utilized in conjunction with the existing lateral flow assays for enabling highly sensitive detection of dilute target analytes.
In vitro dendritic cell activation via exogenous delivery of tumor cell lysate using coacervate
1Dongguk university
Dendritic cells (DCs) can recognize tumor‐associated antigens and initiate antigen‐specific adaptive immune responses by interacting with T cells via surface‐presented antigens on DCs. Tumor cell lysates (TCLs), as major immunomodulatory sources of multiple antigens, effectively induce the activation of DCs. Moreover, the efficacy of TCLs could be facilitated by pre‐treatment of source cells using chemical additives (i.e. squaric acid (SqA)) through increasing oxidative stress. However, exogenously administrated bolus TCLs are easily degraded during the migration in blood and lymph stream and inefficient to reach DCs in vivo. Therefore, we developed coacervate (Coa)‐mediated TCL delivery strategy to strength DC‐mediated immune system. Coa, a colloidal droplet composed of a biodegradable mPEGylated poly(ethylene arginylaspartate diglyceride) cation and counterpart heparin anion, is self‐ assembled in an aqueous environment via electrostatic interaction. A repeated freeze‐thaw cycle for SqA‐ treated CT26 colon cancer cells resulted in cargo TCLs with high mobility group box 1. TCLs were then encapsulated in Coa and a sustained release pattern were obtained over 7 days. Our results also demonstrated that this exogenous delivery sufficiently facilitated the maturation of bone marrow dendritic cells (BMDCs), indicated by (1) the higher expression of MHC and CD80/86, and (2) modulated cytokine secretion pattern of increased IL‐12 and decreased IL‐10, as compared with the treatment of bolus TCLs. Moreover, BMDCs effectively presented AH‐1 peptide that known as antigen of CT26 via MHC I antigen‐presentation pathway. Thus, our Coa carrier could be utilized as an effective exogenous TCL delivery platform for multi‐epitope targeting toward DCs and immune cell activation.
Chemical and biomolecular surface modification of electrospun nanofibers for localized delivery of adeno‐associated viral (AAV) vectors
1Yonsei University
Adeno‐associated viral vectors (AAVs) have received intensive attention for last a couple of decades in both the clinical, and biological science and engineering societies due to their non‐pathogenicity, low immunogenicity, wide tissue tropism, and simple structure enabling tissue/cell‐targeted capsid engineering. AAV‐based therapeutics have been commercialized and utilized in the clinic for a decade, however, there still are unmet needs for more efficient and safer delivery methods of AAVs. Biomaterial‐ mediated gene delivery system is a promising strategy in order to address major limitations of delivering the genetic therapeutics alone, including off‐target effect, high required dosage, high dosage‐related toxicity, and neutralization of the vectors. Amongst various fabrication methods for therapeutic delivery systems, electrospinning technique has been actively employed in order to fabricate ECM‐analogue biomimetic nano/microfirous structures with controllable fiber diameters and a variety of applicable materials including both nature‐derived and synthetic materials. Herein, various design strategies of electrospun fibers we developed for delivering AAVs in a localized manner with diverse administration routes and target tissues are discussed. Electrospun fibers were fabricated with multiple design parameters such as backbone materials, surface properties modulated by chemical and biomolecular engineering, macroscopic fiber dimensions (i.e. 1‐,2‐ or 3‐D), and intra‐fiber structures. The combinatorial utilization of the electrospun fibers and AAVs showed a great potential to reduce spread‐out delivery of AAVs and increased the transgene efficiency, which could contribute to reducing the required dosage and enhancing the safety of the therapeutics.
CRISPR‐based gene manipulation for stem cell engineering and regenerative medicine
1National Tsing Hua University
Gene editing by CRISPR and gene regulation by microRNA or CRISPR activation have dramatically changed the way to manipulate cellular gene expression and cell fate. In recent years, various gene editing and gene manipulation technologies have been applied to control stem cell differentiation to enhance tissue regeneration. This presentation will focus on how to develop CRISPR, CRISPR activation (CRISPRa), CRISPR inhibition (CRISPRi) as well as bi‐directional CRISPR‐AI gene regulation technologies to control cell differentiation and tissue regeneration.
GENE modification of human MSC for increased therapeutic potency via paracrine activity
1Lomonosov Moscow State University, 2National medical research center of cardiology
Human adult multipotent mesenchymal stromal cells (MSC) were in a spotlight of potential therapeutic application throughout the last decade. Discovery of their paracrine activity and use of secretome for “cell therapy without cells” have remained a hot topic in the field.
We summarize and report our extensive development in the field using adeno‐associated virus, baculovirus and plasmid DNA vectors to express growth factors in MSC with a goal to enrich their secretome and thus functionalize for higher therapeutic efficacy.
Besides using gene modification for MSC secretome functionalization we addressed the issue of cell delivery and used cell sheet (CS) technology to facilitate cell survival in models of human disease of ischemic nature. Eventually, our studies of MSC application lead us to a conclusion that we may try to apply cell secretome alone and we used a basic approach expressing human BDNF and uPA by pDNA transfection to producent cells and obtained samples enriched by a neurotrophic growth factors. We evaluated its efficacy and observed increased animal survival, reduction of neurological disorders and brain injury volume in a model of intracerebral hemorrhage in rats.
We suggest a number of directions for future development in the field and believe that in regenerative medicine MSC gene modification are a great option taking into account their paracrine mode of action in human disease.
The study was supported by RSF grant #19‐75‐30007; State assignment and Development Program of the Interdisciplinary Scientific and Educational School of Lomonosov Moscow State University “Molecular technologies of the living systems and synthetic biology.”
CRISPR‐based bidirectional gene regulation for improved chondrogenic differentiation and calvarial bone regeneration in osteoporotic animal
1National Tsing Hua University
Calvarial bone regeneration poses a tremendous challenge in clinical settings due to poor spontaneous healing. Previous studies reported that chondrogenic induction of ASC was able to boost the healing progress via the non‐native endochondral ossification pathway. Non‐osteoporotic and osteoporotic ASC (OVX‐ASC) are inferior in osteogenesis capacity and predisposed to adipogenic commitment, while OVX‐ASC chondrogenic tendency remains elusive. CRISPR‐mediated epigenetic manipulation has emerged as a robust approach for targeted gene activation/silencing. To activate chondrogenesis, we developed CRISPR‐AceTrans harboring a dSpCas9 fusion with the histone acetylation domain p300core with an extra recruitable MCP‐VPR (Bac‐p300‐VPR) targeting the chondrogenic Sox trio (Sox5, Sox6, Sox9). To suppress adipogenesis, we adopted the CRISPRi architecture comprising the dSaCas9 fusion with the DNA methyltransferase 3A (Bac‐D3A) targeting adipogenic regulators (C/ebpa, Pparg). Bac‐ p300‐VPR‐transduced ASC displayed a profound promotion of chondrogenic phenotype as judged by abundant presence of glycosaminoglycan (GAG), while OVX‐ASC remained resistant to chondrogenic differentiation albeit the Sox trio activation. We Strikingly, combination of CRISPR‐AceTrans and CRISPRi generated a bidirectional platform, or CRISPR‐BiD, robustly driving OVX‐ASC towards chondrogenic lineage. 3D construct culture of the CRISPR‐BiD‐engineered OVX‐ASC gave rise to significant amount of GAG and collagen type II over the mock‐transduced construct. Ultimately, implantation of the transduced construct accelerated and enhanced calvarial defect regeneration in osteoporotic rats. Collectively, our results demonstrated the feasibility of CRISPR‐based epigenetic alteration for bidirectional regulation of gene expression in stem cells for regenerative medicine.
Bone regeneration using 3D‐printing hybrid bone scaffold in canine radial bone defect
1Department of Plastic and Reconstructive Surgery, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Korea, 2AI & Mechanical System Center, Institute for Advanced Engineering, Yongin 17180, Korea, 3Yeouido St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Korea, 4Regenerative Engineering Laboratory, Center for Dental and Craniofacial Research, Columbia University Irving Medical Center, New York, USA, 5Department of Mechanical and Design Engineering, College of Engineering, Wonkwang University, Iksan, Republic of Korea, 6Department of Health Sciences and Technology, GAIHST, Gachon University, Republic of Korea, 7Department of Cell Therapy Center of Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Korea, 8Department of Molecular Medicine, College of Medicine, Gachon University, Republic of Korea
Designing bioink, bioresin and multicellular spheroid fusion platforms for musculoskeletal regenerative medicine and disease modelling
1CReaTE Group, Department of Orthopaedic Surgery & Musculoskeletal Medicine, Centre for Bioengineering & Nanomedicine,University of Otago Christchurch, 2Light Activated Biomaterials Group, Department of Orthopaedic Surgery & Musculoskeletal Medicine, Centre for Bioengineering & Nanomedicine,University of Otago Christchurch
Biofabrication technologies, including 3D bioprinting and bioassembly, enable the generation of engineered constructs that replicate the complex 3D organization of native tissues via automated hierarchical placement of cell‐laden bioinks, tissue spheroids, and/or bioactive factors. A major bottleneck lies in designing hydrogel bioinks that are both cell‐instructive and compatible with high resolution 3D‐biofabrication techniques.
This study describes the design of versatile, photo‐clickable and cell‐instructive gelatin‐based bioinks and bioresins (e.g. GelAGE) using ruthenium (Ru)/sodium persulfate (SPS) photoinitiator that are printable across multiple biofabrication technologies, including extrusion‐ and lithography‐based (DLP) bioprinting.
We developed 3D multicellular spheroid bioassembly platforms (e.g. co‐cultured hAC/bmMSC/ucbMSC/HUVEC cells) for probing spheroid fusion kinetics, extracellular matrix (ECM) formation, tissue‐tissue interfaces and fusion mechanisms in healthy and diseased tissue spheroids, with specific examples of new paradigms for high‐throughput screening. We modulated the cell‐instructive musculoskeletal tissue niche for multiple cell types via covalent incorporation of thiolated bioactives (e.g. heparinSH), nanocomposites (e.g. strontium, Laponite) and mechanical stiffness yielding enhanced chondrogenic and osteogenic differentiation and vascular network formation.
Fabrication of clinically relevant sized osteochondral constructs demonstrates the flexibility of the platform – supporting tissue fusion, long‐term cell viability, and deposition of zone‐specific ECM proteins.
[1] Murphy et al; Advanced Materials 2022;10.1002/adma.202107759
[2] Lindberg et al; Advanced Science 8(22);2021
[3] Cui et al; Biofabrication 14(3);2022
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[7] Bertlein et al; Advanced Materials. 29(44);2017
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[9] Mekhileri et al. Biofabrication, 10(2);2017
Volume adaptation in 3D encapsulated cardiac spheroids
1University of Western Australia, 2University of Western Australia, School of Human Sciences
With the adoption of 3D cell culture for in vitro modelling of cardiac function and regenerative medicine applications, there is an increased need to understand cardiomyocyte mechanosensation in 3D. With existing studies of cardiomyocyte mechanosensation primarily focused on behaviour of individual cells in a 2D context, it is unclear whether mechanosensation is the same in a 3D, multicellular context. In this study, H9C2 cardiac‐derived myoblasts were encapsulated as individual cells and as cell spheroids within stiffness gradient gelatin methacryloyl (GelMA) hydrogels to investigate individual‐ and collective‐ cardiac cell mechanosensation in 3D. Over a 3.7 to 17.5kPa stiffness range, H9C2 cells showed a limited capacity to adapt their volume to increasing stiffness, demonstrated by the lack of changes in cell volume and shape across the stiffness gradient. When cultured as spheroids rather than as individual cells, H9C2 cells adopted a distinct morphology with comparably smaller nuclei than individually cultured cells, while retaining the same overall cell volume. As spheroids, H9C2 cells were sensitive to stiffness cues, shown by decreasing YAP nuclear localisation, decreasing MRTF‐A nuclear localisation, increasing Lamin‐A expression, and increasing vinculin expression with increasing stiffness. Like the individually cultured H9C2 cells, mechanomarker expression was dependent on volume adaptation. With increasing cytoplasmic volume, YAP and MRTF‐A became less nuclear localised, Vinculin expression was increased, and with increasing nuclear volume, the expression Lamin‐A was increased. Together, these data suggest that cardiac cell volume adaptation may be enhanced by cell‐cell interactions.
Induction of osteocyte differentiation for mesenchymal stem cells in 3D scaffold‐free spheroid culture
1Nagoya University, 2Kyoto University
The osteocyte plays a significant role as control tower to regulate the bone remodeling in the bone. Inside the hard bone matrix, they were embedded in the fluid space termed lacunae, so that they were not directly attached to the bone matrix and floated through the lacunae fluid. Our group have developed 3D scaffold‐free culture models using osteoblast precursor cells to induce the osteocyte differentiation. We found out that the modulation of culture environment by removing cell‐substrate interaction under the floating culture provoked up‐regulations of osteocyte differentiation markers. In this study, we fabricated scaffold‐free spheroids reconstructed by human mesenchymal stem cells (hMSCs). Compared to the conventional 2D monolayer culture, it up‐regulated the osteocyte differentiation marker (Opn, Phex, and Sost) within 2 days, whereas the osteoblast differentiation markers (Alpl and Col1) in the spheroids were suppressed. To investigate the structural effect on the osteocyte differentiation, we also carried out transferring experiment from the 3D spheroid culture to the 2D adhesive culture (3D‐to‐2D model). Immediate after transferring, the cells started dissociating by spreading over the adhesive dish. The cells from the spheroid eventually became 2D‐like culture model after 2 days. We then examine the gene expression of the 3D‐to‐2D model compared to the monolayer and spheroids, the osteocyte markers in the 3D‐to‐2D were down‐regulated up to the level of the monolayer. Hence, we showed that the cell condensation condition acquired from the 3D spheroid culture is required to maintain the up‐regulations of osteocyte differentiation of hMSCs.
The approval of the ATORM‐C for the first in human trial in Republic of Korea (Adult tissue derived organoid based regenerative dedicine)
1ORGANOIDSCIENCES Ltd.
Organoids, miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro‐anatomy. They include multiple organ‐specific cell types, grouped together and spatially organized like an organ and capable of recapitulating some specific functions of an organ.
Due to its characteristic of excellent regeneration, differentiation, and safety, organoid‐based regenerative medicine may have the potential to dramatically provide a fundamental treatment solution for unmet needs.
In this lecture, the following content will be discussed. First, the current status and strategies of development of organoid‐based regenerative medicines will be introduced. Second, we will disclose the CMC development and non‐clinical results of the pipeline for bowel diseases (ATORM‐C) based on ATORM platform of the ORGANOIDSCIENCES, Ltd. Finally, we would like to share current limitations for clinical and industrial application of organoids and introduce our challenges for the first in human trials.
Full automation system for stem cell spheroid fabrication and mass production
1Seoul National University Hospital
Due to low in vivo efficacy, clinical translation of stem cell therapy has been unsatisfactory thus far. Many studies have been conducted to strengthen the function of stem cells via gene transfection; however, gene modification is associated with a safety concern. As a result, spheroidal formation has gained attention as another strategy for improving function without jeopardizing safety. Spheroids express a high level of vascular endothelial growth factor (VEGF), a cytokine that promotes angiogenesis, as a result of the central hypoxia condition. A spheroid composed of mesenchymal stem cells secretes cytokines that improve cell viability, proliferation, differentiation, and extracellular remodeling.
In our previous experiments, a spheroid with a diameter of 250 micrometers secreted the most VEGF, and they demonstrated improved angiogenesis in both in vitro and in vivo experiments.
We are currently developing a full automation system for standardized fabrication and mass production of spheroid before clinical translation.
We are also collaborating on a faster technique to detect VEGF than the Elisa kit for spheroid quality assurance.
I'd like to talk about these topics, as well as the findings of a series of disease model animal experiments with enhanced regeneration using spheroids.
Highlights of the Austrian cluster for tissue regeneration: The p‐rpS6‐ zone delineates wounding response and the healing process
1Ludwig Boltzmann Research Group SHoW ‐ Senescence and Healing of Wounds
The Austrian Cluster for Tissue Regeneration (ACTR) integrates the Ludwig Boltzmann Institute for Trauma (LBI Trauma) and 31 further research groups of 12 universities and 2 non‐university institutions in Austria. With its fully translational approach, an interdisciplinary team including MD/VetMD, located partly in hospitals, and a GMP facility, it provides an optimal research environment. ACTR covers ‐ Neuroregeneration, Soft Tissue Repair/Wound healing, Cartilage/Tendon and Bone/Ligament Regeneration as well as competence centres for molecular biology, extracellular vesicles (EV‐isolation, characterisation and GMP production), senescent cells, miRNA analysis, polymer synthesis, bioprinting, preclinical in vivo facilities incl. imaging and morphology on multiple levels.
It is unknown what the spatial boundaries of tissue response to wounding are. Here we show that in mammals the ribosomal protein S6 (rpS6) is phosphorylated in response to skin injury forming a zone of activation surrounding the region of the initial insult. This p‐rpS6‐zone forms within minutes after wounding and is present until healing is complete. The zone encapsulates markers of the healing process, including proliferation, senescence, and angiogenesis in wounded skin. A mouse model unable to phosphorylate rpS6 shows an initial acceleration of wound closure, but results in disrupted healing. Finally, the p‐rpS6‐zone accurately reports on the status of dermal vasculature and the effectiveness of healing. In summary, the zone divides an otherwise homogenous tissue into regions with distinct properties.
Mammalian cell aging is associated with the mechanical environment on the hydrogel
1Department of Biomedical Science, College of Life Science, CHA University, Pocheon, 11160, Republic of Korea, 2CHA Fertility Center, Seoul Station, Hangang‐daero, Jung‐gu, Seoul, 04637, Republic of Korea, 3Mitoimmune Therapeutics Inc., Gangnam‐gu, Seoul, Republic of Korea
Aging is not only nonreversible processing but also related to several chronic diseases like dementia, atrophy, et al. Therefore, there is no doubt that aging is obsoletely associated with environmental conditions. Especially mechanical environment conditions involved modulation of aging. However, aging‐related mechanical status was not clearly identified in human cells. Here we investigate that the aging of cells depends on the stiffness of the matrix as the mechanical cure.
We set up 4kPa, 10kPa, and 16kPa hydrogel by acrylamide mixture + bis‐Acrylamide hydrogel coated with collagen solution and cultured the HEK293 cell line with DMEM culture media. Therefore, we observed cell morphology and assessed proliferation depending on the hydrogel stiffness. And we evaluate age‐related gene expression depending on the stiffness environment.
The cell morphology dramatically changed from 4kPa to 16kPa. 4kPa condition significantly decreases cell size compared with 16kPa. cell proliferation and viability also dramatically decrease by 4kPa. Additionally, we analyzed longevity‐related genes such as SIRT1, FOXO, SOD2, and PGC‐1alpha. Longevity‐related genes such as SIRT1 and PGC‐1alpha significantly increase in the cultured cells with 16kPa. But 4kPa culture condition reveals a decrease in longevity genes compared with 16kPa.
In conclusion, the mechanical environment is one of the important effects of cell aging. The biological system is a mechanical environment study requests for the regulation of cell aging and restoration of aging.
A roadmap of colPatchTM acellular skin substitute‐ a potential solution for full‐thickness skin loss
1Centre for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia.
Functional biomaterials is currently an essential component under regenerative medicine approach in complementing future precision medicine. The successful development of authentic biomaterial primarily as medical device would be considering the resources selection, three‐dimensional design using various fabrication techniques, optimum mechanical strength, extensive physico‐chemical properties, biological interactions (toxicity, biocompatibility and cellular interactions) prior to implantation on pre‐clinical model (small and larger mammals). The major challenge of biomaterial development is commonly faced in the translational stage (clinical trials) before product registration & commercialization (potential industry collaboration). Centre for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia developed a natural‐based biomaterials collagen type I from ovine tendon since 2009 and currently known as ColPatchTM has been completed the biological safety through accredited laboratory (GLP & ISO), long‐term storage evaluation, and ongoing clinical trial phase I. ColPatchTM intends to cater the current wound management of full‐thickness skin injury. Briefly, ColPatchTM could be a potential ready‐to‐use product for patients and clinicians to improve cutaneous wound healing and embark new hope near future.
Decellularized extracellular matrix (DECM) reactor
1BTI A*STAR
Decellularization involves removing the cellular components while retaining the functional extracellular matrix (ECM) components. The current decellularization methods use improvised labware or a handful of commercial devices limited in applications. These set‐ups and techniques have low production scales. They cannot track the progress of decellularization, hence resulting in an inefficient process that can be costly, wasting resources and tissue samples. This leads to inadequate handling procedures and difficulty monitoring and optimizing the parameters in real‐time and may also lead to issues with process reproducibility. There is a need for a device to minimize handling steps, advanced monitoring of the decellularization process and potential for scalability. Our novel patent‐pending DECM reactor is a scalable, clean, and compact solution for better process control and decellularization of animal, human, and plant samples. We employ monitoring and adaptive process controls to regulate the decellularization progress and optimize yield. We have demonstrated the utility of our DECM reactor via porcine liver tissue samples. The reactor is now being adapted for animal skin and plant tissues for bioink and cultured meat applications respectively.
Injectable composite hydrogel embedded with eutectic Ga/In liquid metal particles for photothermal and chemotherapy against breast cancer
1Dongguk University
Liquid metal with a low melting temperature and photothermal conversion ability has been widely used in various engineering applications. Especially, compared with other metals, eutectic gallium‐indium alloy (EGaIn) has excellent chemical stability, transformability, and biocompatibility. Under near infrared (NIR) irradiation, the elevation of temperature as well as the formation of reactive oxygen species lead EGaIn to be utilized in cancer treatment platform. Herein, an interpenetrating network (IPN) hydrogel, consisting of thiolated gelatin (Gel‐SH) conjugated with 6‐mercaptopurine (6‐MP) and poly(ethylene glycol) diacrylate, works as an injectable reservoir for EGaIn‐mediated multiple anticancer treatments: (1) increasing temperature by photothermal effect, and (2) additional delivery of anticancer chemo‐drug 6‐ MP. It was found that the temperature of hydrogels embedded with EGaIn particles increased over 50 °C under NIR laser irradiation, with controllable and repeatable thermal stability for 5 on‐off cycles. Moreover, our in vitro results demonstrated the composite hydrogel‐mediated anticancer mechanism for triple negative breast cancers: (1) high temperature by photothermal efficacy killed cancer cells, (2) photothermal‐mediated cancer lysis could induce glutathione leakage, (3) subsequently, GSH cleaved disulfide bonds between 6‐MP and thiolated gelatin, and (4) the release of 6‐MP led to additional cancer cell death. Therefore, our injectable IPN hydrogel platform containing EGaIn liquid metal particles and chemical drug 6‐MP could be applied to a multi‐functional anticancer therapy.
Chitosan‐based nanofibrous polyelectrolyte complex for rapid hemostasis
1National Institute of Technology Rourkela, 2GADVASU Ludhiana, Punjab,
Hemostatic agent is the saviour of emergencies like accidents, gunshots, and critical bleeding. Worldwide, accidents are the leading cause of trauma and prehospital death. There is a need for a safe, effective hemostatic agent to avoid these preventable deaths due to bleeding. Here, we report a nanofibrous hemostatic agent of chitosan and casein loaded with silver nanoparticles. The chitosan and casein nanofiber have fabricated by self‐assembly of polyelectrolyte complex. The self‐assembled nanofibers have fiber diameter of 25 ± 9 nm, surface charge of ‐10 ± 5.3 mv, and haemolysis percentage of 1.23 ± 0.56. The hemostatic agent could clot the goat blood in 9 ± 3 seconds under in vitro conditions. The hemostatic agent also inhibited the growth of both S. aureus and E. coli to 1.05 X 102 and 0.75 X 102 colony‐forming units, respectively, in 24 hours. Platelet adhesion through a scanning electron microscope and the enzymatic activity of platelets resulted in clot formation by the mechanism of rapid fluid absorption, cell coagulation, and platelet activation. The hemostatic agent could also stop the bleeding of the rat femoral artery in 10 ± 2 seconds. Subcutaneous implant in Sprague‐Dawley rat's blood and histopathological parameters did not show any toxic effect. The proposed hemostatic agent can have great potential for emergency care and prehospital haemorrhage
Biocompatible Ag2S nanodots: Mussel protein‐based NIR light‐ responsive for imaging‐guided photodynamic anticancer therapy
1Department of Biomedical Convergence, School of Convergence, Kyungpook National University, Daegu 41566, Korea, 2Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 37673, Korea, 3Cell and Matrix Research Institute, Kyungpook National University, Daegu 41944, Korea
Light‐responsive nanotheranostics are highly desirable for cancer theranostics because they can make it possible to visualize and treat the cancer specifically through precise external modulation of the site via a single injection. Quantum dots (QDs) have been extensively considered to construct light‐responsive nanotheranostics due to excellent optical properties and rich surface chemistry. However, the clinical application of QDs‐based nanotheranositcs is still elusive due to the potential toxicity of contrast agents and the poor retention at the target site. Here, a sticky protein‐based nanodots platform that simultaneously allows near‐infrared (NIR)‐responsive photodynamic therapy (PDT) treatment and real‐ time tracking in a highly biocompatible, site‐specific manner. A suite of proteinic nanoparticles decorated with silver sulfide (Ag2S) QDs were fabricated through electrospraying and subsequent biomineralization of a bioengineered mussel adhesive protein fused with a silver‐binding AgP35 peptide (MAP‐AgP35) under mild condition. The light‐activatable proteinic Ag2S nanodots exhibited excellent anticancer therapeutic effects through intensive release of reactive oxygen species (ROS) as well as effective in vivo imaging ability under irradiation of NIR light (808 nm), while showing a good biocompatibility towards normal cells. Thus, our mussel protein‐based Ag2S nanodots have a great potential as an externally controllable nanotheranostics to realize imaging‐guided therapeutic implications for the complete ablation of cancer.
25 years of commercializing nanomedicine: from tissue engineering to fighting COVID
1Hebei University of Technology
Nanomedicine has already revolutionized medicine through the development of materials that can prolong circulation in the body, avoid immune system clearance, penetrate cells and bacteria, invade tumors, promote tissue growth, inhibit infection, and so much more. New fields have emerged such as 4D printing which can enhance the performance of nanomaterials by 3D printing them into desirable shapes, implant them, and control their shape through external stimuli (such as near infrared excitation, temperature control, and others). This presentation will provide an overview of 25 years of commercializing University based research into real products helping human health. It will cover the promises and pitfalls of commercializing University based research and even discuss if this is the right model to advance science and research into the medical industry. It will also highlight new areas emerging for commcerialization such as the use of 4D printing in medicine for straightening the spine for scoliosis patients, closing of sphincters that weaken as one ages (for example, to decrease acid reflux from the stomach to the esophagus), promote intervertebral tissue growth by increasing pressure on such tissue during regeneration, deliver stem cells on the same materials in which they are cultured to enhance stem cell viability, and more. It will cover additional new areas like picomedicine, implantable sensors and more. It will cover in vitro and in vivo assessment of such materials and discuss what is needed to experience full application of nanomedicine throughout all of medicine.
Advances in cell coculture porous membranes: Recapitulation of in vivo microenvironments
1Korea Institute of Science and Technology
In this talk, we will introduce the advances in cell coculture platforms integrating engineered porous membranes for the recapitulation of in vivo microenvironments, mediating heterogeneous cell‐cell signaling. A porous membrane‐based coculture platform is crucial for cell research since it enables the creation of modular cellular and/or tissue interfaces and helps establish barrier models for tissue‐on‐a‐ chip. The in vitro coculture systems incorporating porous membranes are often used to mimic the in vivo microenvironments of differentiating stem cells by recapitulating the naturally occurring cell‐cell communications. Commercially available coculture membrane, normally a Transwell®, has been well‐ established and conventionally used for fundamental and practical studies in heterogeneous cell coculture systems. However, its relatively thick membrane thickness (∼10 μm) and low porosity resulted in insufficient interactions between cocultured cells. To resolve the limitations, we have developed advanced coculture membranes made from polymers. The new membranes with thin‐thickness and high‐porosity have demonstrated enhanced cell‐cell interactions, mimicking the native tissue microenvironments.
Biomimetic virus‐based de novo soft tissue niche engineering
1Pusan National University
Extracellular niches play a crucial role in overcoming the surrounding harsh pathological environments as well as for stem cell differentiation and other important functions. Nanofibrous arginine‐glycine‐aspartic acid‐engineered M13 phage has recently been considered as an extracellular matrix‐mimicking bioinspired nanofiber that serves as an instructive tissue engineering material, providing a vascular niche and cytoprotective function at injured sites, which is an essential option for stem cell therapy. Herein, the novel and essential therapeutic cues provided by an engineered phage are exploited, contributing to de novo soft tissue niche engineering, where the interplay of biomimetic phage cues with sur‐rounding organ tissues is identified and cells are implanted between tissues to achieve an appropriate soft tissue niche that enables the proper functioning of the implanted stem cells at the injured site. These biomimetic phage nanofiber cues are considerably supportive for cell therapy, as they establish promising therapeutic extracellular de novo soft tissue niches for curing ischemic diseases.
Establishing an attractive regenerative alliance: Human mesenchymal stem cells and nanomagnetic materials
1National Institute of Research and Development in Technical Physics Iasi, 2National Institute of Research and Development in Technical Physics
Magnetic nanoparticles (MNPs) and magnetic nanowires (NW) are versatile tools in regenerative medicine. Remote controlled actuation of MNPs‐loaded cells delivers micro‐mechanical stimulation for stem cell differentiationl increasing osteogenesis and chondrogenesis. Nickel NW can be used as platforms for the delivery of r magneto mechanical stimulation.
Human adipose derived stem cells (ADSCs) and Wharton jelly MSCs (WJMSC) loaded with MNPs were tested for viability, proliferation, culture induced senescence. In vitro osteogenesis adipogenesis of ADSCs‐MNP as well as chondrogenic potential of ADSC and WJMSC –MNP exposed to magnetic field (MF) was tested. ADSC on NW substrates and MF exposure were tested for viability and differentiation potential.
Stem cells retain viability and proliferative capabilities compared to non‐loaded and become remote controllable within MF. MNP presence decrease stem cells culture induced senescence in ADSC but not WJMSCS ADSCs–MNP display increased osteogenic and decreased adipogenesis when exposed to MF. ADSC‐MNP increased chondrogenesis within MF. NW substrate supports attachment and viability of ADSC. Osteogenesis but not adipogenesis of ADSC cultured on NW increased by MF exposure. Moreover, osteogenesis in ADSCs cultured on NW substrate could be detected even without differentiation media and enhanced by MF exposure.ADSCs‐MNP display increased osteogenic and decreased adipogenic potential under MF.l. ADSC‐MNP but not WJMSC display increased chondrogenesis with MF exposure. NW substrate and MF exposure can be used to obtain osteogenic conversionl of ADSC in vitro in the absence of differentiation media. This can be used for bone engineering or for cell preconditioning in the case of cellular therapies.
A pre‐clinical animal study for zonal articular cartilage regeneration using stratified implantation of microcarrier expanded zonal chondrocytes
1National University of Singapore, 2Massachusetts Institute of Technology, 3Singapore‐MIT Alliance in Research and Technology
The zonal properties of articular cartilage critically contribute to the mechanical support and lubrication of the tissue. Current treatments for articular cartilage have yet to regenerate this zonal architecture, thus compromising the functional efficacy of the repaired tissue and leading to tissue degeneration in the long term. In this study, the efficacy of zonal cartilage regeneration through bilayered implantation of expanded autologous zonal chondrocytes was investigated in a porcine chondral defect model. Autologous chondrocytes extracted from articular cartilage in the non‐weight bearing trochlea region of the knee were subjected to our novel expansion‐sorting strategy, integrating dynamic microcarrier (dMC) culture, and spiral microchannel size‐based zonal chondrocyte separation. Zonal chondrocytes were then implanted as bilayered fibrin hydrogel construct in a porcine knee chondral defect model. Repair efficacy was compared with implantation with cell‐free fibrin hydrogel and full thickness (FT) cartilage‐derived heterogenous chondrocytes. Cartilage repair was evaluated 6 months after implantation. Six‐month repair outcomes showed that bilayered implantation of dMC‐expanded zonal chondrocytes resulted in substantial recapitulation of zonal architecture, including chondrocyte arrangement, specific Superficial Zone Protein distribution, and collagen alignment, that was accompanied by healthier underlying subchondral bone. Our results demonstrate that with appropriate expansion and isolation of zonal chondrocytes, the strategy of stratified zonal chondrocyte implantation represents a significant advancement to Autologous Chondrocyte Implantation based cartilage regeneration, with the potential to improve the long‐term integrity of the regenerated tissues.
3D printing of assemblable bespoke scaffold as versatile microcryogel carrier for site‐specific regenerative medicine
1Institute for Biomechanics, Department of Health Science and Technology, ETH Zurich, 2Complex Materials, Department of Materials, ETH Zurich, Switzerland, 3Institute for Biomechanics, Department of Health Science and Technology, ETH Zurich, Switzerland, 4Complex Materials, Department of Materials, ETH Zurich
Through the advent of additive manufacturing techniques, various approaches have been developed to fabricate patient‐specific implant designs based on CT images of the patient. However, this requires intensive work and can bring significant financial burden to the healthcare system. Especially in developing countries, affordable treatment options are required, while still not excluding these patients from potential advances. Here, we used a digital light processing (DLP) 3D‐printing technique to fabricate a hive‐structured assemblable bespoke scaffold (HIVE). HIVE can be manually assembled in any shape/size with ease, so a surgeon can create a scaffold that would best fit a defect before implantation. Simultaneously, it can have site‐specific treatments by working as a carrier filled with microcryogels (MC) incorporating different biological factors in different pockets of HIVE. After fabrication, we analyzed the mechanical characteristics of HIVE, through testing and finite element analysis. The results demonstrated that HIVE has sufficient mechanical properties to withstand loading in a physiological system. We investigated possible site‐specific applications by utilizing HIVE as a versatile carrier with incorporated treatments such as growth (factors GF) (BMP, VEGF), bioceramic (silicon nitride, hydroxyapatite) or (cells hMSC, HUVEC). HIVE as a GF‐carrier showed a controlled release of BMP‐2/VEGF and induced osteogenesis and angiogenesis from hMSC and HUVEC, respectively. Furthermore, as a bioceramic‐carrier, HIVE demonstrated enhanced mineralization and osteogenesis. When loaded with HUVEC, HIVE also upregulated both osteogenic and angiogenic gene expression of hMSCs. Finally, we confirmed successful cell migration in HIVE, even in case of multiple component assemblies and demonstrated possible clinically relevant applications by assembling.
Understanding the role of focal adhesion in regulating dedifferentiation of chondrocytes using a cell traction and intracellular force microscopy
1Soonchunhyang Institute of Medi‐bio Science (SIMS), Soonchunhyang University, Republic of Korea
The native extracellular matrix (ECM) of articular cartilage plays an important role in maintaining chondrocyte phenotypes and homeostasis of cartilage tissue. However, due to the intrinsic nature of cartilage, its regeneration potential is limited, and chondrocytes undergo dedifferentiation during in vitro expansion. Here, we investigate the dynamic role of cell‐matrix interaction of chondrocytes in regulating their cellular phenotypes by employing various matrix stiffnesses and modulating focal adhesion (FA) to support in vitro expansion of chondrocytes, while maintaining their ability to produce cartilage‐specific matrix, including type II collagen and proteoglycans. Our findings demonstrated that cells cultured on soft matrix exhibited a restriction of cell adhesion and spreading, inhibiting FA complex formation, actin cytoskeleton re‐organization, and fibrocartilage‐specific ECM production. Furthermore, our novel approaches to elucidate the role of cell‐matrix interaction in dedifferentiation of chondrocytes harnessed cell traction and intracellular microscopy (TFM‐IFM), demonstrating that dedifferentiated chondrocytes underwent extensive cell spreading, exhibited actin cytoskeleton assembly, and exerted significantly increased cell traction force and intracellular tension. Taken together, our study provides a potential explanation for the role of focal adhesion in regulating chondrocyte phenotypes during dedifferentiation.
A phase I/IIA trial to test safety and feasibility of an autologous IPS cell‐ derived retinal pigment epithelium patch in age‐related macular degeneration patients
1NIH
Induced pluripotent stem cells (iPSCs) can provide autologous and allogeneic replacement tissues, potentially for all degenerative diseases. Autologous tissues have the advantage of not requiring immune‐ suppressive drugs that have deleterious side‐effects. The safety and feasibility of autologous iPSC‐based therapies hasn't been established. Here, we developed an autologous iPSC‐based therapy for age‐related macular degeneration (AMD), a blinding eye disease that affects over 30 million people world‐wide. AMD is caused by the progressive degeneration of retinal pigment epithelium (RPE), a monolayer tissue that maintains photoreceptor function and survival. We tissue engineered a clinical‐grade iPSC‐RPE‐ patch on a biodegradable scaffold using autologous cells from AMD patients. Preclinical investigational new drug (IND)‐enabling studies performed on iRPE‐patch derived from multiple AMD patients demonstrated reproducible manufacturing, validating our manufacturing process ‐ a key requirement for an autologous phase I trial. Functional validation of clinical‐grade iRPE‐patches allowed determination of tissue barrier resistance, purity of RPE cells, and RPE cell shape metrics as key critical quality attributes now used as clinical product release criteria. Preclinical animal studies performed in immune‐ compromised rats confirmed safety of the auto‐iRPE‐patch and efficacy studies performed in a porcine laser‐induced RPE injury model that mimics AMD‐like eye conditions demonstrated integration and functionality of RPE patches. A phase I/IIa IND‐application for an auto‐iRPE‐patch to treat AMD was recently cleared by the FDA. This Phase I/IIa clinical trial will test safety, feasibility, and integration of an auto‐iRPE‐patch in twelve advanced AMD patients.
Omics analytical data sets for quality control of iPSC‐based manufacturing
1Korea National Institute of Health
Korea national stem cell bank was opened in 2012. Currently, 69 research lines and 27 GMP‐compliant lines have been deposited. To evaluate the genetic stability of the banking cell lines, we are using multi‐ omics data such as SNP‐chip, WES, Methyl‐seq, and RNA‐seq data. In this meeting, I will share the recurrent mutations found in the genomes of our banking cell lines and, their derivatives. Mainly, RNA‐ seq‐based broad range genetic quality tests on GMP‐compliant human leucocyte antigen (HLA) ‐ homozygous hiPSCs under post‐distribution conditions will be introduced. Finally, the usefulness of a single cell RNAseq analysis to ensure the quality of the cell therapy products will be addressed. Collectively, omics analytical data sets allow the early screening of candidate hiPSC seed stocks for clinical use by facilitating safety and potential risk evaluation, and multi‐omics based genetic quality testing of cell therapy products would be useful for ‘go’, ‘no‐to decision’.
3D bioprinted choroid and RPE to investigate dry‐AMD mechanism
1NIH
Age‐related macular degeneration (AMD) is a long‐term progressive disease that leads to permanent central vision loss. While wet‐AMD with neovascularization in the outer retina can be treated with anti‐ VEGF approach, dry‐AMD demonstrates degeneration of retinal pigmented epithelial (RPE) and capillary loss of choroid has no treatment. There have been investigations that suggest the role of the alternative complement pathway in lipid accumulation on the basal lamina of RPE and is also linked to progression to advanced stages of dry‐AMD. Among other inhibitors of alternative complement pathways, compromised complement factor H (CFH) expression and activity changes are associated with increased risk of dry‐AMD, but the relative contributions of CFH from the RPE and choroid are unknown. Here we aim to study the effects of CFH in choroid versus RPE using CFH knockout (KO) iPSCs and the 3D bioprinting technique.
Healthy iPSC line (WT‐iPSC) and genetically modified CFH‐KO‐iPSC were differentiated into RPE, endothelial cells (EC), pericytes, and fibroblasts and validated by immunophenotyping, hydrogel capillary formation and 3D‐bioprinted choroid. 3D‐bioprinted choroid was combined with RPE monolayer that matured 5‐6 weeks forming outer retinal blood barrier and Bruch's membrane. All differentiated cell components from WT‐ and CFH‐KO‐iPSC demonstrated compatible cell‐type‐specific phenotypes with slight differential expressions of complement system‐related proteins. When exposure to the complement factors by human serum, lipid deposition, apoptosis, and capillary loss were observed. In summary, we generated 3D‐bioprinted ex‐vivo choroid‐RPE system that provides a tool to investigate the disease mechanism and the role of anaphylatoxins in RPE versus the choroid.
Targeting senescent retinal pigment epithelium for retinal regeneration: An emerging therapy for age‐related macular degeneration
1Konkuk University School of Medicine, 2UNIST, 3Fusion Biotechnology
Although age‐related macular degeneration (AMD) is a multifactorial disorder with angiogenic, immune, and inflammatory components, the most common clinical treatment strategies are antiangiogenic therapies. However, these strategies are only applicable to neovascular AMD, which accounts for less than 20% of all AMD cases, and there are no FDA‐approved drugs for the treatment of atrophic AMD, which accounts for ∼80% of AMD cases. Here, we report that the elimination of senescent cells is a potential novel therapeutic approach for the treatment of all types of AMD. We identified senescent retinal pigment epithelium (RPE) cells in animal models of AMD and determined their contributions to retinal degeneration. We further confirmed that the clearance of senescent RPE cells with the MDM2‐ p53 inhibitor Nutlin‐3a ameliorated retinal degeneration. These findings provide new insights into the use of senescent cells as a therapeutic target for the treatment of AMD.
Cell and gene therapy for Huntington's disease
1CHA University / iPS Bio
Huntington's disease (HD) is a devastating autosomal‐dominant neurodegenerative disease, characterized by progressive motor, cognitive, and psychiatric disturbances associated with neuronal dysfunction and atrophy of the striatum and other brain regions. We investigated the therapeutic effects of neural precursor cells (NPCs) derived from human iPSC lines in the YAC128 transgenic mouse model of HD. HLA‐ homozygous iPSC line was differentiated into neural precursor cells, and then, they were transplanted into 6 months‐old YAC128 mice. Motor and cognitive functions were significantly improved in transplanted animals. Five months after transplantation, the donor cells had differentiated into neurons, oligodendrocytes and astrocytes. Transplantation restored DARPP‐32 expression, synaptophysin density, myelin basic protein expression in the corpus callosum and astrocyte function. In the second study, we employed CRISPR/CAS9 gene editing technology in order to knockdown Spt4 (SUPT4H1) gene, which is a transcription elongation factor, in Q57 HD‐iPSC‐derived NPCs. The Spt4‐Spt5 complex binds RNA polymerase II (Pol II) and regulates transcription elongation of expanded nucleotide repeats. First of all, ex vivo Spt4 knockdown of Q57 iPSC‐NPCs resulted in decreased mutant Htt expression and increased neuronal differentiation. Secondly, transplantation of Q57/Spt4 iPSC‐NPCs gave rise to behavioral improvements and neuroprotective effects (EM48↓, MAP2↑) in YAC128 HD mice. These results strongly suggest that Spt4 knockdown in HD iPSC‐NPCs may provide a novel therapeutic strategy for autologous stem cell therapy in HD.
This research was supported by Korean Fund for Regenerative Medicine funded by Ministry of Science and ICT, and Ministry of Health and Welfare (RS‐2022‐00070674, Republic of Korea)
The immunomodulatory effect of adipose‐derived stem cells in the human‐to‐rat xenograft skin transplantation model
1Seoul National University
Adipose‐derived stem cells (ASCs) have demonstrated the immunomodulatory effects for the treatment of transplant rejection. The angiogenic and anti‐inflammatory properties of ASCs were reported to increase graft survival in a murine model of allotransplantation. The purpose of this study was to evaluate the effects of ASCs on the survival of xenogeneic skin grafts, then compare single and multiple injections of ASCs.
We divided 48 male Sprague‐Dawley rats into control, single, and multiple injections of ASCs int the subcutaneous (SC) plane evenly around the 2.0 x 2.0 cm2 human full‐thickness skin grafts. In one group of 16 rats, 1 ml allogenic rat ASCs (1.0 x 106 cells per rat) were injected once after skin grafting (single
group). In another group of 16 rats, ASCs (1.0 x 106 cells/ml per rat X 1 ml/day x 3 days) were injected on postoperative days (POD) 0, 3 and 5 (multiple group). An additional group of 16 rats received a single injection of phosphate‐buffered saline and served as controls. Human skin grafts were harvested from the remnants of transverse rectus abdominis musculocutaneous (TRAM) flap. The planned analysis of this study included graft survival assessment, histological examination, immunohistochemistry, and flow cytometric analysis of regulatory T cells (isolated from peripheral blood mononuclear cells and skin).
In the present study, local injection of ASCs was evaluated for their ability to effectively suppress immune reaction after human‐to‐rat skin xenograft transplantation. It is hypothesized that multiple injections would be superior to a single injection.
Biomimetic scaffold composition benefits hMSCs differentiation towards the specific lineage
1Tissue Engineering Laboratory, Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong, China
Each tissue has a certain composition of the ECM. Bone is composed of organic (40%) and inorganic compounds (60%), in which 90% of the organic component is collagen and has a low content of glycosaminoglycan (GAG). Cartilage is composed of 60% collagen and 25‐35% PGs, and IVD is composed of 20‐30 % collagen and 36‐70% PGs, respectively. The appropriate microenvironment also supports and regulates MSCs differentiation towards the specific cell linage.As the major components of ECM, collagen, HA and GAG are important candidates to mimic the native microenvironment. We aimed to fabricate scaffolds that mimic the complex ECM of different tissues and we hypothesized that the biomimetic scaffolds will benefit hMSCs differentiated into specific lineages. Herein, we demonstrate that scaffolds mimicking the ECM composition particularly with different GAG content in terms of GAG/HYP ratios, will facilitate multiple differentiation of hMSCs towards specific lineages. In osteogenic differentiation, the Col (GAG/HYP 0) scaffold showed higher calcium (Ca) and phosphorus
(P) deposition and Ca/P ratio, biomimetic ultrastructure, and osteogenic phenotypic expression. In chondrogenic differentiation, aCol‐GAG (GAG/HYP 4.9:1) showed hydration, higher GAG deposition, biomimetic ultrastructure, and higher chondrogenic phenotype expression. In discogenic differentiation, the aCol‐aHA‐GAG (GAG/HYP 19.8:1) showed intensive GAG deposition, biomimetic ultrastructure, and higher phenotypic expression of NPCs. his work demonstrated that the fabrication of scaffold mimicking the natural composition of native tissue is beneficial for promoting the differentiation of hMSCs towards the specific lineages of the resident cells in those particular tissues, facilitating their potential applications in musculoskeletal tissue engineering.
Engineering 3D biomaterials for improved cancer modelling and drug screening
13B's Research Group, I3Bs‐ Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciencia e Tecnologia, Zona Industrial da Gandra, 4805‐017 Barco, Guimaraes, Portugal and ICVS/3 B's PT Government Associate Laboratory, Braga/Guimaraes, Portugal., 23B's Research Group, I3Bs‐ Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciencia e Tecnologia, Zona Industrial da Gandra, 4805‐017 Barco, Guimaraes, Portugal and ICVS/3 B's PT Government Associate Laboratory, Braga/Guimaraes, Portugal.
Cancer‐related deaths remain a significant burden despite the considerable advances in diagnosis and treatment. The therapeutic approaches based on the "one‐size‐fits‐all" paradigm are not efficient and patients respond to drugs differently. More individualized therapies adapted to the molecular features of the tumour can improve the efficacy of cancer treatments. These personalized approaches share certain shortcomings of conventional therapies, mainly associated with systemic drug delivery, including reduced effectiveness or specificity. Efforts are invested in developing novel strategies based on tissue engineering methods [1], advanced biomaterials [2], functionalization strategies [3], and nanotechnology tools [4] to facilitate the early diagnosis of cancer, the relevance of cancer modelling, and the efficiency of cancer drug screening/discovery. These strategies are envisioned to be precise, efficient, standardized, and less expensive. Precisely‐engineered 3D biomaterials have emerged as a powerful interdisciplinary strategy to detect, model, and treat cancer more efficiently [2]. These "precision" biomaterials are accurately designed to contain theranostics functions and bioactive components. We will discuss the advances in biomaterials for 3D cancer modelling, diagnosis, and therapy and describe illustrative examples based on bioengineered 3D hydrogels and microfluidics [4‐7]. [1] Brancato et al., Biomaterials 232, 119744, 2020
A scaffold platform for sinonasal cancers: In‐depth tumor understanding for personalized therapy
1University of Pisa
Sinonasal cancers are rare tumors that account for 3% of all cancers of head and neck district, with prevalence in some geographical areas [e.g., China, Japan, Italy (Tuscany and Eastern Piedmont)], and are associated to specific job categories, like carpentry and tannery. Diverse tissues can be affected, including epithelia, glands, bone, cartilage and olfactory nerve, as the anatomy of nasal sinuses is very complex. Being rare and diversified in more than 200 subtypes, much is still to be understood to treat those tumors in a personalized manner. The goal of this work is to create a platform of scaffolds able to reproduce 3D models of selected sinonasal cancers.
We developed 3 different scaffold types, all based on polyhydroxybutyrate (PHB and PHBV) to mimic three tissues affected by sinonasal cancers, i.e., squamous epithelium (melanoma), mucous epithelium (intestinal‐type adenocarcinoma; ITAC) and bone (osteosarcoma). By studying quaternary polymer‐ solvent systems, we obtained and characterized collagen/PHBV (50/50 w/w%) and chitosan/PHBV (5/95 w/w%) electrospun scaffolds for epithelial tissues, and nano‐BaTiO3/PHB (5/95 w/w%) 3D printed scaffold for bone. We cultured tumor cell lines representative of the selected tumors and their normal counterparts and characterized the obtained 3D models according to cell viability and morphology. We isolated 10 cell lines derived from patients affected by sinonasal tumors, including ITAC, melanoma, and sinonasal undifferentiated carcinoma (SNUC), and tested our scaffold platform with primary cells of melanoma and ITAC, demonstrating that it can replicate the morphologic and genetic features of those sinonasal tumors.
An integrated precision medicine platform CIT‐CoPT toward treating advanced colorectal cancer patients
1Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2Research Center for Tissue Engineering and Regenerative Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 3Huazhong University of Science and Technology, 4Department of Gastrointestinal Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology
Investigating clinical actionable targets in a fast manner is crucial for formulating precise treatment of patients with advanced colorectal cancer suffering from rapid disease progression and few effective drugs. However, a complete platform that can integrate profiling of molecular targets, prediction of drug reactivity and screening of personalized therapeutics is scarce currently.
By combining high‐throughput sequencing with microfluidic‐based organoid drug testing system, we have established an integrated precision medicine platform named CIT‐CoPT (Capture sequencing for Identifying Targets for Colorectal Personalized Therapy) toward screening effective therapeutic agents for the patients. Tumor specimens obtained from biopsy or surgery are divided for next generation sequencing and organoid establishment simultaneously. Exon sequencing of drug‐actionable genes using custom‐designed probes identify the molecular characteristics of the patient. Meanwhile, the patient‐ derived organoids are cultivated and loaded into a microfluidic system for subsequent drug screening. According to the molecular profiling results, various drug candidates are elected and applied in the microfluidic system to evaluate the sensitivity of the organoids. Effective targeted drugs can be filtered as candidates for clinical trials within two weeks.
When applying the CIT‐CoPT platform in clinic, we have sequenced a stage III colorectal cancer patient and identified 6 drug‐targetable genes and 13 candidate drugs. The drug sensitivity has been evaluated using the microfluidic screening system containing patient‐derived organoids. With this integrated precision medicine platform, we have successfully identified optimal drugs including Afatinib, Dacomitinib and Osimertinib for treating this patient.
3D disease modelling of hard and soft cancer using PHA based scaffolds
1Jawaharlal Nehru University, 2University of Westminster, 3University of Sheffield
Tumour cells change shape and lose polarity when they are cultured in 3D, a feature typically associated with tumour progression in vivo, thus making it significant to study cancer cells in an environment that mimics the in vivo milieu. In this study we have established hard and soft 3D cancer tumour models utilizing a blend of P(3HO‐co‐3HD) and P(3HB). P(3HO‐co‐3HD) and P(3HB) belong to a group of natural biodegradable polyesters, PHAs, that are synthesized by microorganisms. The 3D PHA scaffolds produced allow nutrients to diffuse within the scaffold and provide the cells with the flexibility to distribute evenly within the scaffold and grow within the pores. On Day 5, MDA‐MB‐231 showed dispersed growth in clusters, MCF7 formed evenly dispersed dense layer while, HCT116 formed large colonies within the pockets of the scaffold. Our results show Epithelial Mesenchymal Transition marker gene expression profiles in the hard tumour cancer models. In the 3D based PHA scaffolds, MDA‐MB‐ 231 expressed higher levels of Wnt‐11 and mesenchymal markers such as Snail and Vim, while MCF7 cells exhibited no change in their expression. However, MCF7 cells exhibited a significantly increased E‐ Cadherin expression as compared to MDA‐MB‐231. The expression levels of EMT markers were comparative to their expression reported in the tumour samples, making them good representatives of cancer models. In future, these models will be helpful in mimicking hypoxic tumours, in studying gene expression, cellular signalling, angiogenesis and drug response more accurately than 2D and perhaps other 3D models.
Direct reprogramming toward cardiovascular tissue and its cardiac regenerative effects
1Emory University
Direct reprogramming of fibroblasts into cardiomyocytes (CMs), endothelial cells (ECs), or smooth muscle cells (SMCs) has emerged as a promising strategy for cell‐based therapy, and tissue engineering has become a crucial means to enhance the effects of cell therapy. However, simultaneous reprogramming of somatic cells into all three cell types or a tissue‐like structure has not been investigated despite its significance. Here, we aimed to directly reprogram fibroblasts into a cardiovascular tissue containing all three cell types, and determined their regenerative effects on infarcted hearts. A combination of miR‐ 208b‐3p with ascorbic acid and BMP4 directly reprogrammed mouse tail‐tip fibroblasts (MTTFs) into a tissue‐like structure referred to as reprogrammed cardiovascular tissue (rCVT), which contains reprogrammed CM‐, EC‐, and SMC‐like cells, and extracellular matrix (ECM). Implantation of this rCVT onto the infarcted mouse heart reduced regional cardiac strains and improved cardiac function. Histological examination showed migration of reprogrammed cells from rCVT into the infarcted hearts. Migrated rECs and rSMCs contributed to vessel formation in combination with host vascular cells. Migrated rCMs initially displayed immature characteristics but became mature over time and formed gap junctions with host CMs. This approach of direct tissue reprogramming can serve as a novel platform for cell‐based therapy and drug discovery.
Next generation immunomodulatory biomaterials to prevent rejection of transplanted stem cells in the infarcted heart
1Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, Regenerative Medicine Program, Physiology and Pathophysiology, University of Manitoba, 2St. Boniface Hospital Research Centre, University of Manitoba, 3nsareen@sbrc.ca
Donor derived allogeneic mesenchymal stem cells (MSCs) from bone marrow and induced pluripotent stem cells derived cardiomyocytes are considered to be the ideal options for heart regeneration and repair after a cardiac injury. The outcome of experimental animal studies and initial clinical trials was positive, and no side effects were observed after transplantation of stem cells. The implanted cells were able to improve heart function. However, the poor survival of transplanted cells in the recipient heart has significantly impacted the translation of allogeneic stem cells‐based therapies towards clinic. We have demonstrated that after transplantation in the infarcted heart allogeneic stem cells become immunogenic and are rejected by the host immune system. In our ongoing studies we are developing strategies to prevent immune‐rejection and improve survival of transplanted stem cells in the ischemic heart. We synthesized and characterized tantalum carbide MXene quantum dots (MQDs). Our data demonstrate that MQDs possess intrinsic immunomodulatory properties and selectively reduce activation of human CD4+IFN‐γ+ T‐lymphocytes and promote expansion of immunosuppressive CD4+CD25+FoxP3+ regulatory T‐cells in an activated lymphocyte population. The MQDs are biocompatible with stem cells. In our studies we also incorporated MQDs into a chitosan‐based hydrogel to create a 3D platform for stem cell delivery to the heart. This composite immunomodulatory hydrogel‐based platform improved survival of stem cells and mitigated allo‐immune responses. These studies highlight the potential of MXene based next generation biomaterials for cardiac tissue engineering and stem cell‐based therapies for cardiac regeneration.
Exosome‐MFGE8 aids in phagocytosis of dead cells and cardiac regeneration and repair
1University of Alabama at Birmingham
Improper phagocytic clearance of dead cells in the injured myocardium impairs inflammation resolution, thus leads to adverse cardiac remodeling and dysfunction. The goal of this study is to determine if mesenchymal stem cell‐derived exosomes (MSC‐Exo) modulates phagocytosis of dead cells after myocardial ischemic injury and to determine the molecular mechanisms therein. We evaluated MSC‐Exo‐ mediated opsonization of apoptotic cardiomyocytes; invitro and invivo effects of mouse MSC‐Exo on macrophage engulfment of apoptotic cardiomyocytes; and its implications on cardiac remodeling, repair and function. Microscopy and FACS analyses show that opsonization of apoptotic cardiomyocytes with MSC‐Exo enhances their engulfment by macrophages. Furthermore, MSC‐Exo pretreatment reprograms phagocytosis‐related signaling in macrophages, therefore leading to increased expression of pro‐ reparative cytokines. Protein analysis of MSC‐Exo reveals expression of MFGE8, a glycoprotein which bridges externalized phosphatidylserine (PS) on the apoptotic cell surface to avb3 or avb5 integrins on the phagocyte. Interestingly, blocking MFGE8 significantly reduces the MSC‐Exo‐mediated dead cell engulfment, associated signaling and pro‐reparative phenotype. Furthermore, in vivo, intramyocardial administration of MSC‐Exo after myocardial ischemic injury increases macrophage uptake of apoptotic bodies in the border zone of infarct and is associated with reduced proinflammatory response, increased angiogenesis and an improvement in cardiac function. Our data suggests that exosome‐MFGE8 augments phagocytosis by two novel mechanisms: i) by enhancing opsonization of dead cells, and ii) by activating phagocytic signaling in macrophages thus leading to efficient removal of apoptotic cells, resolution of inflammation and cardiac recovery after injury.
Injectable neural stem cell‐laden gelatin‐based bioink for 3D bioprinting an in vitro brain tissue model
1Feng Chia University
Recapturing the features of the three‐dimension (3D) multi‐cells‐based environments, such as cell‐cell configurations and interactions in vitro, is important to provide a reliable drug testing platform for brain diseases. However, 2D models as brain disease models need more accurate specific 3D configurations and 3D neuron‐astrocyte interactions. In this study, we developed a 3D brain‐like construct using a 3D bioprinting technique. By this biorprinting technique, a multi‐cells‐laden brain‐like tissue, including neural stem cells, can differentiate into neurons, recapture the synapse functions and possess the ability to recapitulate the neuron‐astrocyte interaction to regulate the glutamate level in the printed brain. Therefore, we supposed that this 3D biomimetic brain model provides better confidence in drug testing results and could reduce the use of animals in the pre‐clinical process.
Neuroprotective and pro‐angiogenic three‐dimensional stem cell spheroids for treating ischemic stroke
1Institute of Biomedical Engineering, National Tsing Hua University
Ischemic stroke is one of the most frequent causes of death and disability globally. Novel therapeutic strategies for enhancing the repair of neural structures and the recovery of functions are urgently warranted. In this work, we aim to employ a stem cell‐based approach for treating ischemic stroke. Three‐ dimensional (3D) spheroids composed of human mesenchymal stem cells (MSCs), which are capable of secreting neuroprotective agents and inducing therapeutic angiogenesis, and human umbilical vein endothelial cells are constructed using a methylcellulose hydrogel system. Our in vitro results demonstrate that the fabricated 3D stem cell spheroids can protect neurons that are under oxygen glucose deprivation. Additionally, the neurite outgrowth from neurons and tubular network formation by vascular endothelial cells are significantly enhanced. Intracerebral transplantation of 3D stem cell spheroids into the mouse model of middle cerebral artery occlusion effectively reduces lesion area, maintain cerebral structural integrity, and improve post‐stroke motor function. In summary, the developed 3D stem cell spheroids possess significant neuroprotective and pro‐angiogenic potential, thus holding a great promise to be employed as an effective therapeutic agent for treating ischemic stroke.
Real‐time monitoring the dynamics of collagen of extracellular matrix secreted during chondrogenesis using fluorescence labeling with azide‐ proline both in single cell and tissue levels
1Ajou university, 2Department of Orthopedic Surgery, School of Medicine, Ajou University, 3Department of Molecular Science & Technology, Ajou University, 4Cell Therapy Center, Ajou University Medical center
In cartilage tissue engineering, it is important to determine the distribution and degradation profiles of implanted engineered cartilage tissues. Observing collagen dynamics is helpful to understand metabolism of cartilage tissues. However, studies of in vivo follow‐up of collagen have not been conducted enough due to limitations of existing labeling methods. Thus, in this study, we used azide‐proline metabolic labeling method because this method is bioorthogonal and suitable for dense and complex tissues as well as cells cultured in vitro. Firstly, to observe the collagen secretion according to releasing time and density of extracellular matrix in a single cell, the cells were encapsulated in agarose and then cultured in chondrogenic medium. Labeled molecules were widely distributed over time and narrowly distributed as the density of the agarose gel increases. Next, we incorporated azide‐proline into the engineered cartilages. Then fluorescence imaging and collagen contents assays were performed. Dependently on time, the amount of collagen and the fluorescence intensity increased correspondingly. To evaluate real‐ time monitoring the azide‐proline‐labeled collagen in engineered cartilage in vivo, we applied it to a nude mouse subcutaneous window chamber model. The amount of collagen also corresponded to the fluorescence intensity. In conclusion, using bioorthogonal and specific labeling methods, collagen synthesis, distribution, and degradation profiles were observed in real‐time at both the single cell and tissue level. The applications presented in this study is expected to be useful for understanding collagen metabolism in various other tissues containing collagen as well as engineered cartilage tissues in vitro and in vivo.
Deconstructing a complex microenvironment with geometrically structured microtumours
1UNSW Sydney
The tumor microenvironment (TME) consists of a complex assortment of multiple cell types and dynamic extracellular matrices. Model systems that mimic aspects of the TME are useful tools for probing signal transduction underlying progression. In this presentation I will demonstrate how mimicking the biophysical and biochemical attributes of the TME in spatially addressable assays can be used to better probe cancer processes. Hydrogel micropatterning can be used to coordinate the interactions of cancer cells and stroma, where confinement on deformable substrates triggers partitioning of different cell types in ways that mimic organisation in vivo. Regions of perimeter curvature are shown to activate fibroblasts to a contractile state, thereby leading to enhanced matrix deposition and corralling of cancer cells to the center. Using a geometric templating approach brings these assays into 3D, with the possibility of directing bi‐directional cross talk with spatiotemporal control. Leveraging biofabrication can further increase the complexity by allowing cancer spheroids, stromal cells and vessel structures to be integrated within a single system. The fabrication of multiple aspects of the TME allows better control over features responsible for progression, thereby providing improved tools for fundamental studies and drug development.
Engineering tumor slice cultures ex vivo for personalized drug testing
1National University of Singapore, 2National Cancer Centre Singapore
Patients with recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) have a median survival of <12 months, typically exhibiting drug resistance. While immune checkpoint inhibitors (ICI, anti‐PD‐1) is approved for use as first‐line treatment, most patients exhibit primary resistance. Being able to accurately identify patients that would respond to PD‐1 inhibition, and more importantly, identify further therapeutic options to overcome resistance are critical unmet needs in HNSCC. Although predictive biomarkers such as PD‐ L1 expression can somewhat predict the response of patients to ICIs, none are robustly predictive. It is likely that the complex and evolving tumor microenvironment determines efficacy in each patient. While patient‐derived organoid cultures are able to recapitulate cancer heterogeneity of patient tumors in vitro, these models are inherently still too simplistic. Patient tumor‐derived tissue slices represent a very attractive alternative as the model potentially preserves, at least transiently ex vivo, the original tissue architecture and composition of the patient's tumor. In this study, we leverage biomaterials to maintain patient‐derived tumor slices to enable derivation of drug responses in real‐time. Through deep characterization techniques including single‐cell RNA sequencing and multi‐dimensional flow cytometry, we show that these engineered tumor slice cultures well‐preserve the architecture, viability and composition of the original tumor for at least 1 week. Further, we show that these models also retain the original gene expression profile of different cell populations, as well as preserve stromal heterogeneity of the original tumor. Lastly, we demonstrate how this platform allows for discrimination of ICI efficacy across different patients.
A physiologically‐relevant in vitro 3D microfluidic model of glioblastoma and the blood‐brain barrier for studying solid tumour heterogeneity, tumour microenvironment and therapy validation
1Institute of Molecular and Cell Biology A‐STAR, 2IMCB A‐STAR
Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults with a dismal prognosis. Currently the only FDA‐approved agents for primary and recurrent GBM are Temozolomide (TMZ) and Bevacizumab, respectively. Unfortunately, TMZ resistance is frequent and Bevacizumab does not improve overall survival. New therapies such as chimeric antigen receptor (CAR)‐T cell therapy have had some promising clinical results, but much work remains to improve their efficacy and minimise toxic side‐effects. Despite the urgent need to develop novel therapies and understand drug resistance, progress is limited by the highly selectively permeable blood‐brain barrier (BBB) and the heterogeneity of GBM. Additionally, most pre‐clinical models do not recapitulate key features of GBM that are relevant to therapy delivery and resistance, leading to many failing at a later stage of testing. We have developed an in vitro 3D model that recapitulates the dense, hypoxic tumour core and pseudopalisading invasive front of GBM, surrounded by a perfusable BBB with a permeability in the same range as in vivo models. The in vitro cultured GBM microtissues are also compatible with downstream analyses such as histology and flow cytometry. With this model, we investigate GBM‐BBB interactions, drug resistance and novel CAR‐ T cell targeting and cytotoxicity. We present data on the impact of our solid tumour model of GBM on vasculature and the effect of TMZ and Bevacizumab on both the GBM and BBB. We also observe CAR‐ T extravasation and targeting in real‐time, providing new insight into CAR‐T cell states while targeting solid tumours.
3D microphysiological systems for pre‐clinical screening of anti‐tumor therapies
1Agency for Science, Technology and Research (A*STAR)
Although the need for better anticancer therapeutic approaches is evident, about 97% of all oncological drugs in clinical trials are not approved by the FDA. This scenario emphasizes the urgency to improve the preclinical testing of anticancer strategies before they reach the clinical trials to lessen the social and financial burden worldwide. In the past decades, most preclinical research studies have focused on 2D cell culture or in vivo studies, with scarce success in translation to the clinic. However, animal studies are technically challenging and present several limitations, including the cost of animal maintenance, animal ethics, and species differences could often translate into ineffective therapeutics for humans. On the other side, recent studies prove that 2D cell culture does not adequately mimic the cellular and matrix interactions that contribute to the different tumor responses to the therapeutics. Therefore, we develop 3D tumoroids that simulate important TME characteristics to represent better drug resistance and aid in developing effective anticancer therapeutic strategies. 3D culture systems, such as tumoroids, are currently considered very promising for preclinical screening as they can incorporate a variety of cells and extracellular matrix proteins to assure the appropriate structural support to cells to maintain their spatial distribution within the tumor microenvironment. Embedding 3D tumoroids in a microfluidic device provides additional advantages, such as (i) application of gradients of cytokines; (ii) control of ECM stiffness; (iii) reduction of in vitro artifacts such as the gravity‐mediated interaction between cells and (iv) possibility to control fluidic perfusion of the sample.
Recapitulating intratumoral morphological heterogeneity in a bioprinted breast cancer model for personalized medicine
1Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), 2Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard
In vitro cancer models simulating patient‐specific drug responses for personalized medicine are receiving significant attention. However, the conventional methods are hard to recapitulate the heterogeneous morphology in patient cancer tissue, called intra‐tumoral morphological heterogeneity, which eventually led to difficulties in predicting drug responses. Here, we developed a novel 3D bioprinting technique to produce an in vitro breast cancer model with intratumoral morphological heterogeneity in a personalized manner. It allows to precisely mimic the ductal and solid cancer microstructures, the representative morphologies of breast cancer, obtaining similar drug responses to those of humans. The bioprinted ductal and solid cancer cell aggregates showed similar phenotypic characteristics of early and advanced breast cancers, respectively. The bioprinted solid cancer aggregates showed significantly higher hypoxia (>8 times), mesenchymal (>2‐4 times) marker expressions, invasion (>15 times), and drug resistance than the ductal aggregates. Co‐printing the ductal and solid aggregates produced heterogeneous breast cancer tissue models that recapitulated the different stages of breast cancer. The models with a high proportion of solid aggregates, representing the advanced cancer stage, showed higher resistance to doxorubicin and lower resistance to tirapazamine, which are analogous to that of humans. Interestingly, our results show that the spatial organization of the ductal/solid cancer aggregates has a significant effect on drug responses, even though the model recapitulated the same cancer stages. This study demonstrates that cancer models that mimic the morphological pathology of human breast cancer can be bioprinted to produce an in vitro patient‐specific breast cancer tissue representing a patient's clinical cancer stage.
When cancer cells leave their comfort zone: Phenotype switching of breast cancer cells upon crossing tissue interfaces
1Institute of Biochemistry, Leipzig University, 04103 Leipzig, Germany, 2Laboratory for Immuno Bioengineering Research and Applications, Division of Engineering, New York University Abu Dhabi, Abu Dhabi, UAE
Breast cancer tissue and its microenvironment pose distinct cellular and ECM compartments with different density of fibrillar collagen. Specifically, a dense ECM compartments is frequently observed within tumour tissues, whereas the adjacent tissue poses as a less dense compartment. Recent data emphasise a correlation of the material characteristics of the tumour extracellular matrix (ECM) and the invasive properties of tumour cells and even a trigger of tumour aggressiveness. Herein, we report that the invasiveness and aggressiveness of breast cancer cells not only depend on the material characteristics of homogeneous ECM, but may dramatically change during the transmigration of cells across interfaces of two different matrices. Using an in vitro (three‐dimensional) 3D model of defined collagen matrices, we found strong changes in cell morphology, migration directionality, proliferation and transcriptome profile when cells cross a matrix interface from dense to open network microstructure. Moreover, our results indicate stress‐induced nucleus deformation and DNA damage during transmigration of the matrix interface as a possible trigger of the more aggressive phenotype. Our findings suggest that distinct tissue interfaces or altered ECM conditions with differences in ECM microstructure may instruct or even reprogram tumour cells towards more aggressive phenotypes also in vivo. The biomedical relevance of our results is corroborated by additional findings that the transmigrated cells exhibit an increased resistance against a common breast cancer therapeutic.
Poster Sessions
SYIS Poster Sessions
Extracellular vesicles derived from fibroblasts promote wound healing by optimizing fibroblast and endothelial cellular functions
1Kyungpook National University Hospital, 2Kyungpook National University
Extracellular vesicles (EVs) have been exhibited as promising candidates for delivering endogenous therapeutic cargos for regenerative therapies. Fibroblasts could be candidate source cells for EVs, to investigate their therapeutic effects in wound healing. Here we demonstrated the isolation and characterization of fibroblast‐derived (L929 cell line) EVs (L929‐EVs). Furthermore, L929‐EVs treatment showed pro‐wound healing effects in vitro by enhancing proliferation, migration, and scarless wound healing related genes in fibroblast cells. L929‐EVs treatment also enhanced the migration and tube formation of endothelial cells. The combination of L929‐EVs with fibrin glue accelerated wound healing in the mouse skin wound model by enhancing collagen formation, collagen maturation, and blood vessels in the wounded skin. The role of fibroblast‐derived EVs in wound healing could be an important phenomenon, and fibroblast‐derived EVs could be harnessed for wound healing therapies.
Which medium is most suitable for the production of extracellular vesicles for therapeutics?
1Xcell Therapeutics Inc.
Mesenchymal stem cells (MSCs) are multipotent adult cells originate from the mesoderm. MSCs possess the capabilities of self‐renewal, differentiation, colony‐forming, and are gradually gaining attention in regenerative medicine. However, there are some critical issues that need to be addressed before MSCs can be used for clinical therapy in humans, most important of which is the safety issue such as immune reaction and low stability. Recently, numerous studies on ‘alternative treatments' have been reported to solve the side effects of stem cell therapy, and many studies have reported that tissue regeneration is induced by exosomes secreted by MSCs rather than stem cell therapies.
Exosomes are secreted as exosomal protein components that contain cell identity. To uniformly secrete exosomes containing active ingredients, cells itself must be in healthy condition. For effective pharmaceutical research, only the target cell‐derived exosomes must be produced and separated, but contamination of animal/human component‐derived exosomes already contained in cell culture media has reduced the purity of exosomes.
Therefore, this study aims to introduce the most ideal culture environment for the production of exosomes by cultivating cells in a serum‐free chemically defined medium (CDM) and confirming the characteristics of secreted exosomes. By cultivating cells in animal‐derived components free, it is possible to improve the quality of the stem cell culture environment and obtain ‘human stem cell derived exosomes' that completely exclude animal‐derived exosomes from the cells.
Overall, this study shows that serum‐free chemically defined media lead to the best stem cell, stem cell‐ derived exosomes you want to get.
Anti‐inflammatory and immune‐modulation effects of exosomes derived from human fetal cartilage progenitor cells (hFCPCs) in vitro
1Inha university, 2Ajou University, 3Pukyong National University
We investigated anti‐inflammatory and immune‐modulation effects of exosomes derived from hFCPCs primed with different methods (unprimed vs IL‐1b treatment, hypoxia, and 3D culture) in comparison with those of human bone marrow mesenchymal stem cells (hBM‐MSC). Conditioned medium was collected and exosomes were isolated using the ExoQuick isolation kit. Exosomes from hFCPCs and hBM‐MSCs showed round morphology in TEM and similar patterns in size distribution. Both exosomes showed clear expression of CD81 and CD63 but very low levels of CD9. Non‐primed hFCPCs exosomes decreased the expression of IL‐1β, IL‐6, and MCP‐1 in IL‐1b‐treated SW982 cells. Primed exosomes showed stronger inhibitory effects, particularly in the hypoxia and hypoxia/IL‐1b combination groups. hBM‐MSCs exosomes showed similar results in both non‐priming and priming groups. hFCPC exosomes suppressed proliferation of activated PBMCs in MTT assay and induction of IFNg levels by ELISA, while they increased the expression of IL‐10 both in non‐priming and priming groups except the IL‐1β priming group.
Through miRNA sequencing data of exosomes, we could identify miRNA differentially expressed (DE) in hFCPCs. The number of miRNAs with significant DE in hFCPCs versus BM‐MSCs was 291 (99 up‐ regulated & 192 down‐regulated). Among the priming groups versus non‐priming control in hFCPCs, the number of DE miRNA in exosomes was 72 (52 up‐regulated & 20 down‐regulated) by IL‐1b priming, 64 (36up‐regulated & 28 down‐regulated) by hypoxia and 224 (119 up‐regulated & 105 down‐regulated) by 3D culture. We are currently carrying out confirmation and investigate role of DE miRNAs of exosomes in their anti‐inflammatory and immune‐modulation responses.
In vivo migration of mesenchymal stem cells to burn injury sites and their therapeutic effects in a living mouse model
1Department of Plastic and Reconstructive Surgery, School of Medicine, Kyungpook National University, Daegu, South Korea, 2KyungPook National University
Mesenchymal stem cell (MSC)‐based therapy has emerged as a promising therapeutic strategy for tissue regeneration and repair. In this study, we non‐invasively monitored the tracking of MSCs toward burn injury sites using MSCs expressing firefly luciferase (Fluc) gene in living mice, and evaluated the effects of the MSCs at the injury site. Murine MSCs co‐expressing Fluc and green fluorescent protein (GFP) were established using a retroviral system (referred to as MSC/Fluc). To evaluate the ability of MSC migration toward burn injury sites, cutaneous burn injury was induced in the dorsal skin of mice. MSC/Fluc was intravenously administrated into the mice model and bioluminescence imaging (BLI) was performed to monitor MSC tracking at designated time points. BLI signals of MSC/Fluc appeared in burn injury lesions at 4 days after the cell injection and then gradually decreased. Immunoblotting analysis was conducted to determine the expression of neovascularization‐related genes such as TGF‐β1 and VEGF in burnt skin.
The levels of TGF‐β1 and VEGF were higher in the MSC/Fluc‐treated group than in the burn injury group. Our observations suggested that MSCs might assist burn wound healing and that MSCs expressing Fluc could be a useful tool for optimizing MSC‐based therapeutic strategies for burn wound healing.
Shear stress‐driven exosome production and transcriptomic analysis in 3D cultured osteocytic cells
1Kyungpook National University
Appropriate exercise promotes bone health. Dynamic loading generates shear stress on the osteocyte, which in turn stimulating exosomes secretion. Previous reports show that osteocyte‐derived exosomes are involved in the regulation of bone metabolism. However, the underlying molecular mechanism of shear stress‐induced exosome release is still unclear. Here, we established a 3D culture system that partially mimics endogenous conditions and analyzed exosome production patterns and the gene expression changes in response to shear stress. We subjected different shear stress levels in osteocyte and found that as the shear stress level increases, more exosomes are released. Also, exosome genes were increased accordingly. Therefore, we analyzed differential gene expression in the osteocyte stimulated with high shear level compared to static control. Through RNA sequencing‐based transcriptome profiling, we identified 325 up‐regulated and 561 down‐regulated genes. Gene ontology (GO) analysis, KEGG analysis revealed that sterol biosynthetic process and organic acid transmembrane transport were increased. Inhibition of sterol biosynthesis with specific inhibitors substantially suppressed exosome production. In summary, we characterized new shear stress response gene sets in osteocyte, and these results may provide important information on the molecular mechanism of the mechanical stimulation‐driven exosome production.
Effect of cytokine stimulation on human mesenchymal stem/stromal cell responses to exosome secretion
1Faculty of Medicine, Chulalongkorn University
Mesenchymal stem/stromal cell‐derived exosomes are currently evaluated as subcellular therapeutics via functions of proteins and genetic materials in their cargos. The possible way to enhancement of medical treatment efficacy is regulation of functional elements in the exosome vesicles relating to mechanism of action (MoA) to each targeting disease. This study shows the influence of extra cytokines (e.g. IFN‐γ, TNF‐α, Interleukins) on activation of mesenchymal stem/stromal cells (MSCs) to response the particular environment by exosome secretion with specific properties. The exosomes from primed culture conditions were investigated their production size and number as well as the cargo components such as microRNAs, TGF‐β, enzymes, cytoskeleton and transmembrane proteins. Furthermore, this study also presents immunomodulatory effects on promotion of macrophage M2 polarization induced by exosomes from cytokine stimulated culture conditions. In conclusion, the results of this study would be useful for fundamental information of MSC activation for biological drug development of exosome characteristics that can be applied for therapeutic applications and regenerative medicine industry.
Exosome derived from human adipose stem cells (ASC) exosome exert therapeutic effect on inhalation injury with caused by burns in vitro
1Burn Institute, Hallym University
Inhalation burn can lead to bronchoconstriction, airway edema, asthma, bronchiolitis obliterans, and carbon monoxide cuts off the body's oxygen supply, causing severe hypoxia. However, there is no specific treatment to care inhalation burn except for treatments such as relieving bronchospasm and cleaning the airways. Therapeutic applications of stem cell derived‐exosome have been reported in different diseases like cardiovascular, heart, kidney, liver, neurological diseases, and wound healing. In particular, ASC have a similar differential potential and regenerative capacity where the yield of stem cells is higher than that of bone marrow MSC. The purpose of this study is to assess the therapeutic effects of ASC‐derived exosomes by inducing the regeneration of damaged respiratory cells. We tested whether ASC exosomes preserve the respiratory cells from damages by the SO2 derivatives. ASC exosomes increased cell viability at the Human Pulmonary Microvascular Endothelial Cells (HPMECs) compared with the group treated only SO2 derivatives. Also, total tubule length was increased by the ASC exosomes at the in vitro angiogenesis assay. Next, we confirmed cell viability and expression of inflammation markers depend on SO2 derivatives at the primary respiratory cells, like Human Bronchial Epithelial Cells (HBEpCs) and Human Nasal Epithelial Cells (HNEpC). The results showed that cell viability was recovered by the ASC exosome at HBEpCs and HNEpCs. Moreover, expression of inflammation markers which was increased by the SO2 derivatives were decreased through the treatment of ASC exosomes. Thus, these results provide that ASC exosomes can regenerate damages of the respiratory cells caused by SO2 derivatives.
Bioengineered airway organoids using a decellularized extracellular matrix for augmented regeneration of tracheal injuries
1Department of Biomedical Convergence, School of Convergence, Kyungpook National University
Surgical reconstruction of trachea has been considered as one of the greatest challenges due to the distinctive anatomical features of trachea including anatomic location, lengths, structural complexity and blood supply. Transplantation of organoids has emerged as a powerful approach to regenerate the tissue injuries by their retained ability to rebuild the tissues of origin. However, Matrigel, an indispensable component of most organoids cultures has restricted the applicability of organoids for transplantation due to its tumor‐derived origin, ill‐defined and batch‐to‐batch variable composition, and high cost. Here, we propose bioengineered airway organoids using a lung‐derived decellularized extracellular matrix (dECM) as a suitable alternative of Matrigel with the native, lung‐mimetic microenvironments including composition, biochemical and mechanical properties. The development and function of airway organoids grown in dECM hydrogels are comparable to those in Matrigel in terms of morphological features and expression profiles. Thus, our bioengineered airway organoids based on lung‐mimetic dECM hydrogels can be a novel treatment option for tracheal injuries in regenerative therapeutics.
Mouse liver organoids culture under in vivo‐comparable steady‐state flow condition support hepatic functions in mice with acute liver failure
1Asan Medical Center
It is imperative to develop in vitro culture platform that can capture the in vivo cell behavior and provide more physiologically relevant results to in vivo state. Here we present a microfluidic system provides an environment for liver organoids that mimics physiological relevant conditions including oxygen and nutrient supply. In vivo‐comparable steady‐state flow condition which can control the expansion and differentiation of mouse liver organoids within 10‐12 days and shows both phenotypic and functional enhancements in the mouse adult stem cell‐derived liver organoids. To test the ability of the liver organoids to engraft as functional hepatocytes in vivo, we transplanted the mouse liver organoids into the liver of mice undergoing extended hepatectomy (90%). Upon transplantation, there is an improvement in animal survival and biochemical liver function through engraftment of the liver organoids. These results demonstrate the potential of a cell therapy leading to the rescue of animals from acute liver failure.
Generation of a hair follicle‐like organoid model comprising human immortalized cell lines
1Kyungpook National University
Recently, a polar elongated structure was generated using a three dimensional (3D) aggregation of human dermal papilla (DP) cells co‐cultured with human outer root sheath (ORS) cells in an ultra‐low attachment plate. The “two‐cell assemblage (TCA)” assay could serve as a simple in vitro method for screening and validating the hair growth‐promoting compounds. However, it is tedious and tricky to obtain primary cultured DP cells and ORS cells from hard‐to‐obtain human biopsies. In this study, we established a hair follicle‐like organoid model using spheroids made of an immortalized human DP cell line, SV40T‐ hTERT‐DPC, surrounded by Ker‐CT, an immortalized human keratinocyte cell line. We observed that the TCA structures elongated in a unipolar manner mimicking a physiological hair follicle in vivo.
Bioengineered intestinal organoids using a tissue‐adhesive biopolymeric hydrogel for reconstruction of intestinal injuries
1Department of Biomedical Convergence, School of Convergence, Kyungpook National University
Crohn's disease, a type of inflammatory bowel disease, can often lead to loss of intestinal function due to impaired intestinal barrier performance and exposure to noxious antigens that cause chronic inflammation. Organoids transplantation has emerged as a promising strategy for restoration of the damaged intestinal epithelial barrier in treating several intestinal diseases. However, most organoids culture commonly relies on the use of an ill‐defined tumor‐derived basement membrane extract, Matrigel, which restricts practical applications to transplantation with aim of regenerating tissues in vivo. Here, we develop tissue‐adhesive hyaluronic acid (HA) hydrogels for generation and in vivo transplantation of intestinal organoids as an alternative substitute to Matrigel. The morphological and functional features of intestinal organoids cultured in the well‐defined, biocompatible HA hydrogels are comparable to those in Matrigel by providing extracellular matrix‐mimetic microenvironments. Moreover, superior tissue‐ adhesion ability of catechol‐functionalized HA facilitates their use in transplantation of intestinal organoids to target tissues even under wet conditions. These results suggest the possibility of only a single hydrogel in generating and transplanting intestinal organoids to accomplish an effective recovery of small intestinal wounds. Therefore, our chemically‐defined, tissue‐adhesive HA hydrogels can be a novel, simple therapeutic platform for organoids‐based regeneration of intestinal injuries with further expansion to general tissue recovery.
High‐throughput endometrial organoids screening platform using decellularized extracellular matrix microgels
1Department of Biomedical Convergence, School of Convergence, Kyungpook National University
Endometrial diseases, such as Asherman's syndrome and endometrial cancer, are the main cause of infertility. Numerous therapeutic drugs have been developed to overcome the endometrial disease‐derived infertility, which leads to the increasing necessity of highly accurate screening for new drugs. Organoids, in vitro three‐dimensional self‐assembled constructs that mimic the structural and functional features of the corresponding organ, have emerged as a reliable test‐bed to evaluate therapeutic performance and safety of drugs. However, the practical application of organoids‐based drug screening remains challenges due to the undefined composition and batch‐to‐batch variance of the most common option for organoids culture, Matrigel. Here, we propose a microengineered endometrial organoids platform using a decellularized extracellular matrix (dECM) as a reproducible test‐bed for the screening of infertility‐ associated drugs. The uniform endometrial‐mimetic microgels were generated by thermo‐responsive crosslinking of dECM within a microfluidic device. The endometrial organoids cultured in dECM microgels exhibit phenotypic and genotypic features specific to endometrial epithelium, while showing the high level of homogeneity. Moreover, we confirm the hormone‐responsiveness of dECM‐derived endometrial organoids, suggesting the reliability to recapitulate functional features of endometrial tissues. Taken together, our dECM‐derived endometrial organoids can be a powerful high‐throughput screening platform of drugs for infertility treatment with further expansion to precise personalized medicine.
Effect of microplastics on blood‐brain barrier (BBB)
1KIST, 2School of Integrative Engineering, Chung‐Ang University
Microplastics, which refer to small plastic particles of 5 mm or less, quietly occupy the world. Extensive pollution has already been confirmed, from Everest, the highest mountain in the world, to polar regions such as the Antarctic and Arctic. Plastic has not rotted for hundreds of years and is constantly worn and split. Because of its small size, microplastics can be easily inhaled by humans because they are not only mixed with food or water, but also float as particles in the air. The human toxicity of microplastics has not yet been clearly demonstrated. However, particles enter the blood, such as contact with the human respiratory system and epithelial cells, and rotate throughout the body, affecting sensitive organs, leading to cell damage, neurotoxicity, and early death. We focused on the effects of PS particles, which are different in shape from secondary polystyrene (PS) particles, and investigated the effects of these microplastic particles on human health at the cellular level. We evaluated how microplastics pass through the blood‐brain barrier (BBB) and how much they are eaten by cells depending on their size. They also evaluated how it affects other cells around them when they penetrate blood vessels. Discuss the impact of particle size to understand the risks microplastic particles may pose to humans and to provide recommendations for future research.
Modeling myocardial infarction using multi‐cellular cardiac organoids from hiPSCs
1Department of Commercializing Stem Cell Technology, NEXEL Co.,Ltd
Myocardial infarction (MI) is an irreversible myocardial injury resulting in high morbidity and mortality. Many cell‐based cardiac in vitro models have been reported as a complementary approach to preclinical research, however, most approaches are not able to mimic human MI pathology. In this study, we generated a self‐organizing cardiac organoid (COs) from human induced pluripotent stem cells (hiPSCs) with cardiomyocytes, fibroblasts, and endothelial cells (multi‐cellular COs) that recapitulates cellular compositions of the human heart. To model MI, the multi‐cellular COs were cultured in the presence of cobalt chloride (CoCl2), a hypoxia mimetic agent, and we confirmed the increased expression of MI markers at the mRNA and protein levels. Furthermore, the cardiac damage by the treatment of CoCl2 resulted in the defective calcium influx and contractility of the COs with cardiac fibrosis. These results have important implications for the application of in vivo‐like 3D heart and disease modeling, and these MI‐CO models might provide a promising alternative to an animal experimental model for studying cardiac disease, as well as a drug screening system for finding therapeutic targets.
Bone organoid model for studying bone metabolism and disease
1MyongJi Hospital, New Horizon Biomedical Engineering Institute, 2MyongJi Hospital, Hanyang
University College of Medicine, 3Ewha Womans University, College of Medicine, 4MyongJi Hospital, Department of Orthopaedic Surgery
Bone formation is a complex process in which cell differentiation and the generation of mineralized organic matrix occur simultaneously to create bone hierarchical architecture. A functional bone model system that simultaneously captures cellular and matrix formation processes is essential to study the mechanisms of bone formation metabolism and bone disease. Therefore, in this study, bone marrow‐ derived stem cells (BM‐MSC) and xenograft bone decellularized extracellular matrix (B‐dcECM) sponge‐ based bone organoids were implemented to evaluate their potential as bone tissue regeneration and functional in vitro model systems. BM‐MSC‐adherent B‐dcECM sponge were cultured in growth medium (GM) and/or osteogenic differentiation medium (OM) under static and/or shear stress condition, respectively. Cell proliferation was inhibited in the group treated with shear stress and OM at the same time but was improved in the group to which only shear stress. Newly synthesized collagen and calcium production was confirmed to be improved to a statistically insignificant difference compared to the group to which both shear stress and OM induction and only shear stress. Also, pore channels like cancellus bone were observed in the group to which only shear stress through electron microscopy and histological observation. This study was confirmed that the bone organoid structure could be implemented only with the structure of B‐dcECM sponge and the shear stress, and it can be used as a tool to verify the applicability of bone formation mechanism research and bone disease model platform.
Development of a hypoxia‐enhanced kidney organoids model that recapitulates human renal tubular structure and function
1Ulsan National Institute of Science and Technology, 2Pohang University of Science and Technology
Since the first reports of kidney organoids derived from human induced pluripotent stem cells(iPSCs), many protocols for the generation of kidney organoids, which differ in terms of growth factors and small molecules used and culture duration, have been described. The kidney organoids recapitulating complex function of nephron have provided great insights into understanding kidney diseases. However, current protocols cannot create kidney organoids having full‐nephron structure and function, because a developmental lineage of collecting duct is different from other nephron segments. In this study, we developed a protocol for generation of kidney organoids with enhanced formation of collecting ducts using a developmentally inspired induction procedure that includes a period of differentiation in hypoxic stimuli. In early developmental environment of embryo, hypoxic condition is an important factor to induce collecting duct formation. The modulation of WNT signal due to the accumulation of hypoxia inducible factor‐1 in vitro effectively enhanced the generation of 2.5 dimensional tubular kidney organoids. Transcriptome analysis revealed remarkable upregulation of collecting duct markers as well as general tubular markers. The hypoxia‐stimulated kidney organoids exhibited branched renal tubules, similar to adult kidney arcades observed in deep cortex in human adult kidney tissue, indicating that our protocol successfully induced the structural and functional maturation. In addition, kidney organoids having elongated tubules showed higher sensitivity to forskolin used for modeling cystic kidney diseases and higher expression of cystic fibrosis transmembrane conductance regulator. This enhanced kidney may therefore represent a reliable in vitro tool for the study of human kidney development and disease.
Establishment of liver organoid system for comprehensive evaluation of post‐metabolic activity of endocrine‐disrupting chemicals
1Department of Agricultural Biotechnology, Seoul National University, Seoul, 08826, Republic of Korea, 2Department of Food Science and Biotechnology, Chung‐Ang University, Anseong, 17546, Republic of Korea, 3Department of Molecular and Cellular Biology, Sungkyunkwan University School of Medicine,
Suwon, Gyeonggi, 16419, Republic of Korea, 4Department of Agricultural Biotechnology, Seoul National University, Seoul, 08826, Republic of Korea / Center for Food and Bioconvergence, Seoul National
University, Seoul 08826, Republic of Korea / Research Institute for Agricultural and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea
Endocrine‐disrupting chemicals (EDCs) interfere with endocrine functions, which leads to serious diseases such as cancer, disorders of sexual development (DSDs), obesity, and diabetes. Organization for Economic Co‐operation and Development (OECD) guidelines for EDC detection mainly depends on in vitro and in vivo experiments.
With increasing interest at animal welfare and the 3R (Replace, Reduce, Refine) principal, the alternative method to animal experiment has been proposed. S9 fractions and microsomes have been studied for liver model, but the models do not fully mimic the liver metabolism. Organoid model is a three‐dimensional cell complex that retains similar structures and functions with in vivo tissues.
Accordingly, we applied liver organoid system for exploring hepatic metabolism of EDCs. The metabolic profiles were coupled to comprehensive monitoring of hormonal receptor binding affinity (e.g., estrogen and androgen). Novel identification approach was implemented to identify the major contributors to the activity change, including knowledge‐based product prediction and data‐dependent molecular networking. Indeed, we characterized unique metabolic profiles of E2 and DHT bio‐transformed by the organoid system, which were well fitted to the dynamics of the activity changes.
Our current study proposes an EDC evaluating system by integrating liver organoids technology and metabolomics, which may replaces animal experiments.
Retinoic acids reciprocally regulate the luminal and acinar cell differentiation via the RAR‐TGFβ signaling in adult salivary gland organoids
1Department of Otorhinolaryngology, Yonsei University College of Medicine
Glutamate‐inducing MAO‐B as a critical modulator for the astrocytic scar in a human glioblastoma microenvironment organoid and xenograft mouse model
1Institute of Quantum Biophysics, Sungkyunkwan University, 2Center for Cognition and Sociality, Institute for Basic Science, 3Department of Nuclear Medicine, Yonsei University College of Medicine
Glial scar formation is an innate immune response confining injured regions in a central nervous system. It is reported that the astrocytic scar is formed around the glioblastoma (GBM). However, the mechanisms underlying the scar formation in response to GBM is still under investigation. Here, we constructed a human 3D triculture and a mouse xenograft model of the astrocytic scar‐GBM. GBM spheroids were preformed and then co‐cultured with glial cells including microglia and astrocytes in Matrigel. For the xenograft model, U87‐MG cells were subcutaneously injected to the mouse. Activated astrocytes formed a glial scar surrounding the GBM in a polarized shape, while microglia were found inside and outside the tumor in both in vitro and in vivo model. GBM‐deprived IL‐6 induced microglia to M2‐like phenotype and the microglia inducing by IL‐6 released more glutamate to the GBM microenvironment. The glutamate originating from GBM and microglia upregulated the expression of astrocytic monoamine oxidase B (MAO‐B) and chondroitin sulfate proteoglycans (CSPGs), a major brain extracellular matrix released by reactive scar tissues. The astrocytic scar was inhibited with the treatment of glutamate transporter inhibitor (TBOA) or MAO‐B inhibitor (KDS2010) and the scar inhibition increased the drug sensitivity and consequently inhibit GBM growth. Taken together, we envision that our recapitulated brain tumor microenvironment could serve as a platform for assessing therapeutic strategies crossing the astrocytic scar barrier.
In vivo oxygen and temperature for ideal culture condition of human nasal inferior turbinate derived stem cells in human nose
1Catholic university of Korea
Huma nasal turbinate derived mesenchymal stem cells(hMSCs) have been considered as an potent and useful source for regenerative medicine. To most effectively mimic the native environment of inferior turbinate could be very effective to hTMSCs biology. Thus the purpose of this study was to evaluate partial pressure of oxygen (ppO2) and temperature in inferior turbinate.
Ten patients were enrolled who underwent endoscopic endonasal transsphenoidal skull base tumor surgery. The commercially available OxyLab pO2 monitor gauges the ppO2 and temperature using a fluorescence quenching technique. Also hTMSCs were isolated from 10 patients and cultivated under hypercapnic condition (5%, 10%, and 15%) to mimic hypoxic intranasal conditions.
The measured oxygen concentration in submucosa tissue was higher than that at surface of the inferior turbinate and the temperature in submucosa tissue was higher than the value at surface of inferior turbinate. There was a statistical difference between surface and submucosa tissue value of inferior turbinate. The patterns of proliferation were significantly different according to hypercapnic cultivation conditions (Figure. 4) and there was statistically significant decreased proliferation rate after the exposure of higher CO2 over a period of 5 days.
ntransal turbinae tissue showed the hypoxia state in concordance with the result of the other tissue or organ. However, indirectly induced hypoxia influenced the influence on the hTMSCs proliferation negatively. Further study is needed to mimic the real hypoxic state but our results could be used to optimize the culture environment of hTMSCs, thereby producing the stem cells for regenerative therapies.
Optimising somatic cell reprogramming outcomes through mechano‐ modulation
1The University of Queensland, 2CSIRO, 3Einstein College of Medicine, 4Monash University
The mechanics of the cellular microenvioment is a well‐known modulator of stem cell fates. However, the effect of matrix mechanics on the pathway to and efficiency of somatic cell reprogramming to induced Pluripotent Stem Cells (iPSC) remains largely unexplored. In this study, we assessed the influence of ECM‐conjugated polyacrylamide hydrogels of varying stiffness on mouse and human fibroblast reprogramming. Mouse embryonic fibroblasts (MEFs) containing a Dox‐inducible reprogramming transcription factor (OKSM) cassette with endogenous Oct4‐driven GFP expression were first investigated. Among the hydrogels assessed, a specific stiffness produced up to a 4‐fold increase in Oct4 GFP+ miPSC colonies compared to the TCP ‘gold standard’ control. This hydrogel was then assessed for its efficacy in improving Sendai virus‐based human dermal fibroblast reprogramming, confirming that the same hydrogel produced much larger, tighter dome‐shaped ALP+ hiPSC colonies compared to the TCPS, and resulted in 10‐fold higher yields of hiPSCs. Cell growth, phenotype evolution, and gene expression analysis of cells undergoing reprogramming (by FACS) indicated significant differences in reprogramming kinetics on hydrogels. Time‐course RNA‐Seq and subsequent bioinformatic analysis revealed differentially regulated signaling pathways at early and late time points on hydrogels, including many known (but also new!) to reprogramming. This study confirmed that our ‘soft’ culture environment produced higher quality, more naïve‐like iPSCs with improved kinetics, offering a novel approach to address some critical deficiencies in current iPSC biomanufacturing.
Dormant state of quiescent neural stem cells links Shank3 mutation to autism development
1DONGGUK UNIV
Autism spectrum disorders (ASDs) are common neurodevelopmental disorders characterized by deficits in social interactions and communication, restricted interests, and repetitive behaviors. Despite extensive study, the molecular targets that control ASD development remain largely unclear. Here, we report that the dormancy of quiescent neural stem cells (qNSCs) is a therapeutic target for controlling the development of ASD phenotypes driven by Shank3 deficiency. Using single‐cell RNA sequencing (scRNA‐seq) and transposase accessible chromatin profiling (ATAC‐seq), we find that abnormal epigenetic features including H3K4me3 accumulation due to up‐regulation of Kmt2a levels lead to increased dormancy of qNSCs in the absence of Shank3. This result in decreased active neurogenesis in the Shank3 deficient mouse brain. Remarkably, pharmacological and molecular inhibition of qNSC dormancy restored adult neurogenesis and ameliorated the social deficits observed in Shank3‐deficient mice. Moreover, we confirmed restored human qNSC activity rescues abnormal neurogenesis and autism‐ like phenotypes in SHANK3‐targeted human NSCs. Taken together, our results offer a novel strategy to control qNSC activity as a potential therapeutic target for the development of autism.
Metabolic switch in human bone marrow stromal/stem cells and discovery of NR2F1 gene that can promote cell survival and osteogenic differentiation under a glucose‐deprived microenvironment
1Dongguk university, 2Dongguk University Ilsan Hospital
Implanted cells undergo rapid death in an ischemic environment largely because of hypoxia and metabolic stress from glucose deficiency. Understanding the intracellular metabolic processes and finding genes that can improve cell survival in these inhospitable conditions are necessary to enhance the success of cell therapies. Thus, the purpose of this study was to investigate changes of metabolic profile in glucose‐deprived human bone marrow stromal/stem cells (hBMSCs) through metabolomics analysis and discover genes that could promote cell survival and osteogenic differentiation in a glucose‐deprived microenvironment. Metabolomics analysis was performed to determine metabolic changes in a glucose stress metabolic model. In glucose‐deprived osteogenic differentiation, reliance on tricarboxylic acid cycle (TCA)‐predicted oxidative phosphorylation instead of glycolysis as the main mechanism for energy production in osteogenic induction. By comparing differentially expressed genes between glucose‐ deprived and glucose‐supplemented hBMSCs, NR2F1 (Nuclear Receptor Subfamily 2 Group F Member 1) gene was discovered to be associated with enhanced survival and osteogenic differentiation in cells under metabolic stress. NR2F1 overexpression enhanced osteogenic differentiation and cell survival of hBMSCs in glucose‐deprived osteogenic condition via AKT/ERK pathway. NR2F1‐transfected hBMSCs significantly enhanced new bone formation in a critical size long bone defect of rats compared with control‐vector transfected hBMSCs. In conclusion, the results of this study provide an understanding of the metabolic profile of implanted cells in an ischemic microenvironment and demonstrate that NR2F1 treatment may overcome this deprivation by enhancing AKT and ERK regulation. These findings can be utilized in regenerative medicine for bone regeneration.
Discovery of GSTT1 gene that can promote osteogenic differentiation of adipose stem cells
1Research Institute for Integrative Regenerative Biomedical Engineering, Dongguk University,
2Department of Orthopaedics, Dongguk University Ilsan Hospital
In this study, we describe the discovery of a new GSTT1 gene related to the osteogenic differentiation of adipose stem cells. To treat avascular necrosis and critical bone defects using adipose‐derived stem cells by improving bone regeneration capacity, the osteogenic differentiation ability of each adipose stem cell was first identified. We divided these cells into two groups according to their osteogenic differentiation ability, and then mRNA‐seq analysis was performed. We found a specific gene, GSTT1, with a large difference in gene expression between the group with good osteogenic differentiation and the group with weak osteogenic differentiation through mRNA‐seq gene analysis. The glutathione S‐transferase theta‐1 (GSTT1) gene is a member of a protein superfamily that catalyzes the conjugation of reduced glutathione to a variety of hydrophilic and hydrophobic compounds. To date, there are no known studies relating to the GSTT1 gene in the field of bone regeneration. In vitro studies showed that the GSTT1 gene was associated with the enhancement of osteogenic differentiation of adipose stem cells. siRNA against GSTT1 reduced osteogenic differentiation of hADSCs, whereas GSTT1 overexpression enhanced osteogenic differentiation of hADSCs under osteogenic differentiation conditions. In conclusion, our studies suggest that transgenic adipose stem cells using this GSTT1 gene could be used in regenerative medicine to improve bone differentiation effectively. Furthermore, the GSTT1 gene has important significance as a genetic marker for pre‐screening adipose stem cells with the potential for osteogenesis in the development of cell‐based treatments for bone regeneration.
Application of skeletal muscle cell differentiated from human tonsil‐ derived stem cells for Duchenne muscular dystrophy
1Department of Biochemistry, College of Medicine, Ewha Womans University, Seoul 07804, Republic of Korea, 2Graduate Program in System Health Science and Engineering, Ewha Womans University, Seoul 07804, Republic of Korea
Skeletal muscle is one of the most dynamic and plastic tissues in the human body, accounting for approximately 40% of total body weight, and is responsible for movement, posture, body temperature regulation, and various metabolic function. Duchenne muscular dystrophy (DMD) is one of the most common hereditary muscular dystrophies caused by dystrophin deficiency, and there is still no clear treatment. For this reason, tonsil‐derived mesenchymal stem cells (TMSCs) may be used as promising therapeutics for DMD. TMSCs have previously been demonstrated to be able to differentiate into various cells including skeletal muscle cell (SKMC), and their ability confirmed through the morphology and the expression of skeletal muscle markers like myogenin, Myf6, MYH2 and MYH8. Based on this potential, we conducted a transplantation to mdx mice, a mouse model of DMD, to check whether TMSCs help improve alleviation of the disease. TMSC‐SKMCs are used for transplantation was transduced with a red fluorescent protein (RFP) gene, and the same differentiation ability as TMSC‐SKMC was confirmed. RFP+TMSC‐SKMC were transplanted into gastrocnemius of mdx mice. During 10 weeks from transplantation, they had improved behavior on rotarod test and their stride length, but there was no significant difference. 10 weeks after transplantation, the expression of dystrophin, titin, MHC was increased in muscle of transplant group only. Furthermore, muscle regeneration of transplant group has also improved through the H&E staining. These results demonstrated that TMSC‐SKMCs have a positive effect on the recovery muscle function and can be a promising treatment for DMD and other muscle diseases.
Corneal regeneration by autologous limbal stem cells cultured on siloxane‐hydrogel contact lens in a limbal stem cell deficient rabbit model
1Department of Ophthalmology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia., 2Department of Pathology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia. , 3Gleneagles Hospital Kuala Lumpur, Kuala Lumpur, Malaysia., 4Centre for Tissue Engineering & Regenerative Medicine, Faculty of Medicine,
Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia.
Hypo‐immune retinal pigment epithelial cells for retinal regeneration
1Department of Convergence Medicine, Asan Medical Center, South Korea, 2Department of Convergence Medicine, University of Ulsan College of Medicine, South Korea, 3Department of Biomedical Science,
Cha University, 4Department of Ophthalmology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea, 5Bio‐Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, South Korea
Retinal pigment epithelium (RPE) transplantation derived from human induced pluripotent cells (hiPSCs) is a promising regenerative therapeutic approach for patients with age‐related macular disease or degenerative retinal tissue such as retinitis pigmentosa. From the standpoint of immunocompatibility, patient‐specific iPSCs may be the first choice for retinal tissue regeneration. When a patient has retinal disease due to a genetic disorder such as retinitis pigmentosa, there requires several steps for retinal tissue regenerative therapy, including establishment of patient‐specific iPSCs, gene editing of defect site to obtain normal iPSCs, and differentiating normal iPSCs into RPEs. The time required to obtain RPE tissue from patient‐specific iPSCs with or without a gene editing step can be estimated at a minimum of 6 months, which also requires cost and manpower. When immune‐related challenges are overcome, hypo‐ immune iPSC‐derived RPE tissue can be transplanted into patients with retinal disease with off‐the‐shelf allograft concept. The concept of ready‐made tissues for therapy is one of the ultimate goal of regenerative medicine researchers. In this study, a single knockout method for the β‐2 microglobulin gene was used to selectively eliminate highly polymorphic HLA class I. Hypo‐immune iPSCs differentiated into RPE cells in vitro. It was confirmed that the differentiated hypo‐immune RPE cells did not express MHC I class. After differentiation, RPE was confirmed by the expression of RPE‐related genes and markers through immunofluorescence and PCR.
Working cell bank preparation of retinal pigment epithelial cells during differentiation from iPSC
1Department of Convergence Medicine, Asan Medical Center, South Korea, 2Department of Convergence Medicine, University of Ulsan College of Medicine, South Korea
iPSC‐derived RPE replacement therapy is evolving as a feasible approach to treat retinal diseases such as Wet AMD and congenital macular degenerations. Differentiation of iPSCs into RPE cells takes at least 10 weeks according to the current protocol, which is labor intensive, expensive and time consuming and carries a fundamental risk of contamination prior to harvest fully developed RPE layers. In this study, a working cell bank concept was adopted and investigated to reduce the shortcomings of the current protocol and to improve the quality of RPE tissues for clinical applications. Conventional RPE differentiation has two steps: retinal progenitor differentiation (14 days) and RPE differentiation step(8 weeks). Cell banking was tried on week 2 after progenitor cell differentiation and on week 6 during RPE differentiation. At each time point, characteristics of RPE cells such as cell viability, proliferation, and expression of RPE‐related genes and markers were checked. It was found that the upregulation of the genes related RPE biomarker (RPE65) from intermediate RPE differentiation time(week 6) and cell proliferation rate was maintained stable. On differentiation week 6, working cell bank was generated by stocking RPE cells in liquid nitrogen. After thawing stock RPE cells and continuing RPE differentiation for up to 8 weeks from the end of progenitor, they were confirmed to retain their original function. Replated cells differentiated into mature RPE cell and maintained its function. These results offer new possibilities in the preparation of working cell banks of RPE cells for the application of clinical RPE regenerative therapies.
Study on hepatic progenitor cell activation and liver regeneration through the interaction of mesenchymal stem cells and liver sinusoidal endothelial cells
1Yonsei university wonju college of medicine, 2Wonju Severance Christian Hospital, Department of Internal Medicine, Division of Gastroenterology and Hepatology
As it has been proven that hepatic progenitor cells (HPC) can restore liver function, the potential as a therapeutic agent for liver cirrhosis is suggested.
In the activation of HPCs, Wnt‐β‐catenin signaling in liver sinusoidal epithelial cells (LSEC) plays an important role. LSECs inactivation and stabilization are important in accelerating the regression of fibrosis and inhibiting the progression of cirrhosis. Therefore, the purpose of this study is to investigate the functional recovery of LSECs through mesenchymal stem cells (MSCs) and the recovery of the Wnt‐ β‐catenin signaling pathway through this.
Fenestrae of LSECs were confirmed by SEM. Changes of various factors affecting fenestration were confirmed by real‐time polymerase chain reaction and Western blot. The specific cell markers of the isolated mouse LSECs and MSCs were confirmed by FACS.
When MSCs isolated from healthy people and MSCs isolated from cirrhosis patients were incubated with LSECs, it was confirmed that fenestrae, which had decreased with the incubation time, increased. Incubation with LSECs and MSCs increased the expression of VEGF, eNOS, HGF, Wnt2 and Wnt9b. Also, when LSECs and MSCs isolated from mice were co‐cultured, the expression of VEGF and HGF, which play an important role in maintaining the morphology of LSEC, increased. It was confirmed that the expression of Wnt9b, which acts as an angiocrine factor in liver regeneration, was also increased.
Based on these results, damaged fenestrae can be recovered by culturing damaged LSECs with MSCs, and the function of LSECs can be improved by maintaining fenestrae for a long time.
Silencing SIRT5 induces the senescence of UCB‐MSCs exposed to TNF‐α by reduction of fatty acid β‐oxidation and superoxide dismutase 2 activity
1Department of Veterinary Physiology, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University
Tumor necrosis factor‐α (TNF‐α) is an inflammatory cytokine involved in cell survival, apoptosis, and homeostasis. However, the regulatory effect of TNF‐α on mesenchymal stem cell (MSC) senescence remains unknown. The process of delaying the senescence of MSCs is important in transplantation therapy to treat inflammatory diseases that result from restricted immunomodulatory effects of senescent MSCs. Thus, we examined the role of TNF‐α‐mediated signaling and its regulatory mechanisms on the senescence of umbilical cord blood‐derived MSCs (UCB‐MSCs) and identified its therapeutic efficacy in a collagen‐induced arthritis (CIA) mouse model. We found that TNF‐α increased fatty acid synthesis and lipid droplet (LD) formation through NF‐κB/SREBP1‐mediated FASN, SCD1, and DGAT2 expression, which protects UCB‐MSCs from TNF‐α‐induced senescence. Additionally, DGAT2‐mediated LD formation was regulated by TNF‐α‐activated TNF receptor (TNFR)1 signaling. We also found that storage of unsaturated FAs in LDs is regulated by SIRT5‐dependent β‐oxidation of FAs, which reduces mitochondrial ROS (mtROS) accumulation. Particularly, mtROS homeostasis was maintained by superoxide dismutase 2 (SOD2) upregulation through TNFR2‐mediated SIRT5/Nrf2 signaling. In a CIA mouse model, UCB‐MSCs transfected with SIRT5 siRNA exhibited reduced therapeutic effects compared with UCB‐MSCs transfected with NT siRNA. Overall, the results indicated that SIRT5 plays a central role in protecting TNF‐α‐induced UCB‐MSC senescence through FA β‐oxidation and SOD2‐mediated antioxidation.
Effect of mESCs transplanted into the cochlea of animals with acute and chronic neurological hearing loss
1Dankook University
Current therapy for hearing loss uses only rehabilitation devices such as hearing aids and cochlear implants without a definitive cure. In addition, the rehabilitation prognosis of patients with chronic hearing loss who is expected to have secondary auditory nerve degeneration is relatively low. Stem cell therapy for cochlear neural structures would be an easy and feasible strategy compared to cochlear sensory cells. In this study, we established a mouse model that mimics chronic auditory nerve hearing loss. We then transplanted mouse embryonic stem cells (mESCs) into the tympanic membrane system and compared the survival and distribution of transplanted cells between models of acute and chronic auditory nerve hearing loss induced by ouabain or kanamycin (KM), respectively. The mESC survival was similar to the acute model and the perilymphatic distribution of cell aggregates was more prevalent in the chronic model.
Finally, we compared the effect of mESC transplantation on the neural signaling observed in the cochlear nucleus (CN), and a statistical increase was observed in the chronic model compared to other models. These results indicate that post‐transplantation mESCs survived in the cochlea and increased neural signaling towards the central auditory pathway even in a mouse model of chronic (secondary) hearing loss.
Basic fibroblast growth factor and forskolin induce cholinergic neuronal differentiation of tonsil‐derived mesenchymal stem cells
1Department of Molecular Medicine & Graduate Program in System Health Science and Engineering, College of Medicine, Ewha Womans University, 2Department of Nanobiomedical Science, BK21 PLUS NBM Global Research Center for Regenerative Medicine & Department of Pharmacology, College of Pharmacy, Dankook University
Mesenchymal stem cells (MSCs) are considered a potential tool for regenerating damaged tissues due to their great multipotency into various cell types. This study sought to determine the optimal conditions for neuronal differentiation of TMSCs, and to expand the potential applications of the TMSCs for treating neurological disorders. Our initial study demonstrated that DMEM/F12 supplemented with 50 ng/mL basic fibroblast growth factor with 10 μM forskolin was the optimal condition for neuronal differentiation for the TMSCs. Western blotting and immunofluorescence analyses showed that the differentiated TMSCs had higher protein expression of neuronal markers, including neuron enolase, growth‐associated protein‐43, post‐synaptic density protein 95, synaptosomal nerve‐associated protein 25 and synaptophysin, compared to the undifferentiated TMSCs. The protein levels of these markers were dramatically increased at the first week of the neuronal differentiation and further increased thereafter until the third week. The expression of glial fibrillary acidic protein and ionized calcium‐binding adapter molecules 1, the markers of astrocytes and microglia, were also slightly increased. Additionally, the differentiated TMSCs released significantly higher levels of acetylcholine (cholingergic neurotransmitter) as analyzed by the LC‐MS/MS as well as the protein expression of choline acetyltransferase compared to the undifferentiated cells. Further optimization to increase neuronal‐like cell population within a heterogenous TMSC pool and functional validation is required. Our study provides a glimpse of the evidence that TMSC‐derived cholinergic neuron‐like cells could be used as a possible therapeutic tool in treating certain neurodegenerative disorders such as Alzheimer's disease.
Transcriptomic analysis of human tonsil‐derived mesenchymal stem cells exposed to far‐infrared irradiation
1Department of Molecular Medicine, College of Medicine, Ewha Womans University, Seoul 07804;
Graduate Program in System Health Science and Engineering, Ewha Womans University, Seoul 03760,
2Department of Molecular Medicine, College of Medicine, Ewha Womans University, Seoul 07804;
Korean Fund for Regenerative Medicine, Seoul 04637, 3Korea Carbon Industry Promotion Agency (KCARBON), Jeonju‐si, Jeollabuk‐do 54853, 4Department of Biochemistry, College of Medicine, Ewha Womans University, Seoul 07804; Graduate Program in System Health Science and Engineering, Ewha Womans University, Seoul 03760
Osteoporosis is the common bone disorder caused by the imbalance between osteogenic and adipogenic differentiation of the bone marrow cells. Our recent study found that far‐infrared (FIR) irradiation induces the osteogenesis but inhibits the adipogenesis of tonsil‐derived mesenchymal stem cells (TMSCs), potentially restoring the imbalanced differentiation potentials and alleviating signs and symptoms of osteoporosis. To investigate the molecular mode of action mediated by the FIR irradiation on the osteogenesis and adipogenesis of the TMSCs, transcriptomic analyses were used to compare the differentially expressed genes between the FIR‐exposed and non‐exposed control TMSCs. As many as 308 and 532 genes were determined to be significantly up‐regulated and down‐regulated by the transient FIR exposure, respectively (p < 0.05; fold‐change >2.0). The biological process enrichment analysis showed that the up‐regulated genes by FIR irradiation were majorly involved in extracellular matrix organization, negative regulation of cell proliferation and positive regulation of osteoblast differentiation, whereas the down‐regulated genes were involved in cell cycle and negative regulation of cell differentiation. The up‐regulated genes that positively regulate the osteoblast differentiation in the FIR‐ exposed TMSCs include IFITM1, PRKD1, BMP4, FBN2, CEBPD, and CTHRC1, which were further validated by the RT‐qPCR and western blots. We are currently in the process of screening the major target molecules of FIR irradiation that induce the osteogenesis of the TMSCs. The findings of this study could be used as potential evidence in designing an effective treatment for bone disorders such as osteoporosis.
Preclinical study of human bone marrow‐derived mesenchymal stem cells using a three‐dimensional manufacturing setting for enhancing spinal fusion
1CHA Bundang Medical Center, Cha University, 2Incheon National University
Spinal fusion surgery is a surgical technique that connects one or more vertebrae at the same time to prevent movement between the vertebrae. Although synthetic bone substitutes or osteogenesis‐inducing recombinant proteins were introduced to promote bone union, the rate of revision surgery is still high due to pseudarthrosis. To promote successful fusion after surgery, stem cells with or without biomaterials were introduced; however, conventional 2D‐culture environments have resulted in a considerable loss of the innate therapeutic properties of stem cells. Therefore, we conducted a preclinical study applying 3D‐ spheroids of human bone marrow‐derived mesenchymal stem cells (MSCs) to a mouse spinal fusion model. First, we built a large‐scale manufacturing platform for MSC spheroids, which is applicable to good manufacturing practice (GMP). Comprehensive biomolecular examinations, which include liquid chromatography‐mass spectrometry and bioinformatics could suggest a framework of quality control (QC) standards for the MSC spheroid product regarding the identity, purity, viability, and potency. In our animal study, the mass‐produced and quality‐controlled MSC spheroids, either undifferentiated or osteogenically differentiated were well‐integrated into decorticated bone of the lumbar spine, and efficiently improved angiogenesis, bone regeneration, and mechanical stability with statistical significance compared to 2D‐cultured MSCs. This study proposes a GMP‐applicable bioprocessing platform and QC directions of MSC spheroids aiming for their clinical application in spinal fusion surgery as a new bone graft substitute.
Anti‐inflammatory effect of human fetal cartilage‐derived progenitor cells (hFCPCs) on IL‐1b‐mediated osteoarthritis (OA) phenotypes in vitro
1Inha university, 2Ajou University, 3ATEMs Inc
We have previously reported that human fetal cartilage‐derived progenitor cells (hFCPCs) showed immune modulatory activity similar to human mesenchymal stem cells (MSCs). In this study, we have investigated whether hFCPCs have anti‐inflammatory activity and can alleviate osteoarthritis (OA) phenotypes in vitro. hFCPCs were obtained with an IRB approval (AJIRB‐CRO‐07‐139). To find an optimal priming factor, hFCPCs were stimulated with various cytokines and their combinations and we found that treatment with poly(I‐C) (1 mg/mL) significantly induced the expression of paracrine factors such as IDO, HLA‐G, TSG‐6, LIF, TGF‐β and HGF from hFCPCs. Then, activated hFCPCs stimulated with poly(I‐C) were co‐cultured with SW‐982, a synoviocyte cell line or human young chondrocytes (AJIRB‐MED‐SMP‐10‐226) treated with IL‐1β to induce OA phenotypes for 3 days. In the qRT‐PCR, co‐ treatment of activated hFCPCs significantly alleviated the IL‐1β induced expression of inflammatory factors (IL‐6, MCP‐1 and IL‐1β), matrix metalloproteinases (MMP13, MMP1 and ADAMTS4) in SW982, while it increased the expression of cartilage extracellular matrix such as aggrecan and collagen type II in human chondrocytes. We also found that treatment of hFCPCs with poly(I‐C) did not show significant effects on its surface marker profiles, proliferation rate, and differentiation ability. These results suggest that hFCPCs have an ability to modulate inflammatory signals and OA phenotypes and encourage further studies to apply them in animal models of OA in vivo.
Enhancement of the stem cell engraftment and differentiation for cartilage regeneration by using transglutaminase‐4/hydrogel
1Department of Orthopedic Surgery, Seoul National University Hospital
Although mesenchymal stem cells (MSCs) transplantation has been shown to be a promising strategy for the repair of damaged articular cartilage, MSCs‐based cartilage tissue engineering has a number of limitations. These include poor implanted cell adhesion, phenotypic alteration of cells, control of mechanical properties, and engraftment rates after implantation. To achieve effective transplantation of synovium‐derived mesenchymal stem cells (SDSCs) for the repair of osteochondral defects, we aim to investigate SDSCs encapsulated in type I collagen/hyaluronic acid/fibrinogen (COL/HA/FG) composite gel by supplement of recombinant human transglutaminase4 (rhTG‐4). Treatment of rhTG‐4 can induce expression of integrin β1 and dynamic actin fiber, enhancing the SDSCs adhesion to fibronectin. rhTG‐4 significantly induced the proliferation of SDSCs encapsulated in COL/HA/FG composite gel. Supplement of rhTG‐4 in COL/HA/FG composite gel also significantly increased the expressions of aggrecan and type II collagen mRNA. Pretreatment with integrinβ1 markedly decreased TG4‐induced dynamic actin remodeling and type II collagen expression. In the Gel/SDSC+TG‐4 group, the defect lesion was more filled with white tissue and good integration between the newly formed cartilage and the surrounding original cartilage was observed. Moreover, cartilage tissue on the defects had more a hyaline cartilage that was densely stained by safranin‐O in the Gel/SDSC+TG‐4 group. These data suggest that rhTG‐4 enhances cartilage regeneration of SDSCs encapsulated in hydrogel by integrin β‐ mediated actin remodeling. This approach could be used in engineering cartilage tissue, to improve the properties of engineered cartilage and ultimately to improve clinical outcomes.
Hypoxic condition enhances chondrogenesis in synovium‐derived mesenchymal stem cells
1SEOUL NATIONAL UNIVERSITY HOSPITAL
The chondrogenic differentiation of mesenchymal stem cells (MSCs) is regulated by many factors, including oxygen tensions, growth factors, and cytokines. Low oxygen tension seems to be an important regulatory factor in the proliferation, chondrogenic differentiation in various MSCs. But, the effect of low oxygen tension on the chondrogenic differentiation in synovium‐derived mesenchymal stem cells (SDSCs) has not characterized. In this study, we investigated the effects of hypoxia on chondrogenesis in SDSCs. Low oxygen condition significantly increased proliferation and colony‐forming characteristics of SDSCs compared to that of SDSCs under normoxic culture. At 18 days after cultivation, SDSCs cultured under hypoxic conditions had 2.5‐fold higher colony number than those cultured under normoxic conditions. SDSCs were concentrated to form pellets and Pellets of SDSCs were differentiated into the chondrogenic lineage for up to 21 days in the presence of TGF‐β in either normoxic or hypoxic conditions. In the chondrogenic cultures under hypoxia condition, the gene expression of COL2A1, ACAN, and the transcription factor SOX9 was greatly increased in comparison with nomoxia condition. In contrast, the hypertrophic cartilage‐enriched gene transcripts of COL10A1 mRNA levels were strongly down‐regulated under hypoxia conditions compared with nomorxia conditions. Histological sections demonstrated that hypoxic conditions had increased proteoglycan synthesis. Hypoxic culture of SDSCs increased type II collagen expression. In addition, GAG deposition significantly higher in hypoxia compared with normoxia at 21 days of differentiation. These findings show that hypoxia condition has an important role in regulating the synthesis ECM matrix by SDSCs as they undergo chondrogenesis.
Establishment of induced pluripotent stem cells by lentiviral reprogramming of primary dermal fibroblasts
1Institute for Medical Research (IMR), National Institutes for Health (NIH), Ministry of Health (MOH), Malaysia, 2Centre for Tissue Engineering and Regenerative Medicine (CTERM), Universiti Kebangsaan Malaysia (UKM), 3Department of Biomedical Engineering, Faculty of Engineering, Universiti Malaya, Malaysia, 4Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia (USM)
Induced pluripotent stem cell (iPSC) generated using patient‐derived samples are useful resources for studying disease mechanisms in‐vitro. iPSCs are often generated from adult somatic cells, however the efficiency of reprogramming of primary cells differs between samples. Here, we examined the efficiency of iPSCs generation from primary human dermal fibroblasts by lentivirus (LV) reprogramming.
Human dermal fibroblasts were isolated from skin biopsy and cryopreserved at passage 3. Passage 5 fibroblasts and BJ cell lines were transduced with LV encoding transcription factors Oct4, Sox2, Klf4, c‐ Myc, Nanog & Lin28 and grown on inactivated fibroblast feeder layer for 20‐27 days. ES‐like cell colonies were clonally expanded in feeder‐free culture system and characterized by immunophenotyping for pluripotency and differential potential by embryoid body formation.
Transduction with LV produced ES‐like cell colonies. Reprogramming efficiency of primary human dermal fibroblasts were 0.7% & 0.84%, and 0.64% for BJ cell line. Expanded ES‐like colonies expressed pluripotent nuclear markers OCT4, SOX2 and NANOG, and surface markers TRA‐1‐81, TRA‐1‐61 and SSEA4. IPSC showed expression of >90% for pluripotent markers SSEA4 and TRA‐1‐81 and <5% differentiation marker SSEA1. All generated IPSCs formed embryoid bodies that spontaneously differentiated into cells expressing ectoderm (AFP), mesoderm (SMA) and endoderm (BIII‐tubulin) markers.
IPSCS can be reproducibly generated by LV transduction with high reprogramming efficiencies. All iPSC‐lines express pluripotency markers with minimal differentiation and differentiate into cells from three embryological lineages. IPSCs produced can be used for future disease modelling of inherited studies.
Characteristics of mesenchymal stem cells from various tissue source according to culture media type
1Xcell therapeutics Inc.
Stem cells are attractive candidates for the regeneration of tissue and organ. Mesenchymal stem cells (MSCs) have been extensively investigated for their potential applications in regenerative medicine and cell therapy. MSCs have ability to secrete growth factors, differentiate into many types of cells, and can act anti‐inflammatory role during inflammation and tissue injuries. These functions underlie the important physiological roles of MSC and underscore a significant potential for the clinical use of distinct populations from the various tissues. It has been also reported that culture characteristics of MSCs and the function of cultured cells may vary depending on the culture media type.
Thus, this study investigated cell biological properties, proliferative capacities, mRNA expression profile, and protein expression profile of cultured human MSCs obtained from various donor and tissue source (adipose tissue, bone marrow, umbilical cord, Wharton jelly, and iPSC) with different type of media (FBS containing media, serum free media, and serum free chemically defined media). Comparative analysis of MSCs from five different source with three types of media was performed based on proliferative capacities using multipassage assay, stem cell specific surface marker expression using FACS, differentiation potency, genetic stability using cytokinesis block micronucleus assay, senescence analysis using β‐galactosidase staining. And mRNA and protein differential expression patterns were analyzed using qRT‐PCR and western blot.
This study provides comprehensive characteristics of MSC derived from various tissue with different culture media condition to better understand their cellular and molecular biology.
Transcriptomic changes of the tonsil‐derived mesenchymal stem cells upon the inhibition of the rate‐limiting enzymes for nucleotide biosynthesis
1Department of Molecular Medicine, College of Medicine, Ewha Womans University, 2Department of Molecular Medicine, College of Medicine Ewha Womans University; Korean Fund for Regenerative Medicine, 33Department of Biological Sciences and Biotechnology, College of Natural Sciences, Chungbuk National University, 4Department of Molecular Medicine, College of Medicine/Graduate Program in System Health Science and Engineering, Ewha Womans University
Nucleotides are basic building blocks of nucleic acid required for various biological activities. Our previous study found guanosine as a critical nucleotide regulating proliferation and differentiation of tonsil‐derived mesenchymal stem cells (TMSCs). This study investigated the effect on the cellular functions of TMSCs upon the inhibition of the rate‐limiting enzymes for nucleotide biosynthesis. The TMSCs were treated with siRNAs of the rate‐limiting enzymes, including adenyl‐succinate synthetase (ADSS), thymidylate synthase (TS), inosine monophosphate dehydrogenase (IMPDH) and cytidine triphosphate synthase (CTPS), required for the synthesis of the following respective nucleotide products: adenine, thymine, guanine and cytidine. The inhibition of the enzymes was confirmed using western blotting analyses. The differentially expressed genes of each siRNA‐treated TMSCs were compared with the control TMSCs treated with random siRNA. Our comparative analyses found that 5 up‐ and 7 down‐ regulated genes specific for ADSS inhibition; 12 up‐ and 7 down‐regulated genes for TS inhibition; 325 up‐ and 376 down‐regulated genes for IMPDH inhibition; and 229 up‐ and 187 down‐regulated genes for CTPS inhibition. The inhibition of purine biosynthesis decreased CRHR1‐IT1, CD209 and ETV7, whereas the inhibition of pyrimidine increased the gene expression of KIF4B, NCAPD3, ZNF616, TERC, SLCO4A1 but decreased YPEL4, PLCD1, ETV7 and LYNX1. KEGG pathway analyses revealed that cell cycle, cancer regulation and PI3K‐Akt signaling were the most commonly affected pathways from the inhibition of each nucleotide. We are currently investigating the functional involvements of the genes in relation to the cell cycle, proliferation and differentiation of the TMSCs.
Inhibitory effect of tonsil‐derived mesenchymal stem cell proliferation by treatment of non‐structural protein 9 through PI3K/Akt signaling
1Department of Microbiology, School of Biological Sciences, College of Natural Sciences, Chungbuk National University
Severe acute respiratory syndrome coronavirus (SARS‐CoV) possesses high replicative capacity and pathogenicity than the classical coronavirus. However, the factors that lead to enhanced replication and pathogenicity remain unclear. Among the non‐structural proteins of SARS‐CoV, non‐structural proteins 9 (NSP9) is known to most likely be involved with viral RNA synthesis. In this study, we investigated the effect of NSP9 on the human upper respiratory system using tonsil‐derived mesenchymal stem cells (TMSCs). In order to confirm the significant toxic effect, NSP9 were treated to TMSCs in various concentrations as follows; 0.01 ,0.05 ,0.2, 0.4, and 0.8 μg/ml. Cell proliferation assay and LIVE/DEAD assay revealed that NSP9 was most toxic to TMSCs at a concentration of 0.2 μg/ml. In addition, NSP9 treated TMSCs showed a pattern of decreased metabolic efficiency in mitochondria. The phosphoinositide 3‐kinase (PI3K) and phosphorylated Akt protein markers were compared to confirm that it affects cell proliferation by reducing the metabolic rate. These results showed that NSP9 inhibits PI3K/Akt signaling proteins related to cell proliferation in TMSCs and induces toxicity by decreasing the cellular metabolic rate. Taken together, the NSP9, possessing toxicity to TMSCs, can affect to upper respiratory system by inhibiting cell proliferation and metabolic rate.
Changes in the integrin α3 expression controls tonsil derived mesenchymal stem cell proliferation and senescence
1Chungbuk National University, 2Ewha Womans University
A long‐term serial cell expansion can potentially induce replicative senescence, which leads to a progressive decline in stem cell function and stemness, losing multipotent characteristics in mesenchymal stem cells (MSCs). Senescence‐related changes in MSC properties have attracted considerable attention. Here, we established a senescent MSC model using senescence‐associated β‐galactosidase, proliferation, and telomere length gene level. We used transcriptomic analysis to identify novel regulators of distinguishing control and culture‐aged tonsil‐derived MSCs (TMSCs). Differentially expressed genes were grouped into Gene Ontology categories using KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis. This analysis showed that culture‐aged TMSCs had decreased integrin α3 (ITGA3) and phosphorylated AKT protein (p‐AKT‐Ser473) expressions compared to the control TMSCs. Extracellular matrix‐receptor interactions related to genes were also shown to be significantly different. Furthermore, we also found that activation of ECM‐receptor signaling, specifically related to integrin family‐mediated activation of the intracellular cell survival‐signaling molecule AKT, can modulate cell senescence in TMSCs. Taken together, we suggest that ITGA3 was found to be a representative biomarker of the senescent TMSCs. The exclusion of the TMSCs with the senescent TMSCs markers in this study could potentially increase the therapeutic efficacy of TMSCs in clinical applications.
UCHL1 plays a key role for the trans‐differentiation of auditory supporting cells into hair cells in the cochlea
1Ajou University School of Medicine, 2Ajou University Graduate School of Medicine, 3Soonchunhyang University College of Medicine
In mammals, hearing loss is irreversible due to the lack of regenerative capacity of the auditory epithelium. However, the recently identified resident stem/progenitor cell population in mammalian cochleae is a therapeutic target for hearing regeneration. The ubiquitin proteasome system plays an important role in cochlear development and maintenance. However, the role of deubiquitinating enzymes in hair cell (HC) regeneration, through the transdifferentiation of neighboring supporting cells (SCs) into HCs in the cochlea, remains unknown. In this study, we investigated the role of ubiquitin C‐terminal hydrolase L1 (UCHL1) in HC differentiation. UCHL1 was extensively observed in the greater and lesser epithelial ridge of the cochlea on embryonic day 17.5 (E17.5). The expression of UCHL1 gradually decreased as HCs developed, and was restricted to inner pillar cells and third‐row Deiters' cells between P2 and P7, suggesting that UCHL1‐expressing cells are similar to the cell population with leucine‐rich repeat‐containing G‐protein receptor 5‐positive progenitors. UCHL1 expression was decreased even under conditions in which supernumerary HCs were generated with a γ‐secretase inhibitor and Wnt agonist in the neonatal organ of Corti explants. Moreover, the inhibition of UCHL1 by LDN‐57444 led to an increase in HC number. Mechanistically, LDN‐57444 increased mammalian target of rapamycin complex 1 activity and allowed SCs to transdifferentiate into HCs. This study provides new evidence for a role of UCHL1 in the transdifferentiation of SCs and progenitors into HCs in the neonatal cochlea.
Bone regeneration induced by osteoblast‐like cells (CF‐M801) differentiated from umbilical cord stroma cells in a goat partial defect model
1CEFO Co., Ltd., 2KPC
CF‐M801 is an allogeneic osteoblast differentiated from umbilical cord matrix stroma cells by 3D culture on hydrogels whose stiffness was optimized. This study evaluated the ability of CF‐M801 to repair bone defect in a large animal. Open femur partial fractures were created in 28 adult goats (14 male and 14 female) with loss of 0.8cm circular segment of the bone. Capsules for transplantation were prepared by 3D printing a cylindrical mold (diameter 8mm x depth 20mm) to fit the bone defects using the alginate gels with/without CF‐M801. Alginate‐based capsule was transplanted into the bone defects of goat femur. The animals were monitored using micro‐computed tomography (micro‐CT) at 13 and 26 weeks to measure bone formation. Micro‐CT three‐dimensional data showed no significant difference in two groups. However, further histological analysis revealed that bone images of alginate treated‐group in CT analysis were not real bone but alginate remnants. Further histological and histo‐morphometric analysis revealed that only an osteoblast‐like cell‐treated femur was superior to control in bone healing by H&E staining and Masson's Trichrome staining. In control, the alginate scaffold remained to prevent from new bone formation. In contrast, osteoblast‐like cell‐treated group showed that the alginate scaffold was demolished, replaced by newly formed collagen matrices and new bone structures were formed. At 26 weeks osteoblast‐like cell‐treated group showed that the defect area was nearly completely replaced by the new bone tissues and recovered to almost normal level. These data support the continued development of the more clinically relevant osteoblast‐like cells for bone regeneration applications.
Optimization of mesenchymal stem cell spheroids for enhanced angiogenesis
1Seoul National University Hospital, 2Seoul National University
Because of the secretory function of numerous cytokines related to cell viability, proliferation, differentiation, immunomodulation, and so on, stem cells are widely used as cell therapy for tissue regeneration. Many studies have been conducted to strengthen the secretory function of stem cells through gene transfection; however, there is a safety concern associated with gene modification. Thus, spheroidal formation has gained attention as another strategy for strengthening the secretory function without compromising safety. Because of the central hypoxia condition, vascular endothelial growth factor (VEGF), a cytokine that promotes angiogenesis, is highly expressed by spheroids. However, the non‐optimized therapeutic effect of stem cell spheroids makes it difficult to use. First, we fabricated spheroids ranging in diameter from 150 to 300m and quantified secreted VEGF from the spheroids. In addition, tubule formation of endothelial cells in vitro and vascularization of implanted spheroids under the subcutaneous layer of mice in vivo were evaluated to confirm the angiogenic effect. Spheroids with a diameter of 250μm expressed more VEGF than those with other diameters. Similarly, spheroids with a diameter of 250μm induced more tubule formation. After subcutaneous implantation, we also observed vascularization composed of implanted stem cells alone or implanted stem cells and endothelial cells from an animal. In conclusion, 250μm spheroids had the greatest angiogenic effect in vitro. Spheroids also participate in vascularization by transdifferentiating into endothelial cells in vivo, in addition to inducing angiogenesis via VEGF secretion.
AGEs‐induced oxidative stress regulation as a potential target to attenuate the impact of the diabetic microenvironment on mesenchymal stromal cells
1Université Paris Cité, CNRS, INSERM, ENVA, B3OA, F‐75010 Paris, France, 2INSERM U1148, Laboratory for Vascular Translational Science, Cardiovascular Bioengineering, Université Sorbonne Paris Nord, 93430 Villetaneuse, France., 3Université Paris Cité, CNRS, INSERM, ENVA, B3OA, F‐75010 Paris, France ; Department of Periodontology, Service of Odontology‐Pitié Salpêtrière Hospital, AP‐HP et U.F.R. of Odontology, 75013 Paris, France
Bone marrow‐derived mesenchymal stromal cells (MSCs), contribute to tissue repair and are promising candidates to treat Type 2 Diabetes (T2D)‐related‐complications. Advanced‐glycation‐end‐products (AGEs) and oxidative‐stress (OS) play a central role in the T2D‐related‐pathologies but their effects on MSCs remain unclear. The present study aimed at investigating the AGEs/OS implication on MSCs‐ dysfunction considering OS regulation as a potential target to attenuate the T2D‐impact on MSCs.
MSCs were harvested from 24‐week‐old Zucker diabetic fatty rats (ZDF) ‐representing long‐standing diabetes‐ and their Lean littermates (ZL). ZDF‐MSCs and ZL‐MSCs were exposed to 0 ∼ 200 mg/ml of AGEs, for several time points, mimicking in vitro diabetic microenvironment. Cell viability, apoptosis, proliferation, migration, and intracellular reactive oxygen species (iROS) production were assessed according to established methods. OS regulation was studied using di‐tert‐butyl‐peroxide and N‐Acetyl‐ L‐cysteine (NAC).
In both ZDF‐ and ZL‐MSC, exposure to AGEs reduced proliferation(p < 0.001), increased apoptosis rate (by 60%, p < 0.01), and reduced migration (up to 66%,p <0.01). The effect was more pronounced in ZDF‐ MSC than in ZL‐MSC. AGEs increased (by 5‐fold, p < 0.01) iROS production; also observed with di‐tert‐ butyl‐peroxide, which lead to an augmentation of iROS concomitantly to a dose‐dependent reduction of ZDF‐MSC viability. 48h‐NAC‐pre‐treatment reduced significantly the iROS induced by di‐tert‐butyl‐ peroxide in MSCs.
Our results showed that AGEs increased iROS production and impair MSCs‐functions pertinent to their potential use in autologous cell therapies. They highlight the pronounced effect of AGEs on T2DM‐MSCs and suggest that the use of an antioxidant could be a promising approach to protect MSCs from OS in a DT2 microenvironment.
The therapeutic potential of MSCs administration in naturally aged sarcopenia mouse model
1The Chinese University of Hong Kong
Sarcopenia, ageing‐associated reduction in skeletal muscle strength and mass, can lead to loss of functional mobility, increased risk of falling and hospitalization in the elderly population. The progression of sarcopenia is attributed to ageing‐associated chronic inflammation; however, the development of interventions is still in its infancy. Recent clinical trials and preclinical studies have demonstrated the potential of mesenchymal stromal stem cell (MSC) therapy for various skeletal muscle‐associated diseases, suggesting that MSC therapy might have the possibility of attenuating ageing‐associated sarcopenia. In this study, we aim to investigate the therapeutic potential of MSCs in improving sarcopenia‐like phenotypic changes. 3‐month‐old C57BL/6 mice were used as healthy young control, and 23‐month‐old C57BL/6 mice were used as aged control. The aged mice received a dose of MSCs through tail vein injection once a week for 2 months as the MSC treatment group. We performed multidimensional assessments to investigate the therapeutic potential of MSCs in improving sarcopenia and systemic inflammation changes. The results indicated that MSC therapy significantly enhanced muscle weight, fibre size and functions, including grip strength and contraction ability. Meanwhile, systemic inflammation was reversed by MSCs. Due to limited effective treatment options for sarcopenia and considering those not expected to benefit most from exercise, such as bedridden patients, this study provides evidence that MSCs can improve sarcopenia and systemic inflammation, which suggest that MSC might be a new therapeutic option for sarcopenia patients.
Evaluation of the effects of Centipeda minima (L.) on cellular viability, osteogenic differentiation and mineralization of human bone marrow‐ derived stem cells
1The Catholic University of Korea, 2Department of Periodontics, Seoul St Mary's Hospital, College of Medicine, The Catholic University of Korea, 32Ethnobotanical Database of Bangladesh, Tejgaon Dhaka, 1208, Bangladesh
This study aimed to evaluate the effects of Centipeda minima (L.) extracts (CMT) on the osteogenic differentiation and mineralization of human mesenchymal stem cells.
Gingiva‐derived stem cells/ were cultured in the presence of at concentrations ranging from 0, 0.001, 0.01, 0.1, and 1 μg/ml. Evaluations of cell morphology were done on Days 1, 3, 5 and 7. The quantitative cellular viability was performed on Days 1, 3, 5, and 7. Alkaline phosphatase activity assays and Alizarin red S staining were performed to evaluate the osteogenic differentiation of stem cells on Days 7 and 14, respectively. Real‐time polymerase chain reaction was used to determine the expression levels of RUNX2, and COL1A1 on Days 14. Western blot analysis was performed to analyze the protein expression.
Stem cells in the control group showed fibroblast‐like morphology and addition of CMT at final concentrations 0.001, 0.01, 0.1, and 1 μg/ml. There were no noticeable changes in morphology when compared with the untreated control group. The application of did not produce significant changes in cellular viability.
Conclusively, CMT had influenced the osteogenic differentiation of the stem cells derived from the bone marrow/gingiva. The use of CMT may produce beneficiation effects on mineralization of the stem cells.
Development of high elastic biodegradable stent based on PCL copolymer with shape memory effect
1Korea Textile Development Institute, 2Korea Institute of Science and Technology, 3Dankook university, 4M.I.Tech Co., Ltd.
A stent is a structure used to expand and maintain a narrowed area into a space of a certain volume. Materials are extended from metal to biodegradable polymers. Biodegradation stents are alternative to the existing metal stents because of easy to remove and a few side effects. However, biodegradable stents are difficult to maintain the diameters of expanded space due to the lower mechanical strength, especially compression or radial force. In this study, we investigated the novel biodegradable stent that maintain a high radial expansion force during an appropriate degradation time. The high elasticity urethane based
multiblock copolymer consisting of poly(caprolactone) (PCL), poly(dimethylsiloxane) (PDMS), and poly(ethylene glycol)(PEG) were synthesized. The copolymer monofilaments were prepared by melt spinning or wet spinning process as a new biodegradable stent. The stents with different morphological characteristics were manufactured by adjusting the fabrication parameters, and their mechanical properties were investigated. The synthesis results were analyzed using proton NMR, GPC and FTIR. The melt spinning monofilaments had higher tensile strength and radial force compared with those of wet spinning monofilaments, and mechanical properties were improved through stretching and thermal process. These results show that the synthetic multiblock copolymers have effective shape memory properties and high elasticity for a novel biodegradable stent. Considering their mechanical properties, the melt spinning monofilaments were found to be effective. The biodegradable stents will be determined through further research on degradation properties and biocompatibility.
Silk sericin increases BMP‐2 expression in macrophage
1Gangneung‐Wonju National University, 2National Academy of Agricultural Science
Silk sericin is a protein originated from the silkworm. It has been considered as an environmental waste in the textile industry. In this study, silk sericin has been investigated as a potential immuno‐modulating agent for using bone regeneration. Silk sericin has been prepared by the different kinds of degumming technology. Each sericin was administered to the macrophage and the expression level of BMP‐2 was examined.
In results, the silk sericin extracted by low temperature with sonification had higher beta‐sheet conformation. The silk sericin having abundant beta‐sheet structure showed higher level of BMP‐2 expression in the macrophages.
As BMP‐2 is well‐known osteoinductive protein, increased BMP‐2 expression induced by silk sericin application would be helpful for the immunomodulation mediated bone regeneration.
The administration of peptides from silk sericin to the macrophage
1Gangneung‐Wonju National University, 2National Academy of Agricultural Science
In our previous study, the silk sericin having high level of beta‐sheet conformation could increase the expression level of BMP‐2 in macrophage. As degumming process is complicated process, peptide from silk sericin was prepared.
First, the conformation of peptide was predicted using software. They were classified as random coiled structure and beta‐sheet structure. Each of them was administered to macrophage and examined the expression level of BMP‐2.
In results, none of them showed elevated expression level of BMP‐2 in macrophage. The effect of peptide administration was not associated with their predicted structure. However, natural silk sericin having abundant beta‐sheet structure showed elevated expression of BMP‐2.
In conclusion, BMP‐2 expression in macrophage by sericin administration was complicated process. Both protein conformation and its molecular weight might be important.
Lotus‐inspired multifunctional antifouling janus nanofibrous membrane for prevention of postsurgical tissue adhesion
1Kyungpook National University
Postsurgical adhesion refers to the abnormal proliferation and attachment of cells resulting from the inflammatory responses during surgical tissue trauma and healing. Even though minimally‐invasive surgery and anti‐adhesion agent gel treatment have been commonly considered to prevent the postsurgical adhesion, the clinical outcomes of these approaches are still elusive due to the rapid degradation and foreign‐body irritation. Here, we devise chitosan‐based multicomposite Janus membranes consisting of a hydrophilic tissue adhesive layer and an opposite superhydrophobic silica layer. The negatively charged silica nanoparticles uniformly distributed on the surface of positively charged chitosan nanofibers could mimic the rough surface of lotus leaf, allowing an antifouling barrier capable of preventing the non‐ specific adhesion of blood or cells. The electrospun composite nanofibers showed an excellent antibacterial activity by virtue of effective water‐repellency and antifouling effects of the surface‐ decorated silica nanoparticles as well as superior filtration efficacy obtained from their nanoporous architectures. Moreover, the catechol functionalization of the opposite side of nanofibrous membranes enabled an effective tissue adhesion on the target sites. Thus, our lotus‐inspired Janus membranes could be practically applied to prevent undesired bacterial infection and postsurgical adhesion in various tissues of clinical surgery cases.
Evaluation of bone formation of duck‐beak particles by heat treatment procedure in a rat model
1Department of Veterinary Surgery, Chungbuk National University, 2Department of Veterinary Surgery, Chonnam National University
This study was performed to examine the chemical and physical attributes of duck‐beak derived xenografts and to evaluate their osteogenic possibility. In vivo experiments were conducted with 20 rats each (n = 5). Duck‐beak bone endured a heat treatment procedure at 400°C (G2) and 1200°C (G3) using ethylene diamine and at 400°C (G4) using hydrogen peroxide and was then milled into particles of 500– 700 μm. All particles were disinfected with 70% ethyl alcohol for 30 min and rinsed with PBS for three times. Both sides of calvarial bone of rats were holed 5 mm size, and then the particles were transplanted. In cell reproductive integrity, cell integrity of the G3 was higher than that of other groups. In fluorescence microscope and alkaline phosphatase assay, bone regeneration process of the G3 was more than that of other groups. In micro‐CT scan analysis of samples in a rat calvarial defect model, bone regeneration of the G3 was considerably higher than that of the other groups. New bone formation was also particularly higher at 8 weeks in the G3, which observed more new osteocytes in the lacuna compared with the other group. It can be resulted that higher procedure temperature bone particles might supply a more successful graft substance for improving bone formation. Also using ethylene diamine duck‐beak bone particles might supply a more useful graft substance for improving bone formation. Acknowledgement: This work was carried out with the support of "Cooperative Research Program for Agriculture Science and Technology Development (PJ01620801)" Rural Development Administration, ROK
Vertical osteogenesis using a 3D printed nylon cap in a rat model
1Department of Veterinary Surgery, Chungbuk National University, 2Department of Veterinary Surgery, Chonnam National University
As an alternative material to the autogenous bone, duck‐beak bone particle for bone substitute have been attracting great attention due to their biological properties. To deliver the most favorable outcome of medical treatment, it is essential to study the effect of various processing methods of the duck‐beak bone. In this study, we compared the two deproteinizing agents for manufacturing duck‐beak bone. The test group was divided into the conventional ethylenediamine (ED)‐treated group and the hydrogen dioxide (HD)‐treated group. In vitro and in vivo experiments were conducted in parallel to compare the cytocompatibility and osteogenic capability between two processing methods. For in vitro tests, human adipose‐derived mesenchymal stem cells (hAD‐MSCs) were planted onto each sample and their attachment and growing were evaluated. For in vivo biocompatibility and osteogenic properties, the samples were applied on the critical‐sized calvarial bone defect of rats. HD‐treated group showed significantly higher cell attachment but ED‐treated group showed slightly higher cell proliferation. In in vivo tests, all groups have shown biocompatibility and increased level of osteogenic potential. However, HD‐treated group had significantly higher bone regeneration (p < 0.05). This experiment confirmed that HD‐treated group can be an optimal processing method for duck‐beak bone particle.
Acknowledgement: This work was carried out with the support of "Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01620801)" Rural Development Administration, Republic of Korea
Properties of biodegradable fiber stents consist of polydioxanone and polycaprolactone for companion animal application
1Korea Textile Development Institute, 2M.I.Tech, Co. Ltd
Biodegradable fiber stents can alleviate intestinal obstruction and stenosis in companion animal patients. The goal of the present study was to develop a novel biodegradable sheath/core bicomponent monofilament fiber stent using polydioxanone (PDO) as sheath material and polycaprolactone (PCL) as core material. The biodegradable PDO/PCL sheath‐core monofilaments were developed by a conjugated melt spinning method. Parameters that guarantee stable processing of PDO and PCL during coaxial extrusion with different core/sheath volume ratios were explored. The prototype biodegradable fiber stents were designed into tubular shape using the cross‐and‐hook knitting handmade method. The dimension of the fiber stent was 2.5 mm in length and 6 mm in diameter. Microscopic studies of the PDO/PCL sheath‐core monofilament fiber showed that diameter of monofilament was 0.34‐0.38 mm and diameter of PCL of core that ranged from 0.15 to 0.25 mm. The tensile strength of the monofilament fiber with draw ratio 6 were higher than 3 gf/d and elongation at break were lower than 30%. The physical properties such as biodegradation behaviors, deploy force, radial force and others of the PDO/PCL sheath‐core monofilament stents were also investigated. In conclusion, this novel biodegradable fiber stents can find valuable applications in treatment of intestinal obstruction and stenosis clinically for companion animals.
Biodegradable vascular scaffold with modified magnesium hydroxide coating for improved re‐endothelialization and anti‐inflammation
1CHA University
Treatment of cardiovascular disease was demonstrated by implantation of a biodegradable vascular scaffold (BVS) in patients. The most commonly used biodegradable polymer in BVS is poly(L‐lactic acid) (PLLA), which has high biocompatibility, biodegradability, and mechanical properties. However, the implantation of BVS leads to restenosis and non‐infectious inflammatory reactions in the body by acidic degradation byproduct of PLLA. Magnesium hydroxide (MH) can prevent inflammatory reactions by neutralization of acidic byproducts, and everolimus (EVL) is well known as a drug for restraining stenosis. In this study, we fabricated coated BVS with oligo‐D,L‐lactic acid grafted MH (GFMH) and EVL. The acidic neutralization effect of the GFMH was elucidated by measuring pH change in PBS solution of the coated BVS. The coated BVS indicated high biocompatibility and low inflammatory reaction on endothelial cell (EC) and inhibited smooth muscle cell (SMC) proliferation. High blood compatibility was demonstrated in coated BVS through increased albumin adsorption to fibrinogen and decreased adhesion and activation of platelet. Moreover, in in vivo large porcine models, the coated BVS showed excellent re‐endothelialization, anti‐inflammation, and anti‐thrombotic effects. As a result, we suggest the functional BVS for the treatment of cardiovascular disease.
Improvement of mechanical properties and anti‐inflammatory effect of poly(L‐lactic acid) by formation of stereocomplex structure and surface‐ modified magnesium hydroxide
1CHA University, 2Sungkyunkwan University
Biodegradable polymers such as poly(glycolic acid) (PGA), poly(lactic‐co‐glycolic acid) (PLGA), poly(lactide‐co‐caprolactone) (PLCL), and poly(lactic acid) (PLA) have high biocompatibility and biodegradability due to degradation after a certain period in the human body. In particular, poly(L‐lactic acid) (PLLA) is widely studied as biomaterials due to its non‐toxicity, elasticity, and excellent mechanical properties. However, the mechanical properties of PLLA are insufficient for use in biomaterials such as biodegradable cardiovascular scaffolds. Among the current methods for improving mechanical properties of PLLA, the formation of stereocomplex structure by combining enantiomeric PLLA and poly(D‐lactic acid) (PDLA) is adopted to have excellent biocompatibility. Furthermore, the acidic by‐products of PLLA cause inflammatory responses in the body. Magnesium hydroxide (MH), which has a pH neutralizing effect, inhibits inflammatory responses caused by the acidic degradation product of PLLA. In this study, the PLLA composites were combined with PLLA/PDLA stereocomplex microparticles (SC) and surface‐ modified MH nanoparticles with oligolactide. The SC improved mechanical properties formed by stereocomplex structure in the PLLA composites. The surface‐modified MH nanoparticles using oligomer also enhanced mechanical properties and inhibited inflammatory responses by forming a stereocomplex structure with PLLA chains. Furthermore, the PLLA composite containing MH nanoparticles had an antibacterial effect and increased the viability of human vascular endothelial cells. This technology is expected to have a high potential for the development of PLLA composites for various biomedical materials, such as biodegradable cardiovascular scaffolds.
Gelatin based edible 3D‐porous scaffolds for culture meat applications
1School of Chemical Engineering and Research Institute of cell culture, Yeungnam University, Gyeongsan‐38541, Republic of Korea, 2School of Chemical Engineering, Yeungnam University, Gyeongsan‐38541, Republic of Korea
Cell cultured meat is artificial meat created by in vitro growing animal‐derived cells, and it has gained a lot of attention as a potential future protein source. Scaffolds are essential components that can be used as artificial extracellular matrices to provide cell growth and differentiation in developing cultured meat. Herein we developed gelatin based edible scaffold crosslinking with proanthrocyanidins. The scaffolds have pore sizes range from 100‐300 μm with good compressive properties were obtained. Gelatin scaffolds allowed C2C12 myoblasts to proliferate and differentiate without the need for specialized cell adhesion proteins. The low‐cost and edible approach provided the scaffolds with improving the cell growth and differentiation have more beneficial for culture meat applications.
Xeno‐ and serum‐free manufacturing of a hypoxic culture enhanced mesenchymal stem cell (MSC)‐derived extracellular matrix (ECM)‐based biomaterial for therapeutic angiogenesis
1Institute for Tissue Engineering and Regenerative Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, Hong Kong Special Administrative Region of China, 2School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New
Territories, Hong Kong, Hong Kong Special Administrative Region of China
Current therapeutic approaches to achieve revascularisation of ischaemic tissues, such as cell‐based therapies and growth factors therapies have numerous limitations and drawbacks, prompting the development of a new biomaterial for therapeutic angiogenesis. We have recently engineered a MSCs‐ derived ECM‐based biomaterial that was shown to exhibit superior pro‐angiogenic and pro‐healing properties in a skin wound model. Here we seek to employ a xeno/serum‐free system for material manufacturing to pave the way towards clinical application, as well as exploit hypoxic culture to further enhance its pro‐angiogenic properties. In brief, we evaluated 5 commercially available xeno‐ and/or serum‐free media, by examining ECM yield and the pro‐angiogenic properties of the resulting biomaterials. We also hypothesized that synthesis of this biomaterial under hypoxic culture conditions of MSCs, will further enhance their production of pro‐angiogenic factors, which subsequently will be incorporated into the material. Results have shown that the use of all xeno‐ and/or serum‐free media has promoted the deposition in the amount of ECM structural proteins (i.e. fibronectin and collagen I) as compared to serum supplemented basal medium, leading to an increased yield. Biomaterials produced using these media also showed augmented pro‐angiogenic bioactivity. Finally, through the combinational use of xeno‐ and serum‐free medium and hypoxic culture, the pro‐angiogenic potential of the resulting ECM‐based material was further enhanced. In conclusion, we have demonstrated that the use of xeno‐ and serum‐free system combined with hypoxic culture can further enhance the pro‐angiogenic potential of our ECM‐based materials, while paving the way towards clinical application.
Heat‐confined tumor‐docking reversible thermogel potentiates systemic antitumor immune response during near‐infrared triggered photothermal therapy in triple‐negative breast cancer
1Chonnam National University, South Korea
Triple‐negative breast cancer (TNBC) features immunologically “cold” tumor microenvironments with limited cytotoxic T lymphocyte (CTL) infiltration. Although ablation therapies have demonstrated modulation of “cold” TNBC tumors to inflamed “hot” tumors, recruitment of myeloid derived suppressor cells (MDSCs) at the tumors post ablation therapies limits the infiltration of CTLs and challenge the antitumor immune responses. Here, a thermal ablation immunotherapy strategy is developed to prevent the immune suppressive effects of MDSCs during photothermal ablation and induce a durable systemic antitumor immunity to eradicate TNBC tumors. An injectable pluronic F127/hyaluronic acid (HA)‐based hydrogel embedded with manganese dioxide (BM) nanoparticles and TLR7/8 agonist resiquimod (R848) (BAGEL‐R848), is synthesized to induce in situ laser‐assisted gelation of the hydrogel and achieve desired ablation temperatures at a low laser‐exposure time. Upon 808‐nm laser irradiation, a significant reduction in the tumor burden is observed in BAGEL‐R848‐injected 4T1 tumor‐bearing mice. The ablation induced immunogenic cell death and sustained release of R848 from BAGEL‐R848 promotes dendritic cell maturation and reduced MDSCs localization in tumors. In addition, inflammatory M1 macrophages and CD8+IFN+ CTL are enriched in distant tumors in bilateral 4T1 tumor model, preventing metastatic tumor growth and signifying the potential of BAGEL‐R848 to treat TNBC.
Physicochemical properties of dexamethasone‐loaded polydioxanone monofilament fiber with different draw ratio
1Korea Textile Development Institute, 2Korea Institute of Science and Technology, 3M.I.Tech, Co. Ltd.,
4CHA University School of Medicine
Polydioxanone (PDO) is a type of polyesters widely utilized for biodegradable medical devices, showing relatively long‐term bio‐absorbable time and higher tensile strength due to semi‐crystallinity. Despite the wide‐ranged use of PDO, unmatched mechanical and biodegradable properties and inflammatory responses caused by acidic hydrolytic degradation still need to be improved. Our hypothesis was that drug infusion in a PDO matrix and a drawing process can effectively improve the above‐mentioned problems. The aim of this study was to develop PDO monofilaments containing dexamethasone (DEX) as an anti‐ inflammatory drug and to investigate how their mechanical and physical properties, biodegradation, and drug release behavior are affected by a drawing process. A series of DEX‐loaded PDO monofilaments drawn at different draw ratios through a melt spinning process was developed to investigate changes in drug release behavior, mechanical properties, and biodegradation. Drawn PDO monofilaments presented decreased diameters, increased tensile strengths, and reduced elongation at break. The XRD analysis of DEX‐loaded PDO monofilament showed changes in crystallinity due to the drawing process. The drawing process also changed behaviors of degradation and drug release, showing reduced degradation rates and long‐term sustained release rates at higher draw ratios. Taken together, obtained characteristic properties and their controllability are expected to be favorable for practical applications requiring invasive and longer implantation such as non‐vascular stents, surgical sutures, and face‐lifting materials.
Fabrication and mechanical compatibility of beta‐tricalcium phosphate/alumina/polycaprolactone scaffolds for bone tissue regeneration
1Industry 4.0 Convergence Bionics Engineering, Pukyong National University, 2Major of Biomedical Engineering, Division of Smart Healthcare, College of Information Technology and Convergence, Pukyong National University
Recently, three‐dimension printed scaffolds have been probed to repair or regenerate bone defects. The physical properties of scaffolds are promising factors in promoting tissue growth and physiological functions in bone reconstruction. This study fabricated polycaprolactone (PCL) blended with β‐TCP/α‐ Al2O3 composites powders from 10%, 20%, and 30% by extruder‐based three‐dimensional printing. The prepared fabricated b‐TCP/α‐Al2O3/PCL scaffolds were employed for the physicochemical characterization, wettability test, microstructure features, mechanical strength, and biological assessments. From the resultant, the physicochemical characterization and wettability test reveals the structural stability and scaffolds stated hydrophilic nature, which transpires the scaffolds to attract cell adhesion with living bone cells. The morphological features validate that b‐TCP/α‐Al2O3/PCL scaffolds were deposited with interconnected pore structures, making the platform for exchanging nutrients and biological functions. Compressive and flexural mechanical testing shows gradual augmentation in 10%> 20 % >30% of Young's modulus of the b‐TCP/ α‐Al2O3/PCL scaffolds. In the cellular activity test, the presence of b‐TCP and PCL validates its excellent biocompatibility, cell adhesion, and proliferation with the host tissues. This study illustrates the incorporation of ceramics composites in PCL scaffolds, leading to better mechanical strength, cell adhesion, and cellular activity with bone tissue cells.
Hyaluronan‐coated Prussian blue nanoparticles mitigate oxidative stress and inflammation during LPS‐induced peritonitis
1CHONNAM NATIONAL UNIVERSITY
Excessive inflammatory response during sepsis causes irreversible damage to healthy tissues and results in multi‐organ failure. During infection, bacterial endotoxin‐triggered inflammatory responses in macrophages facilitate the recruitment of circulating leukocytes, including neutrophils and monocytes. A key component that aggravates the systemic inflammatory response is the generation of stable reactive oxygen species such as hydrogen peroxide (H2O2). In this study, we present a versatile strategy to reduce the activation of tissue‐resident macrophages and prevent leukocyte infiltration in an LPS‐induced endotoxemia model. We designed and synthesized hyaluronic acid‐stabilized Prussian blue (HAPB) nanoparticles and validated their activity in the dismutation of H2O2 in LPS‐induced tissue‐resident macrophages. Hyaluronic acid provided stability and enhanced the intracellular uptake of insoluble Prussian blue via the CD44 receptor on LPS‐activated macrophages. Following HAPB administration to an LPS‐induced peritonitis murine model, the level of the M1 inflammatory macrophage population decreased, and the infiltration of neutrophils along with monocytes was suppressed. Overall, we have developed biocompatible Prussian blue nanoparticles to ameliorate inflammatory stress in LPS‐induced endotoxemia by scavenging the intracellular peroxide thereby inhibiting inflammatory cascade in tissue‐ resident macrophages. Therefore, HAPB nanoparticles may potentially be used as novel nano‐stress relievers in sepsis. The nanomaterials may have clinical application in sepsis and in other inflammatory diseases involving peroxides as key inflammatory agents.
Inflammation‐sensing catalase‐mimicking nanozymes alleviate acute kidney injury via reversing local oxidative stress
1Chonnam National University
The reactive oxygen species (ROS) and inflammation, a critical contributor to tissue damage, is well‐ known to be associated with various disease. The kidney is susceptible to hypoxia and vulnerable to ROS. Thus, the vicious cycle between oxidative stress and renal hypoxia critically contributes to the progression of chronic kidney disease and finally, end‐stage renal disease. Thus, delivering therapeutic agents to the ROS‐rich inflammation site and releasing the therapeutic agents is a feasible solution. We developed a longer‐circulating, inflammation‐sensing, ROS‐scavenging versatile nanoplatform by stably loading catalase‐mimicking 1‐dodecanethiol stabilized Mn3O4 (dMn3O4) nanoparticles inside ROS‐ sensitive nanomi- celles (PTC), resulting in an ROS‐sensitive nanozyme (PTC‐M). Hydrophobic dMn3O4 nanoparticles were loaded inside PTC micelles to prevent premature release during circulation and act as a therapeutic agent by ROS‐responsive release of loaded dMn3O4 once it reached the inflammation site. The findings of our study demonstrated the successful attenuation of inflammation and apoptosis in the IRI mice kidneys, suggesting that PTC‐M nanozyme could possess promising potential in AKI therapy. This study paves the way for high‐performance ROS depletion in treating various inflammation‐related diseases.
Tumor microenvironment‐regulating immunosenescence‐independent nanostimulant synergizing with near‐infrared light irradiation for antitumor immunity
1Chonnam National University
The combination of photothermal therapy (PTT) and toll‐like receptor (TLR)‐mediated immunotherapy can elicit antitumor immunity and modulate the immunosuppressive tumor microenvironment (TME). Unlike other TLRs, TLR‐5 is a promising target for immune activation, as its expression is well‐ maintained even during immunosenescence. Here, we developed a unique tumor microenvironment‐ regulating immunosenescence‐independent nanostimulant consisting of TLR‐5 adjuvant Vibrio vulnificus flagellin B (FlaB) conjugated onto the surface to an IR‐780 loaded hyaluronic acid‐stearylamine (HIF) micelles. These HIF micelles induced immune‐mediated cell death via PTT when irradiated with a near‐ infrared laser. In comparison with PTT alone, the combination of in situ‐generated tumor‐ associated antigens produced during PTT and the immune adjuvant FlaB demonstrated enhanced vaccine‐ like properties and modulated the TME by suppressing immune‐suppressive regulatory cells (Tregs) and increasing the fraction of CD103+ migratory dendritic cells, which are responsible for trafficking tumor antigens to draining lymph nodes (DLNs). This combinatorial strategy (i.e., applying a TLR‐5 adjuvant targeted to immunosenescence independent
TLR‐5 and the in situ photothermal generation of tumor‐associated antigens) is a robust system for next generation
immunotherapy and could even be applied in elderly patients, thus broadening the clinical scope of immunotherapy strategies.
BMP‐2 immobilized lubricated orthopedic implant coating suppresses bacterial infection and improves osseointegration
1Yonsei University
Orthopedic implant surgeries entail a high risk of bacterial infections. To prevent the infection, various strategies have been studied. One of their main approaches is implant surface coatings, which inhibit bacterial adhesion and biofilm formation. Among them, the recently reported lubricant‐infused implant surface showed excellent repellency against various biomolecules and bacteria. Combining the micro/nanostructured rough and low surface energy of fluoro‐silanized implant surface tightly captures the thin lubricant layer to possess the superior anti‐biofouling capacity. However, although the lubricant‐ infused implant exhibits enhanced anti‐bacterial properties, its extreme repelling nature also hinders osteoblast adhesion to the implant surface. This critically limits the application of lubricant coating to the intramedullary nails since they require significant osteoblast adhesion followed by osseointegration property. Here, we report BMP‐2 immobilized lubricated coating to ensure both anti‐bacterial and osseointegration abilities. The coating induces substantial osteoblast adhesion through immobilized BMP‐ 2 while inhibiting non‐specific biomolecules and bacterial adhesions. We demonstrated the anti‐bacterial and osseointegrative properties both in vitro and in vivo. The in vitro results showed the superior anti‐ biofouling ability of the coating against the various immune‐related proteins, blood, and bacteria. Also, significantly improved osteoblast adhesion and its differentiation were characterized in vitro. The intramedullary nail with coating was investigated in a rabbit model. The in vivo results showed both anti‐ bacterial properties of fully lubricated‐ and BMP‐2 immobilized lubricated implants while only the BMP‐ 2 immobilized implants exhibited enhanced osseointegration with bone tissues. This suggests the coating to be a promising candidate for a successful orthopedic implant.
Electrochemical identification of naïve and primed PSCs based on cellular metabolism
1School of Integrative Engineering, Chung‐Ang University, Seoul 06974, 2College of Pharmacy, Seoul National University, Seoul 08826
Pluripotent stem cells (PSCs) exist in naïve or primed states based on their origin. For in vitro culture, these PSCs require different supplements and growth factors to maintain pluripotency and guide differentiation, which are key functions of stem cells. However, owing to their similar phenotypic features, identifying both cell types without harming cellular functions is challenging. This study reports an electrochemical method that enables simple, label‐free, and non‐destructive detection of naïve mouse embryonic stem cells (ESCs) based on the differences in cellular metabolism. Two major metabolic pathways to generate adenosine triphosphate (ATP)—glycolysis and oxidative phosphorylation (OXPHOS)—were blocked, and it was found that mitochondrial energy generation activity is the origin of the strong electrochemical signals of naïve ESCs. The number of ESCs is quantified when mixed with primed ESCs or converted from naïve–primed switchable metastable ESCs. The mouse PSCs derived from doxycycline‐inducible mouse embryonic fibroblasts (MEFs) are also sensitively identified among other cell types such as unconverted MEFs and primed PSCs. The developed sensing platform operates in a non‐invasive and label‐free manner. Thus, it can be useful in the development of stem cell‐derived therapeutics and the establishment of in vitro organ‐mimicking models for drug screening and toxicity assessment.
Evaluation of cartilage regeneration efficacy of recombinant human transforming growth factor‐beta 3(rhTGF‐beta3) in rabbit knee model
1Research Center, CGBIO Co., Ltd,
Growth factors (GFs) are vital for tissue engineering. TGF‐β3 is known to promote chondrogenic differentiation of MSCs in vitro and in vivo. The transforming growth factor‐β (TGF‐β) family has received considerable attention because of its ability to induce MSCs toward chondrogenesis by activating the SMAD signaling pathway and up‐regulating chondrogenic genes and reported that TGFβ significantly induced the expression of KDM4B in MSCs and therefore enhanced chondrogenic differentiation of MSCs.
In microfracture, the introduction of multiple perforations into the subchondral bone of the cartilage defect leads to bleeding, allows mesenchymal stem and progenitor cells from the bone marrow to enter the defect, and induces formation of cartilaginous repair tissue. Although clinical studies demonstrated that microfracture shows good results in the short term the repair tissue induced by microfractures has been shown to be of a hyaline to fibrous appearance with limited short‐term durability.
In this study, growth factor TGF‐β3 was loaded on a hyaluronic acid gel cross‐linked with BDDE by concentration and produced in an injectable form. Then, by applying the gel prepared to the rabbit femur micro‐fracture model, cartilage regeneration was analyzed visually and histologically by period. Finally, the OARSI system was applied and evaluated to confirm the joint cartilage regeneration ability.
In conclusion, it was confirmed that the cartilage regeneration ability of the hyaluronic acid gel mixed with TGF‐β 3 at a concentration of 60 PPM was the best.
Acknowledgment: This research was supported by the Ministry of Trade, Industry & Energy (MOTIE, Korea) (grant number: 20014222)
Programmed ‘triple‐mode’ anti‐tumor therapy: Improving peritoneal retention, tumor penetration and activatable drug release properties for effective inhibition of peritoneal carcinomatosis
1Chonnam National University
Peritoneal carcinomatosis (PC) is a fatal condition arising in the gastrointestinal tract. Herein, we synthesized a lithocholic acid (LCA)‐ conjugated disulfide‐linked polyethyleneimine (ssPEI) micelle (LAPMi) nanoconstruct by covalently conjugating ssPEI and LCA, thereby forming positive charged nanomicellar structures loaded with paclitaxel (PTX) (LAPMi‐PTX) for intraperitoneal chemotherapy. The incorporation of a positive surface charge aided in prolonging the peritoneal retention time, presumably via ascites‐induced protein corona formation, and the subsequent size expansion caused resistance against undesired clearance through lymphatic openings. Furthermore, preferential tumor penetration by LAPMi‐PTX is attributable to the permeation‐ enhancing properties of LCA, and the subsequent tumor activatable drug release was induced by the presence of disulfide linkages. By integrating these properties, LAPMi exhibited prolonged peritoneal residence time, enhanced tumor penetration and chemotherapeutic effect evidenced by in vitro, tumor spheroid and in vivo studies. In conclusion, we provided a new paradigm of intractable PC treatment by enabling the prolonged residence time of the nanoconstruct, thereby enhancing tumor penetration and anti‐tumor therapy.
Complex coacervate as a localized drug delivery and adhesive coating for wound healing patches
1seoul national university, 2Seoul national university, 3Yonsei university, 4Korea institute of science and technology (KIST)
Complex coacervation is a liquid‐liquid phase separation, or liquid droplets formation, phenomenon that occurs when two oppositely charged macromolecules are interacting for net charge neutralization. From food additives to biotechnology, coacervates are applied in various research areas. From our previous studies, complex coacervate of fucoidan, a marine‐derived glycosaminoglycan, and poly‐l‐lysine (PLL) has shown promising protein delivery abilities. It was able to release cytokines and proteins in controlled manner with superior encapsulation yields for cancer immunotherapy and tissue engineering applications. In this study, to surpass current wound healing patches, fucoidan was functionalized with dopamine (DOPA) to introduce adhesive properties to the coacervate system. DOPA conjugated fucoidan‐PLL coacervate was able to rapidly and homogeneously coat collagen sponges with Fibroblast Growth Factor (FGF), which inhibited contamination from preparation process. Moreover, DOPA conjugation was able to enhance adhesion properties to prevent the patches from interfacial delamination; allowing prolonged FGF release in the wound area. The DOPA conjugated fucoidan‐PLL coacervate coating showed promising adhesive and bioactive properties for enhancing current wound healing patches.
Reactive oxygen species suppressive kraft lignin‐gelatin based antioxidant hydrogels for chronic wound repair
1Department of Chemical and Biomolecular Engineering, College of Engineering, Yonsei University,
2Transdisciplinary Department of Medicine and Advanced Technology, Seoul National University Hospital, 3College of Medicine, Seoul National University, 4Biomedical Research Division, Korea Institute of Science and Technology (KIST)
Chronic wound is difficult to repair because the inflammatory responses generate high level of reactive oxygen species (ROS) at wound sites. ROS delays inflammatory responses and inhibits normal wound healing process. Therefore, ROS scavenging is critical for chronic wound healing. Herein, plant‐derived antioxidant containing hydrogel was developed by cross‐linking kraft lignin and gelatin (Klig‐Gel). High ROS scavenging activities were confirmed by various antioxidant capability evaluations, and in vitro natural antioxidants expression. Superoxide, catalase and glutathione expression tests exhibited reduction of ROS, which indicated the suppression of oxidative stress. Mechanical properties of Klig‐Gel hydrogel were tailorable by introducing different concentrations of kraft lignin to the gelatin matrix. The micro‐ structure, biocompatibility, pH neutralization and antibacterial ability of Klig‐Gel hydrogels was able to provide a supportive environment for chronic wound repair. Klig‐Gel hydrogel is a promising ROS scavenging biomaterial that can be applied in various chronic wound healing.
BORON‐doxorubicin‐chitosan scaffolds as dual functional carriers with antitumor efficacy and bone regeneration ability
1University of Zagreb Faculty of Chemical Engineering and Technology, 2University of Zagreb Faculty of Science
Biologically compatible scaffolds have been considered as promising platforms for tissue regeneration, drug delivery and tumor treatments. Chitosan‐based scaffolds can serve as pH‐responsive drug carriers with targeted delivery resulting in less invasive tumor treatments. Going further, development of dual‐ functional carriers that will provide simultaneous antitumor efficacy and induce tissue regeneration through therapeutic ions can advance current tumor treatments.
Here, boric acid‐containing crosslinked chitosan scaffolds were prepared as pH‐responsive carriers for doxorubicin, a chemotherapy drug used to treat tumors such as osteosarcoma, while simultaneously inducing bone regeneration through therapeutic effect of boron. The encapsulation of boric acid and doxorubicin was confirmed by FTIR and XRD analysis, while doxorubicin release from chitosan‐based scaffold was evaluated in phosphate buffers of different pH (6.0 and 7.4) during 24 hours using HPLC method. The obtained scaffolds exhibit a highly porous and interconnected microstructure capable of high drug encapsulation efficiency. Due to chitosan's polycationic nature, the obtained scaffolds exhibit higher swelling and drug release at lower pH characteristic for tumor microenvironment making them possible candidates for targeted drug delivery. The cytotoxicity of prepared doxorubicin carriers was evaluated on human sarcoma cells at pH 6.0 indicating proposed materials as cytotoxic agents.
Functionalization, preparation and use of stem cell‐laden bio–based photo‐clickable hydrogels for spinal cord injury treatment
1CNRS@CREATE Ltd, CREATE Tower, #08‐01, 1 Create way, Singapore 138602 / School of Chemical and Biomedical Engineering ‐ Nanyang Technological University, 62 Nanyang Drive, Singapore, 637459, 2School of Chemical and Biomedical Engineering ‐ Nanyang Technological University, 62 Nanyang
Drive, Singapore, 637459, 3Universite Claude Bernard, INSA de Lyon, Universite Jean Monet, CNRS, UMR 5223, 15 bd A. Latarjet 69622 Villeurbanne Cedex France/CNRS@CREATE Ltd, CREATE Tower, #08‐01, 1 Create way, Singapore 138602, 4School of Chemical and Biomedical Engineering ‐ Nanyang Technological University, 62 Nanyang Drive, Singapore, 637459/ Lee Kong Chian School of Medicine, Nanyang Technological University, 11 Mandalay Road Singapore 308232/School of Materials Science and Engineering, 50 Nanyang Avenue Singapore 639798, 5Institute of Cell and Molecular Biology, 61 Biopolis Drive, Singapore 138673
Spinal cord injury (SCI) can cause severe irreversible motor, sensory, and functional disorders. The design of biomaterials able to encapsulate neural stem cells (Spinal Cord Progenitor Cells‐SCPCs) and mimic native neural tissue behaviour have been regarded as a promising strategy to restore neurological function. In this study, we propose the functionalization of chitosan and gelatin with photo‐clickable groups, particularly methacryl and thiol groups, allowing the preparation of 3D hydrogels loaded with SCPCs. The functionalized biopolymers were successfully obtained, and then characterized to confirm their macromolecular structure and determine their degree of substitution (DS): methacrylate chitosan (ChMA, DS of ∼26 %), methacrylate gelatin (GelMA, DS of ∼84 %) and thiolate gelatin (GelSH, DS of
∼ 75 %). The concentration of the ChMA:GelMA and ChMA:GelSH mixtures, and conditions of gelation were optimized. In vitro tests showed the possibility of SCPCs encapsulation with high cell viability (∼ 80
%). Moreover, 3D hydrogel structures were prepared by digital light processing enabling specific neural guide designs for the tissue regeneration process. In vivo implantation of these materials is ongoing, and the preliminary results are very promising. The ability to process hydrogels in mild conditions and form a 3D hydrogel structure loaded with cells via photo‐crosslinking, make these materials potentially high impact. Summarizing, these preliminary results demonstrate the potential of these materials for neural tissue regeneration coupled with cell therapy, therefore potentially offering a possible spinal cord injury treatment.
Synergetic effect of dual oxygen and hydrogen peroxide release on enhancing the osteogenic and antibacterial properties of injectable hydrogels
1Department of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of Korea (*kdp@ajou.ac.kr)
Injectable hydrogels have been extensively used as therapeutic vehicles and implants for the treatment of various diseases as well as for tissue repair and regeneration. Particularly, horseradish peroxidase (HRP) and hydrogen peroxide (H2O2)‐catalyzed gelation system has attracted much attention due to their ease of handling and controllable hydrogel properties. In this study, calcium peroxide (CaO2) was introduced as an oxygen (O2) and H2O2 generating reagent that can gradually supply a radical source for HRP‐ catalyzed crosslinking reaction and enhance the biological activity of the hydrogel. The physico‐chemical properties of hydrogels such as gelation time, mechanical strength, microstructure, and degradation rate were well controlled with varying the concentrations of HRP and CaO2. In addition, the controlled and sustained release of bioactive molecules (H2O2, O2, and Ca2+ ions) from hydrogels could synergistically stimulate the cellular behaviors including attachment, proliferation, migration, and differentiation of human mesenchymal stem cells, in comparison with cell treated with conventional HRP/H2O2‐catalyzed hydrogels. Moreover, the hydrogels exhibited antibacterial efficacy against both gram‐negative and gram‐ positive bacteria, dependent on the H2O2 and Ca2+ release amount. These promising results lead us to believe that in situ hydrogel formation induced by HRP/CaO2‐mediated reaction has the potential for a wide range of biomedical applications, such as infection treatment, bone regeneration and tissue regenerative medicines.
Decellularized bovine pericardial patch loaded with stem cells enhance the mechanical strength and biological healing of chronic retracted rotator cuff tear in a rat model
1Asan Institute for Life Sciences, Asan Medical Center, 2Department of Orthopaedic Surgery, Asan Medical Center
Despite current technological advancements, high retear rates remain a concern in the surgical repair of large‐to‐massive rotator cuff tears. The purpose of this study was to determine whether the addition of a decellularized bovine pericardial patch loaded with mesenchymal stromal cells enhanced bone‐to‐tendon healing and improved the biomechanical strength of large‐to‐massive rotator cuff tears in a small animal model. In this study, we fabricated decellularized bovine pericardium patches and loaded them with stem cells (patch‐SC). The rats were allowed free cage activity for 6 weeks after tear creation. A total of 18 rats were randomly allocated to repair‐only (control), repair with patch augmentation, or repair with patch‐SC augmentation groups. After creating chronic retracted rotator cuff tears in rats, we augmented the repair with patch‐SC and evaluated the histology and biomechanical load‐to‐failure rate. Fibrocartilage and tidemark formation at the bone‐to‐tendon interface and collagen fiber density and orientation were better in the patch‐SC group than in the control or patch group. Load‐to‐failure in the patch‐SC and patch groups was higher than that in the control group. Decellularized bovine pericardial patches loaded with stem cells enhanced healing in terms of histology and mechanical strength following rotator cuff repair in a rat model.
Engineering of islets with chitosan microspheres and on‐demand loading of heparin for preventing IBMIR in islet transplantation
1School of Medicine, Sungkyunkwan University, 2066, Seobu‐ro, Jangan‐gu, Suwon‐si, Gyeonggi‐do, Republic of Korea, 2College of Pharmacy, Keimyung University, 42601, Daegu, Republic of Korea
EGCG‐embedded hybrid gelastin injectable hydrogel a provisional bio‐ template for future application: in vitro evaluation
1Centre for Tissue Engineering & Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur 56000, Malaysia, 2Department of Chemistry, CICECO ‐ Aveiro Institute of Materials, University of Aveiro, Campus Universitu00e1rio de Santiago, 3810‐193, Aveiro, Portugal., 3School of Applied Physics, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
Immediate treatment for cutaneous injury is a realistic approach to accelerate the rate of healing and minimize the risk of complications. Functionalized biomaterials have been proven to be a potential strategy to embark the chronic skin wound management e.g. diabetic ulcers. This study aimed to evaluate the effectiveness of gelatin‐elastin (Gelastin) injectable hydrogel incorporated with epigallocatechin gallate (EGCG) to promote wound closure and newly‐formed skin. Briefly, gelatin hydrogel was pre‐ mixed homogenously with EGCG followed by elastin and genipin (GNP) as the crosslinking agent.
MTT Cell Toxicity assay of the fabricated hydrogels was observed, followed by the physicochemical profile via water vapor transmission rate (WVTR) whereas, for cell‐bioscaffold interaction, various assays such as live and dead assay, MTT Cell Viability assay, Cell Migration, and Immunocytochemistry (ICC) has been utilized.
EGCG 0.037mg (w/v) shows the most optimum cell viability (<100%) for MTT Cell toxicity assay whereas the WVTR of the crosslinked hybrid biomatrix demonstrated >1500 g/m−2 h−1, an optimal moisture content for cell proliferation and regular function. Moreover, cell‐scaffold interaction shows a cell viability average of above 90% for live and dead assay, a consistent increase of cell viability from day 1 to day 7 (85% to <150%) for the MTT Cell Viability assay, and cell successfully migrate through from day 0 to day 7 and the embedded human dermal fibroblast successfully express vinculin for the ICC assay.
Hence, the Gelastin‐EGCG hydrogels provide optimum characteristics to be used as a provisional skin biotemplate.
Engineering of biocompatible hybrid gelatin‐PVA bioink for potential chronic wound treatment
1Centre for Tissue Engineering Centre and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Cheras, Kuala Lumpur 56000, Malaysia, 2Department of Biology, Faculty of Science, University of Erciyes, 38039, Turkey
Alginate and gelatin‐based scaffolds for the manufacture of
in‐vitro
reconstructed meat
1YEUNGNAM UNIVERSITY
In vitro meat culturing is regarded as a technological revolution and a novel concept in the field of biotechnology and food science. Since inception the global market for cultured meat have been constantly accelerating under steady pace. The approach for cultured meat production involves the cultivation of skin and muscle cells on a scaffold made of biomaterials. In order to imitate the texture, framework, and physiological attributes of a native meat, and the microstructure of the selected biomaterial is very important. In parallel, efforts are being made to improve the mechanical attributes, porosity, and cell adherence ability of the designed scaffold to recapitulate the three‐dimensional (3D) architect and microenvironment of native meat. Moreover, the designed scaffold should also have the ability to manipulate and proliferate cells without the involvement of any external chemical agents. In the present study edible scaffolds were fabricated using alginate and gelatin and were examined for their morphology and porosity. Further, the scaffolds were tested for their mechanical attributes to be at par with the desired mechanical features to imitate native meat. Skin fibroblast and myogenic cells were grown on the designed scaffolds to investigate their cytocompatibility and adherence property. The appearance of porosity in the scaffolds as a platform for cell growth along with cytocompatibility and ability to proliferate skin and muscle cells indicates the capability of the designed scaffold to recapitulate cultured meat. Further work in this area could open new avenues to design scale up production approach for in vitro meat culturing.
Morphological control of gold nanoparticles to enhances stability of localized surface plasmon resonance signals
1School of Integrative Engineering, Chung‐Ang University, Seoul 06974, Republic of Korea
The mechanism of surface plasmon resonance (SPR) is caused by resonance of the metal surface. Accordingly, the SPR signal properties reflect the height, unevenness, and shape of the sensor surface. We fabricated a spherical gold nanoparticle (GNP)‐based localized SPR (LSPR) sensor by adopting an electrochemistry‐based deposition system for low‐cost and rapid fabrication. However, this method increases the instability of the LSPR signal due to the disadvantage of anisotropic growth of the metal. To solve this drawback, we first stabilized the HAuCl4 solution using TWEEN‐20 as a surfactant, and secured the conditions for gold deposition by optimizing the surfactant conditions. Second, the GNP sphericity was adjusted to the best condition through optimization of the thermal‐annealing method. The value of unevenness of the GNPs on the LSPR sensor was successfully confirmed by field emission‐ scanning electron microscope (FE‐SEM), and height deviation by atomic force microscope (AFM). Additionally, LSPR signal stability of spherical GNPs on LSPR sensors was evaluated through ultraviolet (UV) vis spectrum. As a result, by optimizing surfactant and annealing conditions, the LSPR signal variation was reduced by about 44 times. Therefore, this study suggests that the LSPR signal variation has been successfully reduced to secure the foundation for advanced LSPR sensors research.
Acknowledgement: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (Grant Nos. NRF‐2022R1A4A2000776, NRF‐ 2019M3A9H2031820, NRF‐2022R1A2C4002217) and Korean Fund for Regenerative Medicine funded by Ministry of Science and ICT, and Ministry of Health and Welfare (Grant number RS‐2022‐00070316).
Fabrication and characterization of starch‐based scaffolds for a cultivated meat
1Yeungnam University
It is known that cultured meat is a lab‐grown meat made from stem cells of animals culturing in vitro, which is emerging with a new concept in livestock industry due to growing interest in animal welfare and food security as well as environmental concern. Although many researches have been conducting, it could not be materialized structure, thickness, and texture like real meat.
In this study, we developed an edible scaffold based on starch that is familiar and easy to obtain ingredients and has various food applications. A starch‐based scaffold is appropriate for fabricating a cultured meat owing to be non‐toxic and changeable concentration, density through simple approaches such as addition of water, etc. We added a reinforcing material and D‐sorbitol to reinforce the mechanical properties of starch, and also created an interconnected porous structure to allow adhere, migration, and growth of cells onto fabricated scaffolds. And various characterization was carried out for comparing and analyzing with real meat. In addition, its possibility as a scaffold of cultured meat was discussed through in vitro biocompatibility assay using muscle cells separated from animals.
Cardiomyogenic induction of human mesenchymal stem cells by altered behavior‐driven epigenetic memory on a dendrimer‐immobilized surface
1Osaka University, 2Osaka University, Research Base for Cell Manufacturability
The dynamic cell behavior has a significant impact on the epigenetic profiles that regulate stem cell fate decisions during differentiation. In this study, we found that alterations in migratory behavior‐driven epigenetic memory can lead to enhancement of cardiomyogenic differentiation of human mesenchymal stem cells (hMSCs) during in vitro culture. We employed a dendrimer‐immobilized substrate that displayed d‐glucose to regulate the cell behavior of hMSCs. Cells exhibited the formation of cell aggregates on the 5th‐generation dendrimer (G5) surface through active migration with morphological changes, and these aggregates showed strong expression of the cardiac‐specific marker cardiac troponin T by immunostaining. Analyses of global changes in H3 histone modifications exhibited pattern of increased H3K9ac and H3K27me3, and decreased H3K9me3 compared to those on the PS surface. Using the aggregate‐based passage method on G5 surface, the nuclear response of migration‐driver aggregate behavior lead to the maintenance of modified epigenetic memory, promoting the differentiation of hMSCs into the cardiomyogenic lineage via repetitive migration behaviors on the G5 surface. These results demonstrate that culturing on the G5 surface is an efficient method for the regulation of migratory behavior‐driven epigenetic mechanisms under aggregate‐based passage of hMSCs and is a novel strategy for ensuring cardiomyogenic differentiation.
Development of Scaffold for cultured meat in the form of lumps through edible materials and crosslinking
1School of Chemical Engineering, Yeungnam University, 280 Daehak‐Ro, Gyeongsan, Gyeongbuk 38541, Republic of Korea
Problems with livestock industry has been facing due to carbon emissions as a global problem, the ethical awareness related to animal slaughter of animals. The market of alternative meat is being grown, faced with the necessity of carnivory resulting from giving up eating meat, therefore, the development of cultured meat is being promoted as the possibility of cultured meat became a topic.
Although the minced cultured meat are making progress, It is running up against limitations such as edible ingredients, the process of manufacture in terms of the development of cultured meat. And animal‐ derived materials such as gelatin, collagen, provide the same problems in the same way as above. Here, we developed an edible scaffold based alginate and carrageenan. Their structure and properties were characterized for comparing and analyzing with real meat. Also, their possibility as a substitute of cultured meat was determined via in vitro biocompatibility test using muscle cells separated from animals.
Influence of substrate stiffness on the biomechanical characteristics of hepatocytes and hepatic stellate cells in the context of liver fibrosis
1Indian Institute of Science, 2Indian Institute of Science
Chronic liver diseases, irrespective of their etiology, pass through stages of fibrosis, cirrhosis, and may culminate in hepatocellular carcinoma. Diagnostic techniques show increase in liver stiffness as one progresses through these stages.
Our study focuses on the effect of stiffness of the microenvironment on the characteristics of liver cells like hepatocytes and Hepatic Stellate Cells (HSCs), in the context of fibrosis progression. We further investigate their role as possible accomplices in fibrosis via ECM (extracellular matrix) remodeling processes.
Polyacrylamide gels of different stiffnesses were fabricated to mimic the mechanical stiffness of the matrix under normal (3 kPa), fibrotic (10 kPa), and cirrhotic liver (40 kPa) conditions. The gels and plain coverslips (>1 GPa, control) were coated with Rat‐tail Collagen 1 solution (0.1 mg/mL) using Sulpho SANPAH. Mono‐cultures of Huh7 (Hepatocyte cell‐line) and LX‐2 (HSC cell‐line) cells were incubated on the samples and further experiments were performed.
Atomic Force Microscope (AFM) was used to measure stiffness of the LX‐2 cells incubated on the three different gels and coverslip for 48 h. We observed increase in cell stiffness as substrate stiffness increases. Using confocal microscopy, we observed increase in the cell projected area, nuclear surface area, and nuclear volume of Huh‐7 cells growing on stiffer substrates. MTT assay results showed an increase in the proliferation of LX‐2 and Huh7 cells growing on stiffer substrates. We also observed increase in alpha‐ smooth muscle actin (marker of HSC activation) and MMP‐2 (Matrix Metalloproteinase‐2) gene levels in LX‐2 cells (low in Huh‐7) growing on stiffer substrates.
Development of a mineralized decellularized tissue for soft‐hard inter‐ regional tissue application
1Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2Department of Biomaterials, Okayama University, 3Department of Bioscience and Engineering, Shibaura Institute of Technology
Recent applications of decellularized tissues, which are called as dECM, have included the ectopic use of sheets and powders for three‐dimensional (3D) tissue reconstruction. dECMs are modified (or fabricated) with the desired functions for application to the target tissue including soft‐hard inter‐regional tissues, such as ligament, tendon, and periodontal ligament. The aim of this study was to prepare a mineralized decellularized pericardium to construct a soft‐hard inter‐regional tissue by the 3D fabrication of decellularized pericardium. The decellularized pericardial tissue was prepared using the high hydrostatic pressurization (HHP) method and the surfactant method. The pericardium consisted of the the aligned fibers, and the fibers were slightly disordered in the surfactant decellularized method compared to the HHP decellularization method. The decellularized pericardium was mineralized by using an alternate soaking process with various cycles. The surface of the decellularized pericardium was covered with calcium phosphate precipitates, which were accumulated on the surface with increasing number of soaking cycles. The inside of the HHP decellularized pericardium was mineralized uniformly, whereas the mineralization of the surfactant decellularized pericardium decreased toward the interior. The findings suggest that the decellularization method strongly affects the structure and the mineralized parts of the decellularized pericardium. Also, it was found that fibroblast and osteoblast cells effectively adhered on the decellularized pericaridum with and without mineralization. The mineralized decellularized pericardium could be a candidate material to reconstruct an alternative inter‐regional tissue, such as ligament and tendon.
Plasmid dna nanoparticles for oral therapy
1Korea national university of transportation, 2Hanyang University, 3HanYang University
Herein, a bile acid‐inspired triple padlock oral gene delivery platform is developed, facilitating the protection of the therapeutic gene from gastrointestinal degradation, selective intestinal accumulation through a bile acid‐specific transporter, and transportation of pDNA NPs through the enterohepatic recycling system. This nonviral oral gene delivery nanoparticle exhibits excellent gene expression kinetics in in vitro, in vivo, and ex vivo studies. A single oral dose leads to maintaining normoglycemia for up to 7 days in three different diabetes mouse models and 14 days in diabetic monkeys. Also, the optimized dosage form can reduce nonfast blood glucose levels and hemoglobin A1C within a normal range from the last stage diabetes conditions with a reduction of weight gain from changes of food uptake behavior after treatment once weekly for 20 weeks. Taken together, the current findings could improve the current painful treatment experience of diabetics and thus improve their quality of life.
Process development for nerve regeneration implant using the 3D bio‐ printer
1T&R Biofab
Recently, as the number of neurological and cerebrovascular diseases due to aging has greatly increased, the size of the nerve regeneration market is increasing, and the development of nerve regeneration implant for the treatment of neurological diseases is actively progressing.
Nerve regeneration implant is a medical device that is implanted in a damaged nerve area and helps the regeneration of that area. In this study, a nerve regeneration implant was manufactured using two types of materials, natural and synthetic polymers, and for this purpose, a self‐developed 3D bio‐printing system was applied to the production.
A 3D printing system was used to combine the two materials, by printing a synthetic polymer on a natural polymer material, a biocompatible material with required mechanical strength was produced. Mechanical strength means structural strength required during nerve regeneration, and biocompatibility means creating an environment in which nerves can be quickly regenerated in a short period of time. For this, PCL, a biodegradable polymer, was used for the synthetic polymer, and ADM and v‐dECM were used for the natural polymer.
In conclusion, a 3D bio‐printing system dedicated to nerve regeneration was developed, and a nerve regeneration implant with the required structural strength and biocompatibility was manufactured.
Development of BMP‐loaded bioabsorbable microspheres with calcium‐ binding polymer‐coating for high‐efficiency transport
1Department of Plastic and Reconstructive Surgery, SMG‐SNU Boramae Medical Center, 2Department of Chemistry, Seoul National University
Development of various artificial bone substitutes to overcome the limitations of autologous and allogeneic bone grafts in the treatment of patients due to alveolar bone graft, upper and lower extremity bones, and vertebrae. However, there is no effective treatment for bone defects due to low osteoinduction, osteoconduction, and osteogenesis. Therefore, in this study, PBMP coated Ca2+‐binding poly(lactide‐co‐ glycolide) microparticles (PLGA/PBMP2 microparticles) were treated for bone regeneration effects. In vitro test established optimal conditions for coated low‐dose BMP/PLGA microparticles through cumulative release test, FE‐SEM and X‐ray photoelectron spectroscopy analysis. In vivo test, Sprague Dawley Rat with an 8mm bony defect were produced and they were divided into three groups: Control and HA+ low dose BMP‐2 only, and HA+BMP2 loading PLGA‐PBMP particles groups and measured the degree of morphometric parameters of bone formation in μ‐CT image, and histological characteristics. At 4 weeks, more new bone formation was noted in the HA+BMP2 loading PLGA‐PBMP particles group than in other groups. The present study results indicate that HA+BMP2 loading PLGA‐PBMP particles to produce new bone formation in calvarial defects in a rat model.
Liquid‐type plasma‐controlled in situ crosslinking of silk hydrogel displayed better bioactivities and mechanical properties
1Ajou University, 2Inha University
Silk is one of the promising biomaterials for injectable hydrogel, but its long‐gelation time is the main obstacle for clinical application. Here, we developed of a new in situ crosslinking technique of silk injectable hydrogel using liquid‐type non‐thermal atmospheric plasma (LTP) for wound healing. Scanning electron microscopy, Fourier‐transform infrared spectroscopy, swelling ratio, and rheology test showed that LTP dose dependently modulated the secondary structure of silk. Controlled release of LTP effectors from silk hydrogel was detected over 7 days based on nitrate determination. Cell viability/mobility/favorable morphology and extracellular matrix depositions were increased by the LTP‐ treated silk hydrogel. An in vivo animal model of vocal fold injury or irradiated skin flap was established. The LTP not only changes the mechanical structures of a hydrogel, but also has sustained biochemical effects on the damaged tissue due to controlled release of LTP effectors. These results suggest that LTP precisely controlled in situ crosslinking of silk and LTP‐treated silk hydrogel to enhance wound healing. Cellular and tissue reactions to released LTP from the silk hydrogel were favorable for the regenerative process of the wound; furthermore, mechanochemical properties of the hydrogel were improved by LTP.
Hydroxyl radical generation mediated antibacterial therapeutics by catechol functionalized hyaluronic acid hydrogels and zinc oxide nanoparticles
1Department of Plastic and Reconstructive Surgery, SMG‐SNU Boramae Medical Center, 2Department of Materials Science and Engineering, Seoul National University
Over the past decades, the general approach for antibacterial therapeutics is to utilize photocatalysts including metal oxide nanoparticles such as titanium oxide (TiO2), and ZnO. Catechol, a bioinspired source from the protein of marine mussels, is recognized as one of the best candidates for biomedical applications due to its versatile characteristics. In particular, ZnO nanoparticles have shown a great promise as biomedical applications because of its distinct characteristics. Recently, metal oxide nanoparticles have been introduced to form the metal‐mediated catechol complexes due to obtaining synergetic properties from both metal oxide and catechol characteristics. We aimed to treatment using catechol‐ZnO complex‐based hydrogels (HCZ hydrogel) for antibacterial therapy was demonstrated through the capability to enhance ROS generation and eradicate bacteria. In vitro tests were performed Human dermal fibroblast cells attached on HCZ hydrogel and disk diffusion test against E. coli and S. aureus of HAZ and HCZ hydrogel. In vivo tests were performed bacterial infection model under the subcutaneous skin tissue (BABL/c mice). Site of infection tissue were injected with saline, HAZ, HCZ 3, and HCZ10. Histological analysis and peripheral blood profiles test were performed. By taking advantage of ROS generation during catechol‐ZnO complex formation, this hydrogel platform demonstrated superior ROS generation in vitro and in vivo study as well as superior antibacterial performance in S. aureus compared to HAZ.
Development of cooling system for nerve regeneration implant using biomaterials
1T&R Biofab
In the case of a 3D structure using a thermoplastic material, the more complex the shape, the faster curing is required. In particular, the nerve implant of a complex structure using heterogeneous materials has a precise internal shape, and in order to realize the structure, rapid cooling of the material and bonding of the material itself must be carried out simultaneously in the printing system. In the case of the existing commercialized FDM method, the thermoplastic material is melted and discharged through a nozzle to form a layer, and the structure is manufactured by stacking additional layer on the cured layer. If each layer of the thin and tall structure is not cured and cooled quickly, the structure will collapse and printing the intended shape becomes impossible. This becomes more severe as the thickness of the structure increase and the height of the structure increases. In this study, a method was developed to cool the discharged biomaterial at a desired time after installing a local cooling device around the discharged nozzle. Through this technology, it has the advantage of printing a conduit‐type structure (for example, a nerve regeneration conduit) with a desired thickness and line width and simultaneously controlling the cooling time of different biomaterials.
In conclusion, the 3D bio‐printing system that can produce the desired structure by introducing a cooling device to the ejection part of the 3D printer for precise manufacturing of the complex nerve structure has been developed.
Process development of heterogeneous sheets for nerve regeneration implant
1T&R Biofab
Nerve regeneration implant refers to a medical device that helps tissue regeneration with rapid recovery by implanting the graft material into the damaged nerve area. Recently, as the number of neurological diseases due to aging has greatly increased, and the materials used for nerve regeneration are being actively studied.
This research is developing a nerve regeneration graft using a heterogeneous material. This study includes information on the sheet manufacturing process using natural polymers among heterogeneous materials. Natural polymers were able to produce ultra‐thin sheets using precisely controlled samples through various processing using ADM and v‐dECM. The ultra‐thin sheet is at a level that can be applied to nerve with an inner diameter of 1.5 – 2 mm, and is a soft tissue‐type sheet that has sufficient structural strength as well as biocompatibility. The process of manufacturing an ultra‐thin natural polymer sheet consists of the quality of the raw material, the fine grinding process, compression, and crosslinking, among which the vitrification process that creates a homogeneous sheet shape after fine grinding is the most important.
In conclusion, in this study, a precision sheet for nerve regeneration implant was manufactured using natural polymers, and through this, a graft implant with a fine structure using heterogeneous material could be developed.
Senescent cancer cell‐derived nanovaccine for cancer therapy
1Seoul National University
In recent years, as immunotherapy has shown great effect on cancer treatment, therapeutic cancer vaccines (TCVs) has emerged as a promising technology. However, clinical application of TCVs is challenging due to difficulty of identifying immunogenic tumor antigens and inducing effective antitumor immunity. Also, using exogenous adjuvant to enhance the vaccine immunogenicity has safety concerns. Here, we present senescent cancer cell‐derived nanovesicle (SCCNV) that can provide tumor antigens and induce high antitumor responses without using exogenous adjuvant. SCCNVs are prepared by inducing senescence in cancer cells ex vivo and subsequently extruding the senescent cancer cells through nanoporous membranes. This SCCNVs do not require identification of immunogenic tumor antigen because it contains autologous cancer membrane. Also, SCCNVs contains interferon‐γ and tumor necrosis factor‐α induced during senescence so that it can be act as adjuvants and enhance immunogenicity without using exogenous adjuvants. SCCNVs effectively activate dendritic cells and tumor‐specific T cells, inhibit primary tumor growth, metastasis and tumor recurrence. In clinical settings, it can be used as personalized vaccine because cancer cells are prepared from patient's own cancer cells. SCCNVs also show synergy with immune checkpoint blockades.
Fabrication of poly(HEMA‐co‐MMA) porous scaffold with highly biocompatibility for soft tissue regeneration
1R&D Institute, TE BioS Co., LTD, 194‐41, Osongsaengmyeong 1‐ro, Heungdeok‐gu, Cheongju‐si, KOREA, REPUBLIC OF, 2Department of Biomedical Engineering, Pukyong National University, 45, Yongso‐ro, Nam‐gu, Busan, Korea, REPUBLIC OF
Fabrication of poly(HEMA‐co‐MMA) scaffold having surface roughness and modulus for soft‐tissue engineering
1R&D center, TE BioS Co., Ltd, 194‐41, Osongsaengmyeong 1‐ro, Heungdeok‐gu, Cheongju‐si, KOREA, REPUBLIC OF, 2TE BioS co., Ltd
In situ forming elastin‐like polypeptide hydrogel for injectable drug delivery applications
1Ulsan National Institute of Science and Technology
In situ forming hydrogels are widely explored as injectable drug delivery systems for biomedical applications. It is important for these hydrogels to undergo gelation and deliver the drug in a timely manner upon administration. In this study, lysine‐rich elastin‐like polypeptide (ELP) and aldehyde‐ presenting alginate are crosslinked via Schiff base formation under ambient conditions to generate hydrogel. The physicomechanical and drug release properties of the alginate‐ELP hydrogel are conveniently controlled by the concentrations of alginate and ELP. Furthermore, due to the thermoresponsiveness of ELP, the alginate‐ELP hydrogel undergoes reversible swelling/deswelling at the transition temperature near the physiological temperature. Therefore, the drug release from the alginate‐ ELP hydrogel is expedited via thermoresponsive deswelling. Taken together, the in situ forming alginate‐ ELP hydrogel is a highly attractive injectable drug delivery system capable of programmable release characteristics.
Applicability of chemically‐defined media in the development of cell therapy using keratinocyte
1Xcell Therapeutics Inc.
Keratinocyte culture required that bovine pituitary extract (BPE) or animal‐derived substances. Recently, regulations related to cell therapy have been strengthened not only in Korea but also in major foreign countries, so animal component free media is needed. Some manufacturers have developed chemically‐ defined media and are commercially available, but it has not performed well compared to serum‐ free media.
This study compared the performance of CellCor Kera CD, which is chemically‐defined media, and a BPE‐containing media. We measured the cell establishment rate, cell proliferation, cell characterization and cell activity to confirm the media performance. When keratinocyte was established using the CellCor Kera CD, it was confirmed that harvested cell numbers were similar to or higher than the BPE‐containing media. Similarly, cell growth comparison showed that CellCor CD Kera had a population doubling time (PDT) similar to or higher than BPE‐containing media. Finally, CFU was compared through colony forming assay to confirm the activity of cells cultured in each media. As a result, it was confirmed that cells cultured in CellCor CD Kera more than CFU compared to cells cultured in a BPE‐containing media. In this study, the performance of CellCor Kera CD and commercial BPE‐containing medium was compared to confirm the applicability of chemically‐defined media to cell therapy. Following this result, chemically‐defined media is expected to have a lot of influence on cell therapy development.
Comparative study of mesenchymal stem cell media for culture reproducibility and consistency
1Xcell therapeutics Inc.
The essential of regenerative medicine is cell‐based therapy. Especially, mesenchymal stem cells have been widely studied for therapeutic applications due to their immunomodulatory activity. Such in cell‐ based therapy, culture media is most important thing for quality of cell therapy. Depending on the various raw materials contained in the culture medium, there may be differences in culture performance between production lots. In particular, media containing fetal bovine serum (FBS) or human‐derived extracts have an undefined heterogeneous composition, and thus are more heterogeneous than chemically defined media. In contrast, chemically defined (CD) media has fully defined all components and chemically identified. Thus, CD media scarcely ever Lot‐to‐Lot variation.
To confirm this, the characteristics of adipose‐derived stem cells (ADSCs) according to lot‐to‐lot variations were compared in three different mesenchymal stem cell cultures media (FBS‐containing media, serum‐free media, and CD media). We analyzed ADSCs biological properties, proliferative capacities using multipassage assay, ADSCs specific surface marker expression using FACS, differentiation potency, secretory protein using western blot, genetic stability using karyotyping. As a results, culture using CD media compared to FBS media and serum free media that was little difference in ADSCs biological properties in terms of Lot‐to‐Lot variation.
In conclusion, this study demonstrates that CD media is advantageous in terms of Lot‐to‐Lot variation of culture media and is suitable for mesenchymal stem cell based regenerative medicine.
Applicability of chemically‐defined media in hair follicle cell culture for hair loss cell therapy
1Xcell Therapeutics Inc., 2Epi Biotech Co., Ltd.
Dermal papillary cells (DPCs) of mammalian hair follicles have been known to be important cells for hair follicle development which is a regenerating organ that produces a new hair shaft during each growth cycle. DPCs have been mainly used for transplantation or cell therapy for treatment of hair loss. However, a dedicated chemically‐defined (CD) medium for DPCs has currently yet to be developed.
We developed CellCor DPC CD, an optimized CD media for DPCs. In this study, we compared the performance of serum‐contained medium and CellCor DPC CD by checking the growth rate, cell senescence, the expression of specific markers during DPCs culture and DPC hair inductivity. The growth of DPCs using CellCor DPC CD was faster than a comparative test using serum‐contained media. The testing showed the degree of cellular senescence using Senescence‐associated beta‐galactosidase (SA‐β‐ gal) staining, The use of CellCor DPC CD showed a lower degree of senescence. Additionally, through flow cytometry analysis, the expression of versican (VCAN), a marker indicating the characteristics of DPCs, was also more stable and highly expressed when using CellCor DPC CD. This aspect was also found in the specific gene expression of DPCs. Lastly DPC culture under CellCor DPC CD showed enhanced telogen‐to‐anagen transition.
These results suggest that CellCor DPC CD which was developed by us can be actively used for DPCs for hair loss treatment, and can contribute to the production of high‐quality therapeutic agents. Furthermore, it will provide insights into the growth of the hair loss cell therapy market.
Coating of graphene oxide on a large‐area plastic surface to fabricate a novel cell culture vessel
1Kyungpook national university, 2Kyungpook national university, Kyungpook National University Hospital
In this study, graphene oxide (GO) was coated on the surface of a large‐area polystyrene film by spray coating. The physico‐chemical properties and cell compatibility of the GO coated surface were characterized to evaluate the potential of this coating method for the development of a large‐area cell culture vessel. It was confirmed that 100 nm or more in thickness of GO was coated on the surface through step measurement by name of instrument and Raman Spectroscopy. The transmittance of the GO coated plastic surface was measured to be about 90% or more, enabling observation of the cell morphology, and the coated surface exhibited hydrophilicity due to increased surface energy. As a result, cells hardly grew in the general polystyrene film, but the polystyrene film coated with graphene oxide had cells well attached and grown, and it was found that there was no cytotoxicity. In particular, in the sample spray coated at 93.75mm/s three times, the cell viability was 1.43 times higher than that of the conventional cell culture vessel. It is thought that economical and efficient large‐area cell culture vessel development will be possible if the appropriate surface curvature shape and surface energy are formed through an appropriate graphene oxide coating.
Biological and mechanical properties of xenograft coated with catechol‐ and thiol‐containing binder
1Korea University
Xenograft granules are widely used bone graft materials in clinical practice. It has micro pores and sufficient cancellous bone support to induce adequate vascularization inside the bone defect site. For the clinical success of implanted xenografts, the xenografts need to be agglomerated with a biocompatible binder. Here, catechol and thiol moieties, two key chemical functional groups of the mussel adhesive proteins, were utilized to aggregate xenograft bone granules and bone regeneration. PETMP‐di‐catechol, a binder molecule containing both catechol and thiol, was cross‐linked with xenograft granules and showed higher mechanical properties, blood affinity, and osteoblast cell proliferation and differentiation of the agglomerated xenograft granules than those of the carboxymethylcellulose (CMC) and chitosan catechols, currently used as a clinical bone binder. These results suggest the potential of PETMP‐di‐ catechol as a biocompatible bone graft material in clinical practice.
Octanoyl glycol chitosan activates cell cycle via up‐regulation of cyclin D1 protein and accelerate osteo induction in tonsil derived mesenchymal stem cells
1Department of Biological Sciences and Biotechnology, College of Natural Sciences, Chungbuk National University, 2Department of Polymer Science and Engineering, Chungnam National University, 3Research Group for Biomimetic Advanced Technology, Korea Institute of Toxicology
Biomaterials using polymers have been widely developed to enhance proliferation and functionality of stem cells. recently, a hexanoyl glycol chitosan (HGC) moiety, one of the glycol chitosan (GC) derivatives, was shown to support the proliferation and long‐term self‐renewal capacity of adipose‐ derived mesenchymal stem cells (AD‐MSC). thus developed a new GC derivative, octanoyl glycol chitosan (OGC), which was designed to possess better water solubility, compared to other known GC derivatives such as HGC. we evaluated the efficacy of OGC in improving cellular functions by assessing its effect on the proliferation and differentiation capacity of tonsil‐derived mesenchymal stem cells (TMSCs). Our study showed that OGC treatment (100 μg/ml) significantly increased the cell proliferation of TMSCs, which was due to the upregulated cyclin D1 protein in the G1 phase of the cell cycle. Additionally, OGC induced cell metabolism such as oxygen consumption ratio (OCR) thereby accelerating cell cycle of TMSCs. We further found that the OGC enhanced differentiation potential of TMSCs into adipocytes, chondrocytes. Particularly, OGC showed a significant osteogenic potential via up‐regulation of the RUNX2 protein, which was also confirmed by increased intracellular calcium deposit of OGC treated TMSCs. Taken together, this new polymeric biomaterial OGC has been proven to promote osteogenesis as well as to induce stem cell proliferation by enhancing regulation of cell cycle pathway. The results of our study show that OGC can be considered a potential new additive for improving stem cell function.
Direct conversion of mouse embryonic fibroblasts into mesenchymal stem‐like cells with cell‐permeable Oct4 gene delivery
1Chungbuk National University, 2Seoul National University, 3Nano Intelligent Biomedical Engineering Corporation (NIBEC)
Direct conversion of one cell type into another is considered a trans‐differentiation process. Recent advances in fibroblasts have shown that, in some situations, epithelial cells can generate fibroblasts by epithelial‐mesenchymal transition, and conversely, in some other situations, fibroblasts can generate epithelia by undergoing a mesenchymal to epithelial transition. To elicit stem cell‐like properties on fibroblast, especially Oct4 is a transcription factor, which acts as a master transcriptional regulator for reprogramming of somatic cells. Notably, the production of gene complex comprising of cell‐permeable peptide, such as low molecular weight protamine (LMWP), next to transcription factor and direct treatment to fibroblast to induce reprogramming without toxicity and mutation. Herein, we designed a complex with non‐cytotoxic LMWP to prevent the degradation of Oct4 DNA by complexing with them. When the Oct4‐LMWP complex was delivered into MEF, stemness‐related gene expression was increased along with a decrease in fibroblast intrinsic properties. The Oct4‐LMWP complex developed in this study is expected to be used to reprogram terminally differentiated somatic cells or convert them to stem cell‐like properties without the risk of genomic dysregulation by improving the low productivity and stability problems of existing other technologies.
Effect of HA particle size and quantity on the bioactive and biological behaviour of PU scaffolds
1Dental Technology Department, Applied Medical Sciences, King Khalid University, KSA, 2College of Dentistry, King Khalid University, KSA, 3Bioengineering Department, Lancaster University,
UK., 4INSIGNEO institute for in silico Medicine, University of Sheffield, UK, 5 Interdisciplinary Research Centre in Biomedical Materials (IRCBM), Pakistan
In maxillofacial area, specifically orbital floor, injuries can cause bone deformities that are difficult to repair or regenerate. Treatment methodologies include use of polymers, metal, ceramics on their own and in combinations, but more attention has been paid to identify suitable materials for orbit floor regeneration. The bioactive PU/HA scaffolds, either micro or nanoparticles size, were prepared via the articles leaching technique. Different percentages of HA (25%, 40% and 60%) were used to evaluate the most suitable percentage for orbital floor repair and regeneration. The mechanical, physical, chemical and biological characterisation of scaffolds were investigated by tensile tests, SEM, FTIR, Cell viability (using MG‐63), and CAM assay. The FTIR‐PAS characterisation confirmed the presence of HA in composite scaffolds, while ATR confirmed that a significant amount of HA was located at the scaffolds top surface in micro‐HA. However, nano‐HA had a better disparity than micro‐HA. Increasing the content of HA up to 40% led to improving the mechanical properties, but nano‐HA was more promising than that of micro‐HA. 60% HA made the scaffolds hard and thus easy to be broken. Cell viability showed no significant difference between the PU and PU/HA scaffolds, either micro or nano. For CAM assay, the PU/HA scaffolds had more vascularity compared to PU. The development of new biomaterials with tailored properties will be useful in TE, especially in orbital floor regeneration. Also, the PU/HA nano 40% scaffold might be the most suitable for orbital floor implant, due to the mechanical and biologcal findings.
Design and preparation biodegradable staple for the intestinal suture
1University of Chinese Academy of Sciences
Intestinal suture through surgical alloy staple in intestinal surgery has been widely used which can shorten the operation time and reduce the pain of patients. However, alloy such as titanium is not biodegradable and will be stored in the body for a long time, as well as cause some adverse reactions.
In the research, we used 3D printing technology to design and develop a biodegradable staple, which is mainly composed of polylactic acid (PLA) and chitosan (CS). PLA is considered as one of the most competitive bio‐renewable polymers because of its excellent biocompatibility, good biodegradability and high mechanical resistance, which is often used as a material for 3D printing. As a biopolymer, chitosan has the advantages of antioxidant activity, anti‐inflammatory activity, angiogenesis stimulation, mucosal adhesion, anti‐tumor, hemostatic effect and wound healing stimulation. The suture effect of staple of different shapes is different, and the advantage of 3D printing is that it is convenient to produce staple that can improve the suture ability. The staple we designed has good mechanical properties which can meet the needs of intestinal suture, reduce the occurrence of inflammation and accelerate wound healing. It showed great potential in intestinal suture and substituted for alloy staple.
Development of porous PLLA/DCPA short fiber for bone formation
1Department of Biomedical Engineering, Doshisha University, 2Graduate School of Life and Medical Sciences, Doshisha University
Though PLLA fiber scaffolds have been applied for bone regeneration, its cell adhesion property is inferior due to hydrophobicity. In addition, cells may be locally distributed in the fiber scaffold depending on density of the fiber scaffold. In this study, we developed the porous PLLA short fiber doped with dicalcium phosphate anhydrous (DCPA) particles for promotion of bone formation. When the short fibers are mixed with cells in gel scaffolds, it is expected that the short fibers are affective for bone formation without localizing cells in the gel scaffolds. Porous PLLA/DCPA fibers with fiber diameters of 5, 10 and 15 μm were prepared by an electrospinning method. The frozen fibers in distilled water were crushed using a freeze grinder for 1, 3, 5, and 10 seconds, and then the short fibers were obtained with freeze‐ drying process. In the all case of fiber diameters, length of the short fiber decreased with increasing crush time. Average fiber length was from 291 to 373 μm. Surfaces of the short fibers were covered entirely with hydroxyapatite after 7 days of SBF immersion due to pore structure and addition of DCPA. According to cross‐sectional observation of the agarose gel mixed with the short fibers using a multiphoton microscope, the short fibers dispersed uniformly throughout the gel.
Bile acids containing nanoparticle and oral delivery strategies
1The Catholic University of Korea, Department of Biotechnology
In this study, we have employed bile acid (BA) conjugated, solid fluorescent probe nanoparticles to exclude any potential artifacts and instability issues in observing transport pathways and measuring oral bioavailability. The glycocholic acid conjugated polystyrene nanoparticle (GCA‐PN, 100 nm) exhibited oral bioavailability (oBA) as high as 47 % in SD rats after oral dosing. The area under the curve (AUC) was dose‐dependent up to 20 mg/kg. The PK profile was sustained. The oBA was influenced by the degree of BA conjugation and nanoparticle size. The bioavailability sharply declined when the rats were pretreated with cycloheximide that inhibits chylomicron export from enterocytes, which is a part of fat digestion/transport pathways. This pretreatment did not influence the uptake rate of PN without GCA. When GCA‐PN interacted with SK‐BR‐3 cells, the nanoparticle was internalized and located in the endoplasmic reticulum and possibly in the Golgi body, but neither in the early endosome nor in the lysosome. The GCA‐PN was observed in lymph fluid collected from the thoracic duct, a central lymphatic duct, by TEM. The evidence obtained from this study support the massive uptake of intact nanoparticle in the ileum via ASBT‐mediated endocytosis. The intracellular pathway seems to follow the way of chylomicron transportation. The nanoparticles are, most probably, transported to the lymphatic system first, rather than portal circulation, as chylomicron does. This may provide us with a new oral delivery route directly to the lymphatic system at high bioavailability.
Optimization of chemically defined soluble basement membrane solution as an alternative to matrigel for epithelialization studies
1Tissue Engineering Lab, Department of Mechanical Engineering, The University of Hong Kong, Pokfulum, Hong Kong ; Advanced Biomedical Instrumentation Centre , Hong Kong Science Park, Shatin, Hong Kong
Matrigel is widely used for modelling the extracellular (matrix ECM) in invitro epithelialization studies. However, while Matrigel can mimic the native 3D epithelial environment in a physiologically relevant manner, its applications are limited by its tumorigenicity, batch‐batch variability and presence of non‐ defined growth factors. To overcome these shortcomings, we propose a chemically defined soluble BM solution, with the hypothesis that this solution can give comparable results to that of Matrigel in epithelialization studies, and hence can be used as its alternative.
Different combinations of Collagen IV, Laminin 511 and Collagen I were combined in variable proportions to form chemically defined soluble BM Compositions. Optimized concentrations of epithelial cells were added on top of the gelated BM layer and cultured for 3‐4 days. The epithelialization of three different cell lines – MDCK, MCF7 and CaCo2, in terms of the barrier integrity of the resultant bi‐layered epithelial samples were then evaluated and compared to that of Matrigel, by means of confocal imaging of barrier markers (e‐cadherin and Zo1) and functional assays such as TEER Measurement and Dextran permeability assay. The preliminary findings of the project demonstrated that the barrier integrity of each of the cell lines were comparable, if not better, to that of Matrigel.
The findings of the project can pave the way to optimizing the composition of an ECM solutions that is not only chemically defined, but is also free from tumorigenic components, and hence can be used for epithelialization studies involving both healthy as well as tumor cells.
Effects of an injectable in situ hyaluronic acid hydrogel combined with basic fibroblast growth factor for a dermal filler
1Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei‐ro, Seodaemun‐ gu, Seoul 03722, Republic of Korea, 2Department of Chemical and Biological Engineering, Gachon
University, 1342 Seongnam‐daero, Seongnam‐si, Gyeonggi‐do 13120, Republic of Korea
Hyaluronic acid (HA) has been applied as a biomaterial for injectable dermal filler due to the unique viscoelastic property. Although HA derivatives are promising dermal filler materials, poor durability by degradation is the limitation of HA dermal filler. The chemical crosslinking of HA hydrogel is necessary to restrain the degradation rate. Herein, we present an injectable in situ HA hydrogel containing basic fibroblast growth factor (bFGF) crosslinked via a visible light‐induced thiol‐ene reaction. The thiol‐ene reaction between thiolated HA (SH‐HA) and methacrylated HA (MA‐HA) is triggered by riboflavin phosphate with blue light (BL) exposure. The mechanical property, matrix degradation, and protein release of HA hydrogels were evaluated regarding SH‐HA and MA‐HA ratio. The HA hydrogels improve NIH/3T3 fibroblast proliferation and migration, and the bFGFs incorporated in HA hydrogel additionally induce the cell proliferation and scratch closure rate. In addition, the HA hydrogel precursor solution under a porcine skin forms hydrogel by transmitted BL through the skin, so the gelation can occur under the skin after injection. According to the results, the in situ crosslinked HA hydrogel is a promising injectable and long‐lasting dermal filler that can deliver therapeutic factors.
The effect of biphasic calcium phosphate and demineralized bone matrix on tooth eruption in mongrel dogs
1Division of Pediatric Plastic Surgery, Seoul National University Children's Hospital, 2Department of Plastic Surgery, CHA Bundang Medical Center
Application of hybrid graphene oxide and silver nanoparticles in wound healing
1Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur 56000, Malaysia, 2Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, UnivesitiKebangsaan Malaysia, Bangi 43600, Malaysia,
3Department of Internal Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia,Kuala Lumpur 56000, Malaysia
One of the major setbacks for hard‐to‐heal wounds is the microbial infection which cause prolonged inflammation stage in wound healing. The introduction of nanotechnology especially metal nanoparticles has been intensively used in healthcare due to their high antibacterial activities. However, the metal nanoparticles such as silver nanoparticles are toxic in high concentration and frequently aggregated. The incorporation of the graphene oxide able to stabilise the silver nanoparticles and enhance the antibacterial activity. This project aimed to evaluate the optimal concentration of hybrid graphene oxide‐silver nanoparticle (GO‐AgNP) for antibacterial properties and biological effects towards the human dermal fibroblasts. The biocompatibility of the hybrid GO‐AgNP was determined through dose‐response and live dead assay. The genotoxicity of the hybrid nanoparticles was further analysed using comet assay to determine the DNA damage caused by the nanoparticles. Finally, the antibacterial activity of nanoparticles was performed using the minimum inhibitory concentration (MIC) and turbidity study using
S. aureus (gram‐positive) and E. coli (gram‐negative). The result unraveled that hybrid GO‐AgNP was viable towards human dermal fibroblasts with a range of 0.00625 to 0.025 mg/mL GO‐AgNP with more than 50% cell viability. Additionally, the DNA tail damaged demonstrated an increment along higher concentration of hybrid nanoparticles. Thus, the optimal concentration of GO‐AgNP for antibacterial effect for both gram‐positive and negative bacteria around 0.025 mg/mL. This study concluded that the optimal concentration of hybrid graphene oxide and silver nanoparticles is 0.025 mg/mL for skin wound healing application.
Study on corrosion mechanism of 3D‐printed porous Ti‐Nb‐Zr‐Sn alloy scaffolds immobilized with type I collagen in simulated inflammatory conditions
1Institute of Oral Biology, National Yang Ming Chiao Tung University, 2Department of Dentistry, National Yang Ming Chiao Tung University
Towards allogenizing a xenograft: The use of human induced pluripotent stem cells in recellularizing xenogeneic cardiac scaffolds do not activate human naïve neutrophils
1King faisal hospital and research center
The limitation of suitable human donor organs for transplantation has resulted in global ever‐increasing patient waiting lists. Alternatively, xenotransplantation is considered an option, but is yet to reach clinical practice. Major progress has been archived to overcome immunological rejection, however, issues with functionality are still to be resolved. The approach of tissue engineering has been evolved to create cardiac tissues with optimized functions. The use of decellularized xenogeneic cardiac tissues recellularized with donor‐derived cardiac cells may prove to be a viable option as supporting structures of the native tissue such as vasculature can be utilized. Here we decellularized adult rat heart using sequential perfusion. The acellular scaffolds were re‐seeded with human fibroblasts, human mesenchymal stem cells, human endothelial cells, and cardiac cells derived from human induced pluripotent stem cells. The ability of the resultant re‐cellularized rat scaffolds to activate human naïve neutrophils in vitro was looked over to measure xenogeneic recognition. In contrast to cadaveric hearts, our results demonstrate that acellular and re‐cellularized xenogeneic scaffolds did not activate human naïve neutrophils. This finding extends the observations of others, demonstrating the removal of xenogeneic antigens that lead to human naïve neutrophil activation, by decellularization.
Magnesium‐encapsulated injectable hydrogel and 3D‐engineered polycaprolactone conduit facilitate peripheral nerve regeneration
1The Chinese University of HK, 2South China University of Technology
Biomaterials for enhancing cell survival in low water environment
1UCL, 2University College London Hospitals
Advances in the biotechnological applications and increasing demand of mammalian cells in the recent years have made it imperative to research for more optimal handling/storage techniques while minimizing the negative impact on cell viability and functionality. It has become evident from recent research that physico‐chemical and mechanical properties of the extracellular environment, as well as water retention profiles are crucial in the cell behaviour and survival.
In this study a variety of biomaterials including animal derived and plant‐based materials (i.e. gelatin, alginate, nanofibrillar cellulose) were comparatively characterised for water retention, thermal, mechanical, structural and morphological/topological properties. Techniques used include Scanning Electron Microscopy, Differential Scanning calorimetry, Fourier‐transform infrared spectroscopy, and Thermogravimetric analysis. Biomaterials were also assessed for cell growth‐support and for storage under low water environments for various cell lines (e.g. promonocytes and myoblast cells) using cell metabolic activity, cytotoxicity and differentiation assays.
A comparative profile of all the gels was obtained. Results demonstrate that animal‐free biomaterials exhibited similar water retention profiles, and mechanical and physico‐thermal characteristics to animal derived candidate biomaterials. What is more, systematic optimization approaches showed that cell protection abilities of these biomaterials under assayed conditions are dependent on parameters such as temperature, water content removal method and matrix formulation (i.e. biomaterial dilution and exogenous protectant supplementation).
Overall, this study summarises some of the key characteristics that may be used to protect cells during (low water) storage and delivery and may form the basis of a predictive tool for the fabrication of next generation storage materials.
Synthesis of polysaccharide‐based nanoparticles for delivery of genistein to combat colon cancer treatment
1Dongguk University, 2Department of Chemical & Biochemical Engineering, College of Engineering, Dongguk University, Seoul, South Korea, 3School of Nano Sciences, Central University of Gujarat,
Gandhinagar‐382030
A significant problem is achieving site‐specific and regulated distribution of hydrophobic drug molecules in the colon environment. Therefore, in order to deliver hydrophobic drug at the site‐specific environment of the colon, we have synthesized the genistein (GEN) encapsulated inulin‐stearic acid (INU‐SA) conjugate‐based nanoparticles. INU is a hydrophilic polysaccharide macromolecule that has been combined with hydrophobic SA to generate an amphiphilic bioconjugate (INU‐SA). INU‐SA has interesting features to self‐assemble into spherical genistein‐loaded nanoparticles (GNPs) with a sustained drug release profile. The GNP produced was nanosized (115 nm), had good colloidal dispersibility (0.066 PDI), and was very effective in encapsulating drugs (92.2%). GNP drug release behavior indicated the specific release of GEN from the colon site was only 3.4% at gastric pH 1.2 while 94% at intestinal pH
6.8. The developed GNP showed potential anticancer activity against HCT 116 human colorectal cancer cells, as confirmed by antiproliferation (IC50 = 5.5 μg/mL, GNP and 28.2 μg/mL, GEN) and apoptosis assays. The prepared GNP has a higher rate of cellular internalization than the free GEN, as confirmed by cellular uptake assay. Therefore, the modified and biocompatible polysaccharide INU is suggested as a means of enhancing the therapeutic efficiency of natural biomolecules.
Design of a conductive nerve guidance conduit with silver nanoparticles/poly(vinyl alcohol) hydrogels for peripheral nerve regeneration
1Korea Institute of Science and Technology (KIST)
When a nerve injury is caused by external trauma and nerve tissue has not recovered normally, symptoms such as dysfunction or pain due to neuroma formation may appear. For treating long‐distance nerve injuries, transplantation has been carried out with nerve grafts like autografts, decellularized allografts, or synthetic nerve guidance conduits (NGC). In this study, we fabricated a biodegradable and conductive NGC with poly(L‐lactide‐co‐caprolactone) (PLCL) and silver nanoparticles (AgNPs)/polyvinylalcohol (PVA) conductive hydrogels to accelerate the regeneration of peripheral nerve regeneration. For enhancing the biocompatibility and reducing cytotoxicity of AgNPs, we introduced tannic acid and gelatin‐boronic acid (BA) into the hydrogel. It is confirmed that tannic acid reduced Ag ions by performing metal complexation and gelatin‐BA inhibited the release of tannic acid and AgNPs by complexation with tannic acid and boronate‐catechol. Also, the conductive hydrogels could lead to neuronal differentiation of neuronal cells and the electrical conductivity was in the range of 9.42 x 10−5 and 3.06 x 10−3 S/cm. These results demonstrated the great potential of biodegradable and electroactive NGCs in repairing peripheral nerve regeneration.
Measurement of cell‐ECM, intracellular stresses, and intracellular tensions using gelatin methacryloly (GelMA) hydrogels based traction force microscopy (TFM), intracellular force microscopy (IFM), and monolayer stress microscopy (MSM)
1Soonchunhyang Institute of Medi‐Bio Science (SIMS), Soonchunhyang University, Cheonan‐si 31151, Korea, 2Soonchunhyang University, 3Department of Otorhinolaryngology‐Head and Neck Surgery, Soonchunhyang University Cheonan Hospital, Cheonan, Republic of Korea, 4Department of
Ophthalmology, Soonchunhyang University Seoul Hospital, Seoul, Republic of Korea
Cell‐generating tractional and intracellular forces within physiological and pathological microenvironments and their transduction to regulate various cell behaviors have been of great interest. Although these mechanical forces exerted cells have been traditionally analyzed by utilizing polyacrylamide (PAAm) hydrogel‐based traction force microscopy (TFM) due to its good linear elastic and clear optical properties, PAAm hydrogel failed to support three‐dimensional (3D) cell encapsulation to understand the cellular forces within 3D microenvironment. Here, we aim to develop a novel method to assess cell‐matrix (traction) and intracellular (and intercellular) stresses using an alternative candidate, methacrylated gelatin (GelMA), to overcome the current limitations. Our preliminary results demonstrated that viscoelastic and elastic properties of both PAAm and GelMA‐based hydrogels were successfully matched, which were confirmed by measuring Young's modulus, dynamic modulus (G’, G’’, and tanδ), stress relaxation, and strain recovery. Next, we evaluated various cell‐generating forces of either tonsil‐derived stem cells at a single‐cell level or conjunctiva‐derived epithelial cells as a monolayered cell sheet using GelMA‐based traction force microscopy (TFM), intracellular force microscopy (IFM), and monolayer stress microscopy (MSM), where the results were also validated using PAAm‐based methods. Our initial findings indicate that our GelMA‐based TFM, IFM, and MSM methods exhibited cell‐generating forces and mechanotransduction in a spatio‐temporal manner. Taken together, these results suggest GelMA can be a good candidate for the cell‐ECM and intracellular stress measurement platform and this technique can offer insight into the important roles of dynamic cell‐matrix and intracellular stresses in regulating stem cell lineage commitment and disease progression within 3D microenvironments.
Cytotoxicity effect of SIM‐PLGA chitosan coated scaffolds on human osteoblast (hFOBs) proliferation
1UNIVERSITI TEKNOLOGI MARA, 2Qatar University, College of Pharmacy, 3School of Pharmacy University of Swansea
Simvastatin (SIM) has long been studied as the material that can increase bone regeneration in the form of scaffolding. In addition to scaffold, much work has been devoted to the development of a bone scaffold structure that has appropriate mechanical strength, high biocompatibility, and an acceptable rate of biodegradation. In this study, we have successfully fabricated the injectable scaffold with chitosan as the coating materials of the microparticles. However, we are lacking information on the cytocompatibility with human osteoblast (HOBs). Therefore, this study aims to compare the amount of SIM release from the simvastatin‐loaded PLGA scaffold with chitosan and the effects of the SIM release in HOBs growth. Methods: To investigate release of simvastatin from scaffolds composed of SIM‐PLGA (T1) and PLGA‐ simvastatin∔chitosan (T4) with various concentration (1,2,3 mg/ml). hFOBs cells were cultured at 24,48 and 72 hours respectively and the effects of cells proliferation was investigated with MTT assay. Simvastatin (T2) was used as negative controls and PLGA microparticles (T3) as the positive controls. The electron microscope scanning illustrates the size of porosities and the HOBs integration with the microparticles. Results: In comparison of the simvastatin release, the effect of T1 and T2 treatments increased HOBs cell viability. Scanning electron microscope and Fluorescence study were performed. However, there is inhibitory effect of HOB cells for T2 treatments. The porosities sizes are observed to have the approximate size of 160‐377μm, which is qualified for angiogenesis and cell growth in the bone and makes the scaffold an ideal choice for bone substitution.
Fabrication of tissue specific multi‐layered scaffold for periodontium regeneration
1Sungkyunkwan University
No iIn recent years, there has been a marked increase in periodontal infections such as gingivitis or periodontitis. Thereby, effective periodontium regeneration has been subjected with notable attention. However, due to the multiplex layers of the periodontium (alveolar bone, periodontal ligament, and cementum), it is challenging to effectively regenerate the described layers. To overcome this issue, we have bioprinted a multi‐layered cell‐laden scaffold consisting of cementum, periodontal ligament, and alveolar bone tissue‐specific layers with each layer responsible for appropriate differentiation. In the alveolar bone layer, bone‐derived decellularized extracellular matrix (bdECM) mixed with bioceramics was selected to evoke efficient osteogenesis of human dental pulp stem cells (hDPSCs). In the periodontal ligament layer, a collagen‐based bioink supplemented with fibrogenic growth factor (FGF) was used to induce fibrogenic and demineralization ability of the human periodontal ligament fibroblasts (hPDLFCs). The cementum layer is complemented with a cementum matrix named “nature hydroxyapatite” to promote cementum differentiation of the hDPSCs. Moreover, various structural designs (mixed, interlinked, and parallel) of the interfaces between the above‐described layers have been investigated via gene expressions and mechanical properties. Based on the results, we believe that the multiplex bioconstructs would provide a platform for future periodontium regeneration.
mages/photos/figures/ tables.
Optimization of printability of alginate‐based bioinks using rheology‐ informed machine learning
1Major of Biomedical Engineering, Division of Smart Healthcare, College of Information Technology and Convergence, Pukyong National University
Printability is one of the important variables of extrusion‐based bioprinting research to improve the accuracy, rapidness, reliability, and adaptability of 3D printed scaffolds. The correlation between printability and extrusion process parameters, including viscoelastic properties, pressure, nozzle diameter, and nozzle length, has been explored by many studies. For instance, mathematical models based on rheology and microfluidics have been applied for the prediction of printability of various bioinks. However, these approaches were confined to the bioink with simple composition whose rheological properties can be modeled by power‐law fluids. Recently, several machine learning studies for optimization of bioprinting parameters have been reported, but they are not versatile due to the confined input layer including the concentrations and types of bioink materials. Therefore, the novel model needs to be created and trained with dataset which is identical to the input layer although various bioink composition is applied. In this study, 3D bioprinting of hydrogel composite is optimized with machine learning by datasets consisting of rheological measurements which were quantitatively analyzed with printing parameters. The prediction of the rheology‐informed machine learning was validated and compared with the conventional methods.
Sea anemone‐derived silk‐like protein‐based nanoparticles for systemic cancer therapy
1POSTECH
In recent years, as the incidence and mortality of cancer continue to increase, demand for cancer therapy is also increasing. Nanotechnology‐based drug delivery system for cancer therapy has been developed for increased efficacy of therapeutics through passive‐targeting effects by enhanced permeability and retention (EPR) effect around cancer cells [1]. Nanoparticles for drug delivery should be biocompatible and have a variety of potentials, including appropriate size, morphology, and rate of biodegradation. Aneroin is the bioengineered silk‐like protein biomaterial derived from sea anemone and has low cytotoxicity, high biocompatibility, and high resistance and flexibility with appropriate mechanical properties [2, 3]. In this work, aneroin nanoparticles (ANPs) were successfully fabricated using dityrosine crosslinking through photo‐oxidative reaction. It was found that ANPs are negatively charged which can prevent the formation of agglomerate during circulation through intravenous injection. Anti‐cancer chemical drug was successfully encapsulated in the aneroin NPs with high capacity. It was clearly demonstrated that ANPs have no cytotoxicity against cells, and drug‐loaded ANPs showed effective in vitro and in vitro anticancer activities. Therefore, our novel aneroin‐based NPs have a potential to be the effective drug carrier for systemic chemotherapy.
Study of mini‐cornea model for eye irritation test
1Industry 4.0 Convergence Bionics Engineering, Pukyong National University, Busan, 2Major of Biomedical Engineering, Division of Smart Healthcare, College of Information Technology and Convergence, Pukyong National University, Busan
This study was to fabricate a cornea‐like in vitro tissue model based on tissue engineering technology and to evaluate the chemical irritancy as an alternative testing method for animal experiments. For reconstructing the collagen‐rich matrix‐like corneal stroma layer, immortalized human corneal keratocytes (iHCKs) were cultured in a modified medium with a high concentration of ascorbic acid. Immortalized human corneal epithelial cells (iHCECs) have been seeded on top of the pre‐cultured stromal matrix and cultured at an air‐liquid interface for keratinization like corneal epithelium. The mini‐ cornea model with two types of corneal cells that verified expressing cornea‐specific markers with immunocytochemistry and western blot analysis was utilized for the eye irritation test. At the 2D level, cell‐matrix production has been observed through macro‐and microscopic images and measured total collagen content. At the 3D level, histological observation and immunohistochemistry revealed the whole shape, thickness, cellular morphology, and distribution. In the irritation test, the viability of the cornea model treated with 2‐Phenoxyethanol, Triton X‐100, sodium lauryl sulfate, and benzalkonium chloride reduced with increasing chemical concentrations. An eye irritation test with animals has been conducted for a long time, but physiological dissimilarity and ethical issues are significant hurdles in representing humans. In this study, we performed and observed irritation tests according to chemicals and can be applied as an alternative approach for replacing animal tests.
Guided assembly of spheroids for complex tissue formation using functionalized magnetic nanofibers
1Hanyang University
Strategies for biofabrication of artificial 3D tissues have been highly investigated in tissue engineering due to the demand on organ model. Although a number of methods have been developed, mimicking complex hierarchical structure of human organs remains challenging. In addition, strategies to control function of cells in organ model have been highly considered for better recapitulation of complexity in body. In this study, we developed a method to engineer complex tissue structure by magnetic assembly of multi‐functional spheroids. First, we fabricated magnetic nanoparticle (MNP) embedded poly‐L‐lactic acid nanofibers (MF) and coated the fibers with polydopamine (PMF). Then, spheroids were fabricated by incorporating the fibers with human dermal fibroblasts (HDFBs) and PMF containing spheroids showed reactive oxygen species (ROS) scavenging capacity. The spheroids were fused using external magnetic field (EMF) to evaluate the effect of fibers on spheroid formation and fusion. Longer fibers were effective for maintaining spheroid size and viability of cells as well as regulated fusion of the spheroids. For complex tissue formation, spheroids with either MFs or PMFs were assembled under EMF and only PMF spheroids showed ROS scavenging effect in the assembly after hydrogen peroxide treatment. Finally, we fabricated vessel‐like microtissues via guided assembly of spheroids using EMF. In conclusion, we expect that this method to fabricate complex tissue structure via guided assembly of spheroids would be a feasible method for engineering artificial organs.
Fabrication of biodegradable polycarbonate 3D printing scaffold and evaluation of biocompatibility
1Ajou university
3D printing technology is suitable for tissue regeneration and artificial organ manufacturing because it can quickly and easily fabricate a scaffold with a sophisticated 3D structure. Biomaterials used for 3D printing should be biodegradable, as well as have low toxicity and immunogenic reaction. Aliphatic polycarbonate(APC) has been studied a lot in recent years due to its biodegradability, appropriate physical properties, and very low immunogenic reaction. In this study, the applicability of aliphatic polycarbonate (APC) as a material for 3D printing was evaluated. Using an FDM‐type 3D printer, APC 3D printing scaffold (APC‐PS) could be manufactured, and it was confirmed that it had the same pore and line diameter as the designed structure with excellent thermal stability. Through the in vitro evaluation results, it was confirmed that APC‐PS had excellent cell adhesion and proliferation and low cytotoxicity. Through the in vivo experiment results, it was confirmed that the transplanted APC‐PS was biodegraded for 16 weeks, and tissues including blood vessels were filled inside the APC‐PS. When calculated as a rate constant, the scaffold degradation rate and tissue growth rate were almost the same, and it was found to be excellent as a scaffold for transplantation. In addition, since the biodegradation by‐product of APC‐PS is based on CO2, the inflammatory response was very small, and excellent biocompatibility was confirmed. Summarizing the research results, APC is promising as a new biomaterial for 3D printing and can be applied as custom artificial organs and tissues.
3D bioprinted kidney‐derived bioink constructs for renal tissue regeneration
1Wake Forest University School of Medicine
One of the challenges in 3D bioprinting is finding an appropriate bioink that provides a tissue‐specific microenvironment supporting cellular growth and maturation. We hypothesize that a kidney‐derived extracellular matrix (dECM) bioink can provide renal‐specific molecules and structural and biomechanical signals to regulate renal cell behavior and accelerate tissue maturation and formation. We expect that the encapsulated cells can organize and create the appropriate architecture and functionality of renal tissue. We developed a photo‐crosslinkable kidney ECM‐derived bioink (KdECMMA) that could provide a kidney‐specific microenvironment for renal tissue bioprinting. Porcine whole kidneys were decellularized through a perfusion method, dissolved in an acid solution, and chemically modified by a methacrylate reaction. This KdECMMA‐based bioink was formulated and evaluated for rheological properties and printability for the printing process. Afterward, the bioprinted cell‐laden constructs were implanted in the kidneys of rats for subsequent analysis. The results showed that the bioprinted human kidney cells in the KdECMMA bioink were highly viable and matured with time. Moreover, the bioprinted renal constructs exhibited the structural and functional characteristics of the native renal tissue. More importantly, the cell‐laden KdECMMA bioink constructs supported renal tissue regeneration in vivo. After 1 and 2 months of implantation, there were newly formed glomerular (podocin+) and tubular (aquaporin+) structures revealed in the KdECMMA constructs both with and without human kidney cells. We demonstrated the potential of the tissue‐specific ECM‐derived bioink for cell‐based bioprinting that could enhance cellular maturation and eventually renal tissue formation.
IPN bioink for 3D printing macro‐scale, complex tissue analogues
Murugan Ramalingam*1, Guang‐Zhen Jin1, Deepti Rana2, Jung‐Hwan Lee1, Hae‐Won Kim1
1Dankook University, 2University of Twente
Functional tissue or organ engineering requires multi‐materials along with heterotypic cell types to recapitulate the complex structural and functional properties of native tissues. Three‐dimensional (3D) bioprinting serves as one of the powerful tools to design and fabricate functional tissue analogues suitable for regenerative medicine. However, 3D printing complex and macro tissues, such as heart or ear, with high design fidelity as well as biological functionality is highly challengeable. In our rational approach, we employed an interpenetrating hydrogel formulation based on albumen/alginate/gelatin hydrogels (IPN bioink) that facilitates 3D printing of self‐standing and vascular‐supportive tissue analogues. The IPN bioinks were studied for their viscoelastic and relaxation behavior prior to 3D printing. The in‐house formulated IPN bioink demonstrated high printability and shape‐fidelity of self‐standing macro structures, including a human ear model. Furthermore, the printed tissue analogues were examined for various structural, mechanical, and physicochemical properties. The in‐vitro cell culture studies (human umbilical vein derived endothelial cells, HUVECs) of the 3D printed tissue analogues showed high biocompatibility and vascular network formation. Altogether, these results demonstrate the suitability of the developed IPN bioinks in biofabricating complex shapes and structures that have great potential for engineering tissues and whole organs.
Development of a flexible vascular external stent for the treatment of aortic aneurysm
1Gachon University, 2Gachon University Gil Medical Center
A hump‐shaped deformation of the blood vessel wall is called 'Aneurysm', and it is one of the cardiovascular diseases, commonly occurs in the aorta. To treat an aneurysm, vascular replacement using an artificial blood vessel has been performed. However, since the mortality is high, a vascular wrapping using the artificial vessel is used as an alternative. However, this wrapping method causes a new aneurysm outside the wrapped blood vessel due to the compliance difference between the artificial and the original vessel. In this study, to overcome the limitations of the wrapping method, an external stent was developed based on 3D printing technology considering the compliance of the human aorta. Expansion rates of five patterned stents were analyzed through finite element analysis, pressure tests using a catheter were performed on the 3D printed stents. Based on a positive Poisson's ratio stent with patterns similar to that of the artificial blood vessels, the negative Poisson's ratio stents of ‘Cut missing rip’ and ‘Sand glass' patterns, and zero Poisson's ratio structures such as ‘Wave’ and ‘Leaf’ patterns were measured in order. Finally, the performance of the negative Poisson's ratio stent was up to 17.7% higher than that of the positive Poisson's ratio stent. In addition, it was confirmed that the results of the finite element analysis and the results of the internal pressure test using the catheter were similar. The structure with a negative Poisson's ratio proposed in this study is expected to be used in the treatment of aortic aneurysms.
Extrusion‐based 3D bioprinting from bench to bedside applications in bone tissue engineering using platelet rich plasma and scaffolding materials
1Novaprint Therapeutics
Extrusion‐based 3D bioprinting is a promising technique to create patients' specific implants for bone reconstruction, however, the current bone tissue engineering scaffolds produced by this method lack osteoinductivity which is crucial for bone reconstruction. In this study, we utilized Platelet Rich Plasma (PRP) as bioactive bone‐inducing content in our proprietary bioink for bone formation. In addition, polycaprolactone and tricalcium phosphate were used as the scaffold material to provide mechanical support. The structure of patients' specific implants was created based on their CT images, engineers' designs, and the surgeon's input in performing surgeries. After the concurrence from the engineering and clinical team, patients' specific implants were printed in a GMP lab with an ISO 5 environment. The printed constructs showed a very low level of endotoxin with zero occurrences of contamination. Three cases of clinical trials were conducted using these patients' specific implants. Results showed excellent osteointegration and osteoinduction between implants and host tissues. All patients could release from the hospital after one week of observation in the hospital without any complications. This bench to bedside application of 3D bioprinting and regenerative medicine is promising and should be widely promoted in more hospitals.
Development of functional insulin‐producing cell clusters from iPSCs using microgravity bioreactor
1Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine,
2UNIVERSITY OF ULSAN, 3Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine
Although pancreatic islet transplantation is an ideal treatment for insulin‐dependent diabetes, a shortage of donor sources is a major hurdle. Stem cell‐derived insulin‐producing cells (IPCs) are a potential approach to overcoming these limitations. However, they still exhibit limited glucose control in vivo due to their low physiological functions, different from natural islets. It is well known that the cell–cell aggregation structure and microenvironment of islets is essential for maintaining their physiological functionality. Floating culture system with microgravity could form a IPC clusters and induce extracellular matrix production in vitro. Differentiation from iPSCs to IPCs was induced using three stages based on the developmental process of the pancreas. A random positioning machine system was used in this study to prepare microgravity culture system. This machine is composed of two rotational axes and the rotation speed of each axis was fixed to 5 rpm. Pancreas progenitor cells were seeded in an oxygen‐permeable soft chamber. The soft chambers were fixed to the rotating panel of the machine. The IPC clusters in microgravity systems showed higher gene expression of insulin, PDX1, collagen IV, fibronectin and insulin secretion in response to high glucose than static culture system. Cell viability was not affected but ECM expression was increased in the bioreactor system. Enhancement of IPC differentiation was directly associated with increased ECM expression as demonstrated by gene expression and cytoplasmic staining.
Highly scalable and automation compatible organ‐on‐chip platform for biological barriers modeling
1Finnadvance
Human body protects its most vital systems with impenetrable barriers like the vascular‐, blood‐brain‐, gut‐, lung‐, and epithelial‐barriers. These barriers though defensive, also prevent passage of several drugs and therapies, most notably across the BBB (blood‐brain‐barrier), for treatment of neurodegenerative disorders and neurooncology.
The current invitro models relies extensively on standard 2D static monolayer cultures or animal models to mimic the disease phenotype. These platforms fail to mimic the complex vascular shear‐stress and biomechanical microenvironment.
Organ‐on‐chip microfluidic devices have been developed to answer this demand, and they are known to recapitulate sophisticated human physiology with high fidelity. To speed up the discovery of novel pharmacological and immune‐oncological strategies for neuro‐degenerative and ‐oncology, we present a highly scalable AKITA organ‐on‐chip platform which permits microphysiological culture of neuronal (astrocytes and neurons) or epithelial (gut and alveolar epithelial) cells cultured across a tunable pore‐size membrane housing barrier‐specific endothelial cells (brain microvascular ECs, lung ECs, gut ECs) cultured under constant flow conditions.
With a throughput of 96 parallel barrier cultures on a standard 384‐well plate SBS standard platform, we show BBB‐on‐chip permeabilities approaching 1E‐7 cm/s for 70kDa dextran. Lung‐on‐chip barriers with A549 cells show intact barrier formation under flow, with mucus generation, which could be inflamed with LPS and TNF treatment‐on‐chip.
With full liquid handling robotics compatibility, the AKITA platform is truly applicable for high content screening of peptides/antibodies/exosome and cellular therapies. Further development of the platform for accommodating organoid introduction will permit development of individualized responses in a patient‐ relevant manner.
Novel GBR method for cranial bone defect healing using dual scaffolds of BMP‐2 and FGF‐2
1Yonsei University College of Dentistry, 2Seoul National University School of Dentistry, 3TaeWoong Medical Co., Ltd, 4Wonkwang University College of Dentistry,
The purpose of this study was to investigate the enhancement of the osteogenic ability of the dual scaffold complex, identifying the most effective growth (factors GF) concentration for new bone formation within the newly designed novel guided bone regeneration (GBR) concept. This study used 24 New Zealand white rabbits. Four bone defects with a diameter of 8mm were formed in the calvaria of each individual to perform GBR. When applying GBR, collagen membrane and BCP were applied to the control group, and collagen membrane + BMP‐2 (0.5, 1.0 mg/ml) and BCP + FGF‐2 (0.5, 1.0 mg/ml) were applied to the four experimental groups, respectively. After 2, 4, and 8 weeks of healing, the rabbits were sacrificed for histological and histomorphological analysis. In the histological analysis, continuous forms of new bones were observed in the upper part of bone defect in the experimental groups, whereas no continuous forms were observed in the control group. In the histomorphological analysis, Group 3 (BMP‐2 0.5 mg/ml, FGF‐2 1.0 mg/ml) showed statistically significantly higher new bone formation (30.65 ± 11.39%, p < 0.05). Also, there was no statistically significant difference in new bone formation according to the healing period at 2 and 4 weeks, but it was statistically significantly higher at 8 weeks (2 = 4 < 8 weeks, p < 0.05). Using collagen membrane + BMP‐2 and BCP + FGF‐2 for bone regeneration in bone defects using the new GBR technique was effective in new bone formation. The effective concentrations of growth factors for new bone formation were BMP‐2 0.5 mg/ml and FGF‐2 1.0 mg/ml.
The effect of extracellular matrix glycation on mechanoresponsiveness of cells
1KAIST, 2Korea Basic Science Institute
The extracellular matrix has various mechano‐physical properties, such as rigidity, confinement, adhesion, and topology, playing a pivotal role in cell fate and function. Most studies, however, have focused on a single variable, and little is known about the interplay of ECM physical modules. Here, based on the glycation of the cell‐derived matrix (cd‐ECM), we investigate the effect of increased stiffness on the mechano‐responsiveness of cells in ECM with low adhesivity. The reaction with high ribose, known as glycation, caused the formation of several types of advanced glycation endproducts (AGEs) in the ECM. The deposition of AGEs resulted in multiple changes in ECM mechanics, including structural changes, increased stiffness, and decreased fibronectin (FN) adhesivity. Due to these alterations, cells in AGE‐deposited ECM exhibited higher levels of RAGE and lower levels of integrin α5 and β1 than those in native cd‐ECM. Weak focal adhesion was observed in different expression patterns of multiple receptors, leading to a decrease in both velocity and directionality in cell migration. Our findings reveal that stiff and slippery ECM can induce pathological mechanosensing of cells; thus, adhesion trumps stiffness in controlling cellular mechano‐response.
On the way to a bio‐printed intervertebral disc (IVD) model: In vitro evaluation of the interactions of ovine IVD cells with a collagen/hyaluronic acid bio‐ink
1Inserm, Regenerative Medicine and Skeleton, Nantes Universite, France, 2AO Research Institute, Switzerland, 3LCMCP, Sorbonne Universite, France
Intervertebral disc (IVD) degeneration is a major cause of low back pain, and it remains poorly characterized, with no adequate IVD models. To mimic its complex structure, with a gelatinous core Nucleus Pulposus (NP) surrounded by lamellae of the Annulus Fibrosus (AF), we selected collagen type I (Col) and Tyramine‐conjugated Hyaluronic Acid (THA), and evaluated their interactions with ovine NP and AF cells.
Col 4%‐THA 6% (ratio 1:1.5) and Col 4%‐THA 1% (ratio 1:0.25) were selected to mimic the NP and AF tissues, respectively. The bioinks Young's modulus were measured with a MicroTester equipment (CellScale). NP and AF cells were harvested from healthy discs of lambs (6 month‐old), in collaboration with the Nantes Veterinary school. NP and AF cells were cultured for up to 28 days. Cell viability (Live/Dead), morphology (actin staining), proliferation (PicoGreen and EdU incorporation) were evaluated.
The bioinks exhibited different mechanical properties with a Young's modulus of 2.0 kPa and 0.6 kPa for the 1:1.5 and 1:0.25 ratios, respectively. The bioinks were cytocompatible, with NP and AF cells remaining alive for 28 days, with a round morphology at day 1, and an elongated morphology afterwards. No proliferation was observed, as shown by DNA content and EdU assay. Interestingly, we evidenced a significant contraction of the constructs, with a significant increase of their Young's modulus, depending of the initial cell density. It suggests a cell‐mediated remodeling and the deposition of extracellular matrix, allowing to control the final dimensions of the construct.
Acellular skin patch of collagen hydrogel fortified with dermal fibroblast conditioned medium (DFCM) for skin therapeutic application: in vivo Study
1Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000 Cheras, Kuala Lumpur, Malaysia, 2Department of Physiology, Faculty of Medicine,
Universiti Kebangsaan Malaysia, Jalan Yaccob Latiff, 56000 Cheras, Kuala Lumpur, Malaysia
Having a skin substitute that is readily available would be the best treatment. However, majority of cell‐ based skin substitutes require a long production time, therefore long waiting time for patients. Secreted proteins from cells and tissues play vital roles in promoting wound healing. Ultimately, the aim was to develop an acellular three‐dimensional (3D) skin patch with dermal fibroblast conditioned medium (DFCM) and collagen hydrogel for immediate treatment of skin loss. Fibroblasts isolated from skin samples were cultured using the serum‐free medium (Epilife, KM) to collect DFCM. Subsequently, the DFCM was mixed with collagen (Col) hydrogel to fabricate acellular 3D constructs (Col/DFCM) and implanted on full‐thickness wound in animal models (BALB/c mice and Ovine). The construct successfully formed soft, semi‐solid, and translucent hydrogels within 1 h of incubation at 37 ˚C with strength of <2.5 Newton (N). The Col/DFCM demonstrated significantly lower turbidity and percentage of porosity (Col/DFCM: 35.15 ± 9.76%; Col:(105.14 ± 11.87%) compared to the collagen alone. Fourier transform infrared spectrometry showed that the construct consisted of oxygen–hydrogen bonds (O‐H) and amide I, II, and III. The implantation of the 3D skin patch on an animal model demonstrated significantly faster healing rate compared to no treatment group. The histological analysis with Hematoxylin & Eosin, Masson's trichrome and Immunohistochemistry analysis confirmed the structure, integrity, and maturity of the regenerated skin. These findings highlight the possibility of using DFCM together with collagen hydrogel in an acellular 3D skin patch that can be used allogeneically for immediate treatment of full‐thickness skin loss.
Enhancing fat graft survival using biodegradable scaffolds with acellular adipose matrix and magnesium hydroxide‐incorporated PLGA microsphere
1CHA University, 2CHA Gangnam Medical Center, 3Seoul National University
Atrophy or depressed deformity of soft tissue may occur due to trauma and surgical treatments (debridement and removal of damaged soft tissue) or aging. Several methods are being used to make up for the soft tissue deficiency such as filler injection or autologous fat grafting which is one of the most common procedures in plastic surgery. However, its clinical outcomes are often suboptimal, and a lack of metabolic and architectural support at recipient sites affects fat graft survival leading to complications such as cyst formation and calcification. To remedy their shortcomings, various scaffolds and growth factors that induce tissue regeneration in the body are developed and used in the tissue engineering field. Poly(lactic‐co‐glycolic acid) (PLGA) and extracellular matrix (ECM)‐based scaffolds such as allograft adipose matrix (AAM), have shown exceptional clinical promise as regenerative scaffolds. AAM is composed of extracellular matrices which can be obtained by decellularization of adipose tissue. PLGA is one of the most commonly used biopolymers owing to its biocompatibility and biodegradability for tissue regeneration and has been approved by FDA. Magnesium hydroxide (MH), an alkaline ceramic, is a non‐ toxic and biocompatible additive with the chemical formula Mg(OH)2. In this study, we combined fat graft with injectable regenerative scaffolds: natural AAM and synthetic PLGA/MH microspheres. Comparing the volume retention changes and viabilities on injected autologous fat graft, injectable natural (AAM) and synthetic (PLGA/MH microsphere) would suggest the better direction for fat transplantation.
Co‐culture model of 3D renal proximal tubule and human immune cells elucidating progression of acute kidney injury involved with hyperactivation of immune system
1Korea Institute of Toxicology
Great attention has been placed on the development of in vitro model describing the relationship between immune hyperactivity and acute renal injury. However, most studies still remain in two‐dimensional (2D) culture conditions, presenting limitations on the recapitulation of the phenomenon in 3D kidneys. In this study, 3D renal proximal tubules of RPTEC/TERT1 were formed within Matrigel on transwell insert (0.4 μm pore), and human peripheral blood monocytes were co‐cultured underneath of the inserts. To induce hyperactivation of immune cells and examine their effect on renal tubules, the system was treated with LPS (1 μg/mL) or polyI:C (20 μg/mL). Encapsulated cells forming 3D tubular structures expressed higher levels of renal function markers than 2D cultured cells. The inflammatory response of kidney cells was highly increased only in the case of co‐culture after drug treatment, and genes related to the development of ciliary and renal function were significantly downregulated. NGS data further described the relationship between the induced immune hyperactivity and the tubules by presenting progression of signaling pathways as “inflammatory response ➝ programmed cell death ➝ Hedgehog signaling ➝ cilium development inhibition ➝ Ciliopathies”. This is the first study of an in vitro 3D model clarifying that immune hyperactivity results in ciliopathy. This study also implicates that kidney damage associated with overactivated human immunity may be caused by biopharmaceuticals with potentials to induce immune hyperactivity. Collectively, we believe that this model is a promising tool to detect potential renal toxicity of biopharmaceuticals that affect the human immune system.
Effects of fibrous collagen/CDHA/hUCS biocomposites on bone tissue regeneration
1Chonnam National University, 2Department of Biomechatronic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan University (SKKU), Suwon, 3Department of Biomechatronic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan University (SKKU)
Collagen‐ and bioceramic‐based composites have been widely used in hard tissue engineering because they are analogous to the organic/inorganic constituents of native bones. However, biocomposites based on collagen and bioceramics show low mechanical stiffness and limited osteogenic activities. To elevate the low biophysical and biological activities, we have introduced a new biocomposite structure. Herein, we propose a biocomposite mimicking not only the physical structure of the extracellular matrix (ECM) structure but also the biochemical components of native bone tissues. Several components including fibrillated collagen, calcium‐deficient hydroxyapatite (CDHA) obtained from α‐tricalcium phosphate hydrolysis, and human umbilical cord serum (hUCS) were used to generate a unique structure of the biocomposite. The 3D‐printed composites were topographically similar to the nanofibrous ECM and exhibited a mechanically stable structure. We also evaluated the in vitro biocompatibilities of the biocomposite using human adipose stem cells and found that the collagen/hUCS/CDHA scaffold accelerated the in vitro osteogenic differentiation of human adipose‐derived stem cells and in vivo osteogenesis in a mastoid obliterated rat model.
Oxygen‐carrying nanomedicine for bone tissue engineering
1Sunmoon Univ., 2University of California Los Angeles, 3Dankook Univ.
Oxygen transport and supply in cell‐encapsulation devices is a pivotal requirement for supporting cell survival and function since inadequate oxygen delivery can lead to adverse effects on cellular metabolism and induce cell death. Although current hydrogels for cell delivery consist of around 90% water with a porous structure, mass transport limitations become apparent as the number of encapsulated cells increase and endogenous repair cells are recruited into the hydrogel, which often leads to a hypoxic condition that shifts to anaerobic metabolic processes. We developed osteoinductive oxygen‐carrying nanomedicine (O2‐NM) to enhance cell survival and osteogenic activity in hydrogels for bone regeneration. The O2‐ NM consists of an osteoinductive cholesterol derivative and perfluorocarbon by emulsifying with a membrane from red blood cells. The oxygen level was monitored using a dissolved oxygen meter. Evaluations for biocompatibility and osteogenic ability of the O2‐NMs were performed. The O2‐NM‐ laden hydrogel showed good cell support and proliferation. The O2‐NMs showed an excellent ability of oxygen supply. In addition, in vitro and in vivo results demonstrated osteoinductive activity and bone repair. The O2‐NMs have been investigated for supplying oxygen and enhancing osteogenic activity for bone tissue engineering.
Hyaluronic acid hydrogel with gradient mechanical properties for biomedical engineering
1School of Chemical Engineering, Pusan National University
Controlling the mechanical properties of hydrogels is of key importance in enabling various cell‐hydrogel interactions. Natural tissues and organs exhibit gradient physical cues depending on the depth or location and it regulates various cellular behaviors such as proliferation, differentiation, and migration during development, inflammation, and wound healing. However conventional hydrogels have a homogeneous environment with an isotropic distribution limiting complete mimicking of the natural extracellular matrix (ECM). In this work, we report hydrogel with gradient mechanical properties using hyaluronic acid which is a natural polymer and an excellent candidate for use in tissue engineering due to its excellent biocompatibility and bio‐functions. We successfully prepared the gradient hydrogel via photocrosslinking method and the gradient hydrogel was characterized and assessed the feasibility of tissue engineering application. The successful formation of the gradient in crosslinking density is confirmed by NMR spectrum. Scanning electron microscopy confirmed the different cross‐sectional morphology and the pore size in our hydrogel system. For in vitro test, L929 cells were encapsulated in gradient hydrogel and cellular behaviors specific to the degree of crosslinking are monitored using an optical microscope. In all samples, cells are highly active and spread well along the hydrogel substrate, and enhanced cell spreading was confirmed as the degree of crosslinking decreases. Taken together, this gradient hydrogel will be exploited to a tissue engineering platform to mimic naturalistic tissues with gradient stiffness.
Milk derived protein based scaffold enhanced ectopic and orthotopic bone formation
1Dankook University
Recently, several studies reported that casein was composed of various multifunctional bioactive peptides such as casein phosphopeptide and β‐casochemotide‐1 that bind calcium ions and induce macrophage chemotaxis, which is crucial for bone homeostasis and bone fracture repair by cytokines secreted in the process. We hypothesized that the effects of the multifunctional biopeptides in casein would contribute to improving bone regeneration. Thus, we designed a tissue engineering platform that consisted of casein and polyvinyl alcohol, which was a physical‐crosslinked scaffold (milk‐derived protein; MDP), via simple freeze‐thaw cycles and performed surface modification using 3,4‐dihydroxy‐L‐phenylalanine (DOPA), a mussel adhesive protein, for immobilizing adhesive proteins and cytokines for recruiting cells in vivo (MDP‐DOPA). Both the MDP and MDP‐DOPA groups proved indirectly contribution of macrophages migration as RAW 264.7 cells were highly migrated toward materials by contained bioactive peptides. We implanted MDP and MDP‐DOPA in a mouse calvarial defect orthotopic model and evaluated whether MDP‐DOPA showed much faster mineral deposition and higher bone density than that of the no‐treatment and MDP groups. The MDP‐DOPA group showed the accumulation of host M2 macrophages and mesenchymal stem cells (MSCs) around the scaffold, whereas MDP presented mostly M1 macrophages in the early stage. This work was supported by the Technology Innovation Program (20008734) funded By the Ministry of Trade, Industry & Energy (MOTIE, Korea).
Non‐destructive measurement of stiffness of tissue‐engineered constructs using ultrasound shear wave elastography
1Major of Biomedical Engineering, Division of Smart Healthcare, College of Information Technology and Convergence, Pukyong National University, 2Department of Biomedical Engineering, Inje University
In tissue engineering, quantitative measurement of tissue mechanical properties including tissue stiffness has an important role because tissue‐engineered structures should endure various conditions with different stress after implantation and be matched with native tissue. Conventionally, destructive methods using rheometry or tensile tests have been used to assess the mechanical property of the tissue‐engineered constructs. However, they require needed multiple constructs for the measurement and hardly be used continuously with the same sample, leading to an increase in cost and time. Also, they are not available with monitoring the constructs with complicated designs or in in‐vivo conditions. To overcome these limitations, we propose ultrasound shear wave elastography (SWE) to non‐destructively measure the mechanical property and monitor tissue‐engineered constructs continuously in real‐time. SWE can assess shear wave speed using an advanced ultrasound imaging system with enhanced acoustic radiation and high‐frame rate, and it has been widely applied in clinical applications to find pathologic lesions such as liver fibrosis using noninvasive quantification of different elasticities between malignant and benign regions. In this study, we validated the accuracy of shear wave speed in the tissue‐mimicking phantom and tissue‐engineered constructs. Assessment of shear modulus using SWE and destructive conventional methods were compared. The study demonstrated the feasibility of the non‐destructive method which can be applied to the original structure of the tissue‐engineered constructs and the in‐vivo models.
Decellularized corneal extracellular matrix scaffold for corneal endothelium regeneration
1Biomedical Engineering Research Center, Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea, 2Biomedical Engineering Research Center, Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea /Department of Convergence Medicine, University of Ulsan College of Medicine, Seoul,
Korea
The worldwide shortage of donor cornea for transplants is a serious problem to the patients in the waiting list and the researchers in tissue engineering have long been tried to build transplantable tissue engineered corneal tissues. The underlining issues in previous tissue engineered cornea tissues are the mechanical stability against intra‐ocular pressure, which is closely related mechanical toughness and biodegradability after long‐term implantation. From the mechanical point of view, our group adopted decellularized corneal extracellular matrix (dECM) for corneal endothelium engineering. Decellularized cornea ECM is known to have less immunogenic, similar microstructure to native cornea, and high biocompatibility. To minimize the risks of conventional corneal scaffolds and elevate safety, the corneal dECM scaffolds should go through various tests prior to in vivo applications. In this study, we confirmed the physical and chemical stability of scaffolds from decellularized porcine corneal ECM. Porcine cornea was separated and decellularized using 0.3% SDS, and then a thin film‐type dECM was prepared using a cryotome with thickness 30‐60 mm. Then the surface was observed by scanning electron microscopy, underwent tensile test, and accelerated biodegradation test. From SEM, the wet state dECM showed irregular pores on the surface whereas the dry state scaffold with a smooth surface without pores. Chemical fixation of dry dECM increased tensile strength. Also, lower decomposition rate was observed in the fixed dECM after biological degradation test using collagenase. Therefore, dECM of porcine cornea may be used as a scaffold for corneal regeneration replacing the donated cornea.
The development of a novel white matter hyperintensity model by mimicking blood‐brain barrier‐oligodendrocytes interface using 3D cell printing
1Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong S.A.R, China
White matter hyperintensity (WMH) is strongly associated with blood‐brain barrier (BBB) disintegration in sporadic Alzheimer disease (AD), by the mechanism remains obscure. We aim to develop a WMH model by reconstructing BBB in vitro using 3D cell printing in order to investigate the interactions between oligodendrocyte (OL), endothelial cells and astrocytes at BBB. Here, we first investigated the effects of APOE4, the strongest AD genetic risk factor, on the interactions between OL and astrocytes in traditional culture. To develop the WMH model, a microfluidic bilayer construct was designed and fabricated using extrusion‐based 3D cell printing in a CELLINK Inkredible+ system with BioInk GelMA (gelatin methacryloyl 44‐45%) or alginate gel. As a proof‐of‐concept experiment, we showed that primary murine astrocytes carrying APOE3, but not APOE4, rescued the differentiation of a OL progenitor cell line, Oli‐Neu, during cholesterol depletion. To optimize the cell printing process, the reproducibility was compared between BioInks. The success rate of GelMA 146/160 (91.13%) is higher than that of alginate gel 178/208 (85.57%) in printing small square pits (1000 x 1000 μm). The biocompatibility of printed prototype is tested with murine astrocytes, bEnd.3 (brain endothelial cell line) and Oli‐Neu using a Live/Dead fluorescence staining. The microfluidic bilayer construct mimicking the interface between cerebrovasculature (astrocyte‐bEnd.3) and WM (Oli‐Neu) is being examined for the establishment of tight junctions and associated permeability using confocal microscopy. This WMH model will help understanding the effects of APOE4 on the cell‐cell interaction at BBB and facilitate a high throughput screening for drug discovery.
Mussel adhesive protein‐based adhesive to retain stem cells for cartilage regeneration
1Dongguk University, 2Pohang University of Science and Technology
In this study, we developed biocompatible adhesive which enables implanted chondrogenic‐enhanced hASCs being strongly fixed to the lesion site of defected cartilage.
The bioengineered mussel adhesive protein (MAP) was produced and purified using a bacterial expression system as previously reported. The cell encapsulated coacervate was formulated with two polyelectrolyte, the MAP and hyaluronic acid(HA). MAP formed liquid microdroplets with HA and subsequently gelated into microparticles, which is highly viscous and strongly adhesive.
The MAP with chondro‐induced hASCs were implanted on the osteochondral defect created in the patellar groove/condyle of OA‐induced rabbits. Rabbits were allocated to three different groups as follows: Group1‐ Fibrin only; Group2‐Fibrin with hASCs; Group3‐MAP with hASCs.
The histological assessment showed that Group3 (MAP+hASCs) had the best healing and covered with hyaline cartilage‐like tissue. The staining image shows that Group3 were filled with perfectly differentiated chondrocytes. Group3 has higher possibility of differentiating to complete cartilage, whereas Group1 (Fibin only) and Group2 (Fibrin+hASCs) have failed to treat OA by rehabilitating cartilage. In order to clarify the evidence of remaining human cell proving efficacy of newly developed bioadhesive, human nuclear staining was proceeded with sectioned rabbit cartilage tissue. The results explicitly showed Group3 (MAP+hASCs) have retained more human cells than Group1 and 2.
We investigated the waterproof bioadhesive supporting transplanted cells to attach to defect lengthily in harsh environment, which prevents cells from leaked to other region of cartilage. Collectively, the bio‐ adhesive, MAP, could be successfully applied in OA treatment as a waterproof bioadhesive with the capability of the strong adhesion to target defect sites.
Biomimetic composite gelatin methacryloyl hydrogels for improving survival and osteogenesis of human adipose derived stem cells in 3D microenvironment
1Hanyang university
In tissue engineering, gelatin methacryloyl (GelMA) hydrogels have been widely utilized for stem cell encapsulation because they provide biocompatible 3D environment and exhibit extracellular matrix‐like properties in terms of polymer structure and water contents. However, GelMA hydrogels have some limitations such as difficulty to modulate encapsulated stem cell functions, and cell damage caused by excessive reactive oxygen species (ROS) and resulting radical species generated during polymerization. It is known that ROS induces inflammation, promotes the maturation of osteoclasts, and delays bone regeneration. Thus, it is necessary to engineer GelMA hydrogels to effectively remove ROS and to induce osteogenesis of stem cells for bone tissue engineering. In this study, we developed composite GelMA hydrogels (G‐TMN) incorporating biomimetic tannic acid mineral nanoparticles (TMN) with ROS scavenging and osteoinductive functions. GelMA hydrogels encapsulated with human adipose derived stem cells (hADSCs) were prepared, and the effect of nanoparticles on hADSCs within 3D environment was investigated. G‐TMN demonstrated greater radical scavenging effect than GelMA hydrogels without TMN, so that hADSCs encapsulated in G‐TMN showed greater cell viability due to the protection against ROS. In addition, osteogenic differentiation of hADSCs in G‐TMN was improved due to the mineral composition of TMN. The encapsulated stem cells in G‐TMN showed significantly greater calcium deposition then the other groups. In conclusion, we expect this composite hydrogel system would be useful for potential enhancement of encapsulated cell viability and osteoinduction.
Scaled‐up hypertrophic cartilage tissue engineering for bone regeneration applications
1Bioprocessing Technology Institute
Hypertrophic cartilage, such as those in the growth plate or fracture callus, is the template that guides natural bone growth and regeneration via endochondral ossification. This cartilage template is a potential material for utilization in many bone regeneration applications. Due to limited quantity, hypertrophic cartilage is often cultured in vitro for research purposes. Many culturing methods have been explored, including microgravity (e.g., hanging drops, micro‐wells), solid scaffolds (e.g., transwells, 3D natural/synthetic scaffolds), and hydrogel. Due to the complexity involved, these methods can only produce small‐scale prototypes, sufficient for a few experiments and perhaps an animal study. We have recently developed a novel method to culture hypertrophic cartilage from human mesenchymal stem cells (hMSCs) in vitro. Our proprietary method allows simple handling of the culturing process, similar to the traditional 2D culture protocol while maintaining the 3D cell‐cell interaction required for chondrogenesis. Our method allows the production of large hypertrophic cartilage tissue which is highly relevant to the clinical requirement. We have characterized our hypertrophic cartilage tissue by gene expression analysis. Our data showed the expected upregulation in gene expression for chondrogenic and hypertrophic markers. Our future studies will validate the efficacy of the hypertrophic cartilage tissue in an animal model for bone regeneration.
Label‐free 3D analysis of odontogenic differentiation of stem cell spheroids using raman spectroscopy
1Chung‐Ang University, 2Jeonbuk National University, 3Nanobase, Inc
Recent research of stem cell area leads to the new innovation such as establish three‐dimensional (3D) cell cultures that mimic the structure, cell composition, and functions of actual tissues and organs in vitro. 3D‐structure precise characterization of the stem cell spheroid (SCS) is also extremely necessary, yet most of the biological analysis is destructive and invasive to the live samples. In this study, we report two issues as follows: 1) the comparison of two type methods for human dental pulp stem cells (hDPSCs) spheroid generation and 2) a non‐destructive and label‐free monitoring of odontogenesis of hDPSCs spheroids based on Raman spectroscopy. The increase of cell density reduced the growth medium diffusion as proven by the decrease of Raman signal at 3,400 cm−1. Also, the nutrient distribution upon odontogenesis was investigated by performing Raman mapping analysis, as to reveal the correlation between cell density and the successful ratio of differentiation. The differentiation rate was found to be higher in low‐density spheroids following on the Raman maps at 960 cm−1 corresponding to hydroxyapatite. Hence, Raman‐based analysis is advantageous for analyzing 3D cell culture (e.g., spheroid or organoid) in a high‐quality manner, which thus will be useful for regenerative therapies and drug discovery.
Polymeric composite hydrogel for mimicking mechanochemical microenvironment of cartilage tissue
1Department of Biomedical Engineering, Indian Institute of Technology Ropar
Articular cartilage exhibits little or no ability for regeneration, its repair and renewal have become a topic of much interest in tissue engineering of articular cartilage for physicians and scientists. A polymeric composite has been developed to mimic the native ECM microenvironment of cartilage tissue using gelatin as collagen equivalent and kappa‐carrageenan as GAG. In this study, the dry carrageenan and gelatin powder were dissolved in distilled water, and then heated and stirred until a homogeneous solution was achieved. The solution was consolidated and dried to a consistent weight at room temperature. Samples were prepared by this process using Gelatin and K‐Carrageenan in different ratios and checked their performance in terms of physical and chemical attributes. These compositions were prepared crosslinking (covalent crosslinking) by soaking them in glutaraldehyde solution containing 0.1N HCl, then dried at 110°C for 25 minutes and air‐dried to a consistent weight at room temperature. Crosslinking procedure was also followed utilizing potassium chloride (ionic crosslinking) and a combination of KCl and Glutaraldehyde. These composites have been tested for comparison of ECM of articular cartilage via nanoindentation, FTIR, SEM‐EDS analysis, and UTM characterization. Further stem cell proliferation and differentiation have been conducted using cellular assay and PCR analysis. The composite has been observed to be capable of mimicking the cartilage cellular microenvironment.
Edible starch‐based three‐dimensional scaffolds for the construction of millimeter‐thick cultured meat
1Yeungnam University
Owing to the growing global demand for meat, the development and innovations in cultured meat technology become paramount to avoiding future meat shortages. The formation of millimeter thick cultured bovine muscle tissue with highly aligned myotubes needs to be produced using biocompatible 3D biomaterials. In our study, we fabricated edible starch‐based hydrogel scaffolds for the culturing of millimeter‐thick bovine muscle tissue. These scaffolds were cultured with bovine muscle cells and stacked to construct muscle tissue with the formation of fully matured myotubes. These scaffolds support the attachment and proliferation of bovine muscle cells to construct 3D bovine muscle tissue. The alpha‐ actinin immunostaining was performed to evaluate the maturation of bovine muscle tissue by estimating the occupancy rate and rate of myotube formation. The microbial contamination of the muscle tissue cultured for 21 days was also evaluated. Thus, the lab‐grown clean cultured bovine muscle tissue can be used as an alternative to real meat.
Engineered tendon nano‐constructs for repair of chronic rotator cuff tears in large‐animal model
1Chonnam National University
Chronic rotator cuff tears (RCTs) are one of the most common injuries of shoulder pain. Despite the recent advances in surgical techniques and improved clinical outcomes of arthroscopically repaired rotator cuffs (RCs), complete functional recovery—without retear—of the RC tendon through tendon‐to‐ bone interface (TBI) regeneration remains a key clinical goal to be achieved. Inspired by the highly organized nanostructured extracellular matrix in RC tendon tissue, we propose herein a tissue engineered tendon nano‐construct (TNC) for RC tendon regeneration. When compared with two currently used strategies (i.e., transosseous sutures and stem cell injections), our nano‐construct facilitated more significant healing of all parts of the TBI (i.e., tendon, fibrocartilages, and bone) in both rabbit and pig RCT models owing to its enhancements in cell proliferation and differentiation, protein expression, and growth factor secretion. Overall, our findings demonstrate the high potential of this transplantable tendon nano‐construct for clinical repair of chronic RCTs.
Heat and pressure‐assisted soft lithography‐ and plasma‐based multiscale structures for soft and hard tissue engineering
1Chonnam National University
Here, we present an analysis of nanoscale deformation of PDMS molds in response to heat and pressure during the repetitive molding process of thermoplastic polymers. The width and height of the nano‐sized ridges of PDMS molds decreased as the number of replications of thermoplastic polymers increased. Using the precisely controlled deformation of nanostructures in PDMS molds, we demonstrated that nanostructures of different sizes can be fabricated on representative thermoplastic and UV‐curable polymers consistently. Using the precisely tunable methodologies of nanoscale structures, we propose a methodology to fabricate hierarchical multiscale scaffolds with controlled hydrophilic and hydrophobic properties by employing capillary force lithography in combination with oxygen plasma modification. In response to multiscale nanotopographic and chemically modified surface cues, the O‐FMN patch enhanced regeneration of the mineralized fibrocartilage tissue of the tendon–bone interface and the calvarial bone tissue in vivo in rat models.
Differentiation of endogenous stem cells migrated with novel chemoattractant into vascular endothelial cells in hybrid hydrogel
1Ajou University
Endogenous stem cells are tissue‐specific adult stem cells that are capable of self‐renewal and can differentiate into specific cells. These cells migrate to the particular regions of the body that require tissue healing. Because Endogenous stem cells can be involved in the process of healing and regeneration of wounded or diseased tissues or organs, in situ tissue regeneration using endogenous stem cells could get over several of the problems, caused by ex vivo stem cell manipulation in conventional tissue engineering. Chemoattractants such as Substance‐P (SP), and stromal‐derived factor powerful endogenous stem cell activating factors and can thus be used to lengthen and improve endogenous stem cell assembly. However, the number of chemoattractant‐guided endogenous stem cells that migrate during the endogenous repair process is generally so low that it can't achieve full in situ tissue regeneration. Thus, new Chemoattractant should be developed for increasing the migration of endogenous stem cells to the impaired area. Substance‐P1 (SP1), a neurokinin‐1 receptor (NK1R) binding peptide ligand, is loaded into injectable electrostatic hydrogel (Chi/HA) using cationic chitosan (Chi) and anionic hyaluronic acid (HA) to migrate stem cells. In an environment where blood vessels can be formed, migrated endogenous stem cells differentiate into new blood vessels using vascular differentiation peptide (VEGF‐Mimic peptide). In conclusion, we successfully provide an environment to generate blood vessels, and differentiates stem cells migrated using Substance‐P1 (SP1) and VP (VEGF‐Mimic peptide).
Wound healing through recruiting stem cell with Substance P loaded electrospun‐biomaterials sheet
1Ajou university
Something important for wound healing is the regeneration of skin tissue. In particular, for the treatment of deep wounds caused by accidents or skin tissue that has fallen off to the dermis or subcutaneous fat, the regeneration of skin tissue using the seeding of therapeutic cells in the wound dressing is groundbreaking. In this study, Substance P (SP) was loaded to small intestinal submucosa (SIS)/poly(ɛ‐ caprolactone)‐ran‐poly(L‐lactide) (PCLA) sheet (S/P sheet) and wound was treated through MSCs recruiting. First, we made drug delivery scaffold using electrospinning with SIS and PCLA. SIS contains various cytokines such as VEGF/EGF etc, which can facilitate the migration of MSCs towards SIS. However, electrospinning of SIS alone leads to weak mechanical properties. For this reason, we designed electrospinning sheet with not only SIS, but also PCLA copolymer which can improve natural material's shortcoming. To S/P sheet, we loaded SP, a neuropeptide which can promotes cell growth and proliferation, and thereby it has effect of recruiting stem cells. Wound healing was confirmed through injection of MSCs using SP‐S/P sheet. We conducted assays like in vitro/in vivo migration analysis and histological/statistical analysis, etc. Overall, SP‐S/P sheets significantly enhanced cell migration in vitro/in vivo and through histological/statistical analysis, results showed that use of an SP‐S/P sheet could provoke a minimal inflammatory response and effectively enhance wound healing through MSCs recruitment. According to our research, SP‐S/P sheet should be a faster and more effective treatment method for people with deep wounds.
Development of injectable click crosslinked hydrogel formulation containing Substance P analog and BMP‐2 mimetic peptide to induce migration and bone formation of stem cell
1Ajou university
Bone tissue engineering is considered a promising approach for effective treatment of damaged bone tissue. In this study, we attempted to design a suitable scaffold for bone tissue engineering with the appropriate combination of biological elements. We attempted to use hyaluronic acid (Ha)‐based hydrogel for bone tissue engineering based on the fact that Ha is biocompatible, biodegradable, and nontoxic. Tetrazine‐modified Ha (Ha‐tet) and transcyclooctene‐modified Ha (Ha‐tco) were prepared to form an injectable click crosslinked hydrogel (Cx‐Ha). Substance P1 (SP1) is one of the Substance P peptide analog, which can promote stem cell migration. SP1 effectively induced migration of human mesenchymal stem cells (hMSCs) in vitro, as evidenced by trans‐well plate migration assay and wound healing assay. Through fluorescence imaging, it was confirmed that SP1 from the injected Cx‐Ha+SP1 hydrogel into mouse body can effectively promote the migration of hMSCs. B2P is one of the bone morphogenetic protein‐2 (BMP‐2) mimicking short peptide. B2P induced osteogenic differentiation of hMSCs in vitro, as evidenced by Von Kossa and Alizarin Red S staining. Also, B2P shows greater increases in the expression of RUNX2, osteonectin and osteocalcin mRNA levels compared to control group. Thus, we are about to design an injectable formulation of Cx‐Ha‐B2P+SP1 hydrogel to have the potential to recruit endogenous stem cells to scaffold and induce osteogenic differentiation of stem cells to meet the unmet need for clinical treatment of damaged bone tissue.
The senolytic drug JQ1 removes senescent cells via ferroptosis
1Seoul National University, 2Korea University of College of Medicine, 3Korea Institute of Science and Technology, 4Harvard Medical School
Ferroptosis is an iron‐dependent, non‐apoptotic programmed cell death. Cellular senescence contributes to aging and various age‐related diseases through the expression of a senescence‐associated secretory phenotype (SASP). Senescent cells are often resistant to ferroptosis via increased ferritin and impaired ferritinophagy. In this study, we investigated whether treatment with JQ1 could remove senescent cells by inducing ferroptosis. We treated bleomycin‐induced senescent human dermal (fibroblasts HDFs) with JQ1 and determined whether JQ1 has senolytic effects with ferroptosis. At a certain range of JQ1 concentrations, JQ1 treatment reduced the viability and decreased SASP expression of senescent cells but did not reduce that of non‐senescent cells, indicating that JQ1 treatment can selectively eliminate senescent cells. Subsequently, JQ1 treatment reduced the expression of ferroptosis‐resistance genes and induced lipid peroxidation only in senescent cells. It suggested ferroptosis occurred in JQ1's senolytic pathway. So, these data indicate that JQ1 can eliminate senescent cells via ferroptosis. This study suggests ferroptosis as a new mechanism of senolytic therapy.
Development of bioreactor system for full‐thickness corneal tissue engineering
1Univ. Of Ulsan College Of Medicine, 2Department of Ophthalmology, Asan Medical Center, 3Biomedical Engineering Research Center, Asan Institute for Life Science, Asan Medical Center
In this study, a bioreactor system was developed for the formation of an implantable tissue‐engineered full‐thickness cornea. When designing the bioreactor, a flow path was set so that the cell culture solution required for corneal epithelial cells and endothelial cells could be supplied respectively. Each chamber filled with corneal epithelial cells and with corneal endothelial cell culture medium has a cylindrical structure with a diameter of 10 mm and a height of 20 mm. Cell cultured scaffolds are layered and inserted in the middle region. A flow control system using microchannels was applied to supply the culture media to the bioreactor. A thin‐film scaffold was prepared with decellularized extracellular matrix (dECM) of the porcine cornea. For dECM scaffold, fresh porcine corneas were explanted and stirred in 1% SDS for 48 h and rinsed with PBS containing DNase and RNase subsequently. Epithelial cells and endothelial cells of the donated human cornea were isolated and cultured respectively. Instead of keratocytes present in the corneal stroma, mesenchymal stem cells were used for this study. Before assembling the bioreactor, dECM scaffolds were seeded with corneal endothelial cells, corneal epithelial cells, and mesenchymal stem cells respectively, and 1 mL of culture solution was supplied per day. After a predetermined period, tissues were histologically stained with H&E and Masson's Trichrome Staining.
Multimodal therapy strategy based on highly functional hydrogels for the repair of spinal cord injury
1CHA University School of Medicine, 2CHA University School of Medicine, Chung‐Ang University
Traumatic spinal cord injury (SCI), in which the axon is physically damaged, is a serious disease that causes permanent motor dysfunction and pain as a secondary injury after a primary injury. In this study, we designed a hyaluronic acid (HA)‐based hydrogel combined with decellularized brain matrix (DBM), polydeoxyribonucleotide (PDRN), TNF‐α/IFN‐γ primed mesenchymal stem cell‐derived extracellular vesicles (TI‐EVs), and neural progenitor cells (NPCs). The formulation of injectable substances could provide structural support within nerves, and it was predicted that nerve regeneration would be possible due to the synergistic effect of each substance. As a result, it was possible to promote the proliferation and differentiation of NPCs in vitro. In addition, it was confirmed that functional motor recovery was possible through reduction of inflammatory response and formation of neurons and oligodendrocytes in vivo experiments. A novel combination of injectable HA‐based hydrogels has been identified as an effective candidate for SCI regeneration.
TGF‐β‐induced transglutaminase‐2 triggers catabolic response in osteoarthritic chondrocytes by modulating MMP‐13 and collagen II
1Seoul National University Hospital
TGF‐β plays an essential role in maintaining cartilage homeostasis. TGF‐β is known to upregulate anabolic processes in articular cartilage, but the role of TGF‐β in chondrocyte catabolism remains unclear. Thus, we examined whether TGF‐β increases catabolic processes in the osteoarthritic joint via transglutaminase 2 (TG2). To investigate whether TGF‐β and TG2 activity is relevant to OA pathogenesis, we examined the level of TGF‐β and TG2 in the osteoarthritic joint. Also, we assessed the level of MMP‐13 in chondrocytes treated with TGF‐β and investigated whether TG2 inhibition prevents induction of MMP‐13. We confirmed overexpression of TGF‐β and TG2 in human OA cartilage compared to non‐OA cartilage. TGF‐β‐induced TG2 expression reduced the level of type II collagen while increased the level of MMP‐13 via NF‐κB activation and expression of type X collagen in chondrocytes. Safranin‐O staining revealed minor observed differences between cartilage of TG2‐/‐ mice and wild type mice, and the extent of cartilage damage was significantly less in TG2‐/‐ mice than that in wild type mice after OA induction. Therefore, our findings show TGF‐β‐mediated TG2 induction may increase chondrocyte catabolism and facilitate degeneration of articular cartilage, suggesting TG2 may be a potential therapeutic target for osteoarthritis.
The possibility of vitronectin‐derived peptide as a treatment for pulp capping
1Seoul National university, 2Dankook University
Dental caries is one of the most common oral diseases and the primary cause of tooth loss. If the pulp is exposed during dental caries treatment, pulp capping is performed to protect the pulp. Our previous study demonstrated that human vitronectin‐derived peptide (VnP‐16) plays a dual role of promoting bone formation by osteoblast and inhibiting bone resorption by osteoclast. The present study investigated the effects of VnP‐16 on reparative dentinogenesis in rat pulp exposure models. Similar to vitronectin, VnP‐ 16 showed no cytotoxicity and promoted cellular behavior in human dental pulp cells (hDPCs), enhancing their differentiation into odontoblast‐like cells and mineralization. In a rat pulp exposure model, Mineral trioxide aggregate (MTA) showed a tendency of early formation of reparative dentin at 2 weeks compared to recombinant human bone morphogenetic protein‐2 (rhBMP‐2) and VnP‐16. However, VnP‐16 induced reparative dentin formation similar to MTA and rhBMP‐2 without inflammation at 4 weeks. In addition, VnP‐16 showed a thicker and homogeneous reparative dentin formation compared to MTA and rhBMP‐2. Collectively, these results suggest that VnP‐16 has potential as a therapeutic agent due to highly qualified reparative dentin formation by promoting cell behavior and odontoblastic differentiation of hDPCs.
Fabrication and evaluation of rhBMP‐2 incorporated 3D printed scaffold as a novel bone substitute in calvarial defect mouse model
1Kyungpook National University School of Medicine, Kyungpook National University Hospital,
2Kyungpook National University Hospital, 3Department of Chemistry, Kyungpook National University
Natural bone is a hard and dense type of connective tissue with excellent mechanical properties. Normally, bone has an innate capacity to heal from damage, however, self‐repair is challenging when there are massive bone defects. Despite the emergence of scaffold‐free tissue engineering as a powerful strategy, the use of biomaterial scaffolds remains the classical approach to regenerating bone due to the good degradation profile and advantageous mechanical properties, as well as to deliver important biomolecules such as growth factors on the scaffold surface.
Current bone repair biomaterial scaffolds are designed to reproduce such a microenvironment to promote cell ingrowth and differentiation, and vascularization for osteogenesis. Thus, biomaterial scaffolds having 3D hierarchical structures with porous nanostructures are the most promising bone substitutes. Biomaterials have been optimized by incorporating additional chemical groups or bioactive factors such as BMP, TGF, FGF, and PDGF. To this end, there is increasing research on modifying the surface architecture and components of 3D biomaterial scaffolds to enhance cell adhesion, growth, differentiation, and migration, and consequently efficient bone regeneration.
In this study, we developed a PLLA‐based bioactive scaffold of 3D hierarchical structures using 3D printing technology. A further modification was made on the surface of the 3D biomaterial scaffold by binding rhBMP‐2 for long‐term release and extracellular matrix by both chemical and physical methods. Bone regeneration efficiency of the thus developed bioactive biomaterial was evaluated in vitro and in vivo.
Changes of characteristics of mouse embryonic fibroblasts according to cryopreservation period for tissue engineering
1Chungbuk National University
Fibroblasts are widely used as feeder cells that are co‐cultured with stem cells to support embryonic stem cells in tissue engineering fields. Several recent tissue engineering studies have shown that fibroblasts that cannot differentiate into other cells can re‐differentiate into other cells using a reprogramming mechanism. Mouse embryonic fibroblasts (MEFs) are commonly used with their advantages of easy isolation and abundant yield, however, the characteristics of cells may not be maintained from the original cells due to aging. Nevertheless, there are not many studies that have investigate the characteristics of MEF according to the period of cryopreservation. In this study, the cryopreservation effect of MEFs on changes in morphology and functional properties according to the period of cryopreservation were investigated. We isolated primary MEFs and compared their cellular characteristics and functional gene expression levels using freshly isolated MEFs and short‐term (1 d – 1 week) and long‐term (8 – 20 weeks) cryopreserved MEFs. As a result of functional gene expression for cryopreserved fibroblasts, the expression levels of gene were different depending on the storage period. The expression levels of vimentin and a‐SMA increased after cryopreservation, whereas FSP1 and fibronectin were maintained constant over short‐term storage, while increased over long‐term storage. Although there are not known data on the functional genes in MEF according to the cryopreservation period, this study provides the basic data to improve the usefulness of MEFs in tissue engineering research according to the purpose of each study.
Optimization of the purification method for skeletal muscle‐derived fibroblast
1Chungbuk National University
Fibroblasts are widely used as feeder cells that are co‐cultured with stem cells to support embryonic stem cells in tissue engineering field. However, the tissue origin is important because the expression of extracellular matrix proteins and cell surface markers are differently secreted depending on the tissue origin of fibroblasts. Commonly used fibroblasts use embryonic origin, but the characteristics of skeletal muscle‐derived fibroblasts may not be reflected due to differences in tissue origin. In particular, it is necessary to selectively isolate only fibroblasts with high purity because of mixing with fibroblasts and myoblasts in skeletal muscle tissue. In this study, fibroblasts from mouse skeletal muscle tissue were isolated using the preplating technique, and their functional and the morphological characteristics were investigated to optimize the effective purification method of fibroblasts. Cell morphology and doubling time were not significantly associated with preplating. The optimized preplating method did not cause significant functional changes in fibroblast‐specific genes such as Vim and Fsp1 and myoblast‐specific genes such as Myod and Myog expressions until passage number 5. Additionally, skeletal muscle‐derived fibroblasts before and after cryopreservation maintained their morphological and functional properties until passage 5. Based on the results, the characteristics of isolated skeletal muscle‐derived fibroblasts were maintained up to passage 5 with or without preplating, and the characteristics of the fibroblasts were maintained even after cryopreservation. Here, this study has optimized the isolation and purification method for skeletal muscle‐derived fibroblast, and is expected to be used in various applications in tissue engineering.
Thermoresponsive nanofiber‐based microwell scaffolds capable of formation and retrieval of salivary gland stem cell spheroids for the regeneration of irradiation‐damaged salivary glands
1Department of Otorhinolaryngology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea, 2Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Republic of Korea
Three‐dimensional spheroid culture enhances cell‐to‐cell interactions among stem cells and promotes the expression of stem cell properties; however, subsequent retrieval and delivery of these cells remain a challenge. We fabricated a thermoresponsive fiber‐based microwell scaffold by combining electrospinning and photolithography. The resultant scaffold appeared to facilitate the formation of uniform‐sized cellular spheroids and enabled the expression of more stem cell‐secreting growth factor genes, pluripotent stem cell‐related genes, and adult epithelial stem cell‐related genes than salivary gland stem cells in a monolayer (culture SGSCmonolayer). The spheroids could be retrieved by decreasing temperature. SGSC‐derived (spheroid SGSCspheroid) cells were implanted into the submandibular glands of mice at 2 weeks after fractionated X‐ray irradiation. At 16 weeks post‐irradiation, restoration of salivary function was detected only in SGSCspheroid‐implanted mice. The production of submandibular acini specific mucin increased in SGSCspheroid‐implanted mice, compared with PBS control. More MIST1+ mature acinar cells were preserved in the SGSCspheroid‐implanted group than in the PBS control group. Intriguingly, SGSCspheroid‐implanted mice exhibited greater amelioration of tissue damage and preservation of KRT7+ terminally differentiated luminal ductal cells than SGSCspheroid‐ implanted mice. The SGSCspheroid‐implanted mice also showed less DNA damage and apoptotic cell death than the SGSCspheroid‐implanted mice at 2 weeks post‐implantation. A significant increase in Ki67+AQP5+ proliferative acinar cells was noted only in SGSCspheroid‐implanted mice. Our results suggest that a thermoresponsive fiber‐based scaffold could be of use to facilitate the production of function‐enhanced SGSCspheroid cells and their subsequent retrieval and delivery to damaged salivary glands to alleviate radiation‐induced apoptotic cell death and promote salivary gland regeneration.
Wound healing applications of sericin/CMC/MSM incorporated hydrogel
1Innoregen,Inc
Natural‐based silk sericin has excellent properties such as biocompatibility, biodegradability, and antioxidant action, and helps in cell proliferation, collagen (type 1) production, skin regeneration and wound healing. In this study, we aim to develop a cross‐linkable multifunctional carboxymethyl cellulose/sericin‐MSM (CMC/S‐M) hydrogel to help patients with tissue regeneration. The effects of CMC/S‐M hydrogel on cell proliferation, wound healing, macrophages and the release of inflammatory cytokines from the wound were analyzed. As a result, CMC/S‐M hydrogel promoted cell proliferation, wound healing and reduced inflammation.
The role of mechanotransduction in tissue remodeling by TGF‐β induced fibroblast activation
1Korea University
Cardiac fibroblasts are the main cells responsible for the homeostasis of the extracellular matrix (ECM) in the heart, leading to physiological and pathological remodeling of tissues depending on the activity of fibroblasts. Activation of fibroblasts is induced by biochemical factors such as TGF‐β or physical stimuli such as ECM stiffness, and activated fibroblasts develop contractile cytoskeletons and fibrous ECMs to change the physical environment, leading to mechanotransduction of surrounding cells. However, the role of mechanotransduction in tissue remodeling by fibroblast activation is still unclear. Here, we implemented 3D fibroblast bundle tissue using human cardiac fibroblasts and an engineered heart tissue system to evaluate the effect of TGF‐β induced fibroblast activity on tissue remodeling and gene expression. As a result, in the TGF‐β treated group, tissue contraction and force generation were increased by about 30% compared to the control group. The mRNA expression of the fibroblast activation markers, collagen type I, α‐SMA and LOX, also increased more than 2‐fold in the TGF‐β treated group. Such tissue remodeling and the gene expression were significantly disrupted by the inhibition of TGF‐β signaling with SB431542, an inhibitor of TGF‐β receptor. To determine the involvement of mechanotransduction, the ROCK inhibitor, Y27632, was treated to hinder actin cytoskeleton formation, and this led to decreased tissue remodeling and the force generation, which was partially recovered by treatment of TGF‐β. These results suggest that TGFβ signaling is one of the key participants of tissue remodeling, and it is partially mediated by actin cytoskeleton implying mechanotransduction in the process.
Myoblast alignment and differentiation in tissue‐engineered skeletal muscle fiber
1Department of Biomedical Engineering, Doshisha University, 2Graduate School of Life and Sciences, Doshisha University
Tissue‐engineered skeletal muscle requires high contractile force to recover mechanical function of skeletal muscle, and high alignment of myotubes is important to obtain high contractile force. In this study, we developed the tissue‐engineered skeletal muscle with the fiber‐shaped structure to improve contractile force. Though the fiber‐shaped structure of the tissue‐engineered skeletal muscle is expected to enhance cell orientation along the longitudinal direction, it is necessary to evaluate effects of initial diameter on alignment and differentiation of myoblasts. Type‐I collagen solution mixed with C2C12 cells was extruded from the nozzles into DMEM with 10% FBS at 37 °C to fabricate the tissue‐engineered skeletal muscle fibers with three different initial diameters. Initial diameters of the tissue‐engineered skeletal muscle fibers were 228, 550 and 950 mm. After the tissue‐engineered skeletal muscle fibers were cultured in DMEM containing 10% FBS for 2 days with both ends fixed, they were cultured in DMEM containing 7% horse serum for 12 days. According to optical microscope observation, diameters of the tissue‐engineered skeletal muscle fibers decreased with cultivation day, and myoblasts aligned along longitudinal direction. Highly aligned cells were confirmed near the surfaces of the tissue‐engineered skeletal muscle fibers with HE staining. The ratio of fiber diameter divided by the initial diameter increased with decreasing the initial diameter. According to fluorescent observation of myosin heavy chain expression after 14 days of cultivation, the ratio of myosin heavy chain expression area divided by cross‐sectional area of the tissue‐engineered skeletal muscle fiber increased with decreasing the initial diameter.
Application of developmental signaling regulations in alveolar bone regeneration after the periodontitis
1Kyungpook National University
OBJECTIVE: We evaluated the role of developmental signaling molecules in bone formation after tooth loss by periodontitis.
BACKGROUND: Periodontitis causes the sequential degradation of the alveolar bone and associated structures, resulting in tooth loss. Several studies have attempted to regenerate the bone for implantation following tooth loss.
METHODS: Maxillary left second molar was extracted from 8‐week‐old male mice following induction of periodontitis by ligature for 5 days. The extraction socket was treated with developmental signaling molecules for 1, 2, and 3 weeks. Detailed morphological changes were examined using Masson's trichrome staining, and the precise localization patterns of various signaling molecules, including CD31, F4/80, interleukin (IL)‐6, and osteocalcin, were observed. The volume of bone formation was examined by Micro‐CT. Osteoclasts were enumerated using tartrate‐resistant acid phosphatase (TRAP) staining. For molecular dissection of signaling molecules, we employed the hanging‐drop in vitro cultivation method at E14 for 1 day and examined the expression pattern of transforming growth factor (TGF)‐β superfamily and Wnt signaling genes.
RESULTS: Histomorphometrical examinations showed facilitated bone formation in the extraction socket following developmental signaling molecule treatments. In addition, treated specimens showed the altered localization patterns of inflammatory and bone formation‐related signaling molecules including CD31, F4/80, IL‐6, and osteocalcin. Also, embryonic tooth germ mesenchymal tissue cultivation with treatment showed the significant altered expression patterns of signaling molecules.
CONCLUSIONS: Developmental signaling molecules would induce bone formation and remodeling through proper modulation of osteoblast, osteoclast, and inflammation with regulations of TGF‐β and RUNX2 signalings.
Functional evaluation of developmental signaling molecules for dentin regeneration
1Kyungpook National University
Introduction
The aim of this study was to evaluate in vitro and ex vivo roles of developmental signaling molecules in tertiary dentin formation.
Methods
We established pulpal access cavity preparation that was treated gain or loss of functional evaluation of signaling molecules before direct pulp capping. We also analyzed altered signaling regulations using quantitative real‐time polymerase chain reaction and immunohistochemistry.
Results
In the short‐term observation period, the siRNA and AAV treated pulp specimens showed the period‐ altered immunolocalization patterns of nestin, CD31, and myeloperoxidase, whereas the control specimens did not. The experimental group showed a complete dentin bridge with very few irregular tubules after 42 days. The micro–computed tomographic images showed more apparent dentin bridge structures in the treated specimens than were in the controls. Quantitative real‐time polymerase chain reaction analysis showed up‐regulated Bmp and Wnt.
Conclusions
These findings revealed that treatment of developmental signaling molecules induced reparative dentin formation that facilitated the maintenance of the integrity of the remaining pulpal tissue via early vascularization and regulation of Bmp and Wnt signaling.
Expression pattern and developmental function of Piezo1 in salivary gland morphogenesis
1Kyungpook National University, 2Gachon University
Recent researches showed the importance of mechanically‐activated factors in the condition of organogenesis, especially in formation of secretory organs including salivary glands. Piezo type mechanosensitive ion channel component 1 (Piezo1) is a sensor and transducer of mechanical stimuli which has critical roles in various mechano‐transduction processes in touch, pain, proprioception, vascular development, and blood pressure regulation. In this study, firstly, we examined the detailed expression pattern of Piezo1 using in situ hybridization and RT‐qPCR during mice submandibular gland (SMG) development. The developmental stage and acinar cell specific expression pattern was examined at E15. To understand the precise signaling regulations of Piezo1 in SMG development, siRNA Piezo1 (siPiezo1) was employed as a loss of function while in vitro organ cultivation of SMG at E14. Alterations in histomorphology and expression patterns were examined in acinar forming cells after 1 and 2 days cultivation. Particularly altered localizations of Aquaporin, E‐Cadherin, Vimentin, and Cytokeratins suggest that Piezo1 would modulate the differentiation of acinar cells in SMG through regulation of signaling molecules including mechanosensitive molecules.
Smart piezoelectric nano hybrid scaffold as noninvasively controllable self‐powering mechanoelectrical stimulator for enhanced bone tissue engineering
1Department of Bionanotechnology and Bioconvergence Engineering, Graduate School, Jeonbuk National University, Jeonju 54896, Republic of Korea., 2Mechanical Design Engineering, Jeonbuk National University, Jeonju 54896, Republic of Korea.
Endogenous electric fields naturally exist in our body and play an important role in development and regeneration. In the case of bone, these endogenous electric fields arise due to the mechanoelectrical coupling properties exhibited by the non‐centrosymmetric spiral structured collagen fibril, the main structural protein in the extracellular matrix (ECM) of bone. However, developing implants with natural polymers and bio ceramic composites cannot deliver endogenous electric potential due to the weak mechanoelectrical coupling effect. Here we report a noninvasively controllable robust piezoelectric barium titanate nanoparticles (BTO NPs) loaded polyacrylonitrile (PAN) smart nanohybrid scaffold as a mechanoelectrical stimulator for bone tissue engineering. Concentration‐dependent PAN/BTO nanohybrid scaffold was developed, and their physiochemical and mechanical properties were analyzed. Systematic electrical studies reveal that PAN/BTO nanohybrid scaffolds exhibit superior ferroelectric properties and generate electrical signals akin to native tissues when mechanically triggered using Food and Drug Administration (FDA) approved Low‐Intensity Pulsed Ultrasound (LIPUS) noninvasively. The energy harvesting performance of the PAN/BTO nanohybrid scaffold was further proved by the real‐time powering of commercial LED upon LIPUS stimulation. The in‐vitro studies with pre‐osteoblast MC3T3‐ E1 cells cultured on the piezoelectric PAN/BTO nanohybrid scaffold exhibited increased ALP activity, calcium mineralization, and osteogenic markers for LIPUS triggered scaffolds than non‐stimulated control groups. Even though the developed PAN/BTO nanohybrid scaffold is nonbiodegradable, this study emphasizes the need to develop a potential bioactive piezoelectric nanohybrid implant that could be employed for noninvasively stimulate the cell microenvironment to achieve functional native tissues and to enhance the therapeutic efficacy.
PTD‐BMP2 enhances the wound healing in type 1 diabetic mice
1Department of Orthopaedic Surgery, Yonsei University College of Medicine, 2Oral Cancer Research Institute, Yonsei University College of Dentistry, Seoul, Korea, 3Yonsei university college of medicine, 4Department of Orthopaedic Surgery, Dankook University Hospital
Decreased angiogenesis contributes to delayed wound healing in diabetic patients. Recombinant human bone morphogenetic protein 2 (rhBMP‐2), which has been approved by the FDA for inducing bone regeneration, has also been shown to promote angiogenesis. However, the short half‐lives of soluble growth factors such as rhBMP‐2 limit their use in wound‐healing applications. To overcome this limitation, we propose a novel delivery model utilizing a protein transduction domain (PTD) formulated in lipid nanoparticle (LNP). And we aim to determine whether a gelatin hydrogel dressing loaded with LNP formulated PTD‐BMP‐2 (LNP‐PTD‐BMP‐2) can improve the angiogenic function of BMP‐2 and improve diabetic wound healing. In vitro, we found that compared to the control and rhBMP‐2, LNP‐ PTD‐BMP‐2 induced higher tube formation of human umbilical vein endothelial cells and increased the cell recruitment capacity of HaCaT cells in a scratch wound assay. Large, full‐thickness back skin wounds were inflicted on streptozotocin‐induced diabetic mice. Gelatin hydrogel cross‐linked by microbial transglutaminase containing rhBMP‐2, LNP‐PTD‐BMP‐2, or a control was applied to diabetic wounds. Wounds treated with LNP‐PTD‐BMP‐2 exhibited enhanced wound closure, increased re‐epithelialization rates, and higher collagen deposition than the rhBMP‐2 or control treatments. In an immunofluorescence study, the LNP‐PTD‐BMP‐2 treatment showed more CD31‐ and α‐SMA‐positive cells, thus demonstrating a higher neovascularization capacity than rhBMP‐2 or control treatments. Additionally, in vivo near‐infrared fluorescent images showed that LNP‐PTD‐BMP‐2 has a longer half‐life than rhBMP‐2 and that BMP‐2 localizes around wounds. These findings suggest that LNP‐PTD‐BMP‐2‐loaded gelatin hydrogel is a viable treatment for diabetic wounds.
Nanotrap‐mediated enhanced cell coating on the tubular scaffold for trachea regeneration
1Seoul National University Hospital
Mesenchymal stem cells (MSCs) are a promising tool in tissue engineering for their paracrine effect and differentiation capacity into various cell types. However, cell delivery to a tracheal scaffold should overcome the big hurdles such as low cell attachment, uneven cell coating on artificial tubular scaffolds, dry and septic environment, and insufficient blood vessels. To fabricate a new nanofiber with enhanced cell attachment (nanotrap), polycaprolactone (PCL) was wet‐electrospun in an ethanol bath and collected on a rotating drum. Nanotraps were characterized using scanning electron microscopy, Raman spectroscopy, and protein adsorption studies, and compared with conventionally electrospun nanofiber (NFs). Cell adhesion and morphology were evaluated with mesenchymal stem cells (MSCs). Nanotrap with MSCs was wrapped onto a PCL tubular scaffold (Nanotrap/MSCs/Scaffold), and was implanted in a partial tracheal defect animal model. At 4 weeks after implantation, histological analysis, endoscopy, and micro‐computed tomography were performed. Nanotraps had a less dense nanofiber network and more hydrophilic surfaces than nanofiber sheets collected via dry electrospinning. In the cell adhesion test, MSCs attached better and spread faster on the nanotrap. The extracellular matrix formed by stem cells spread easily along the surface of single nanotrap fibers, as confirmed by SEM and vinculin staining. MSC‐implanted nanotraps led to complete mucosal layer regeneration and better angiogenesis. Moreover, the inflammatory reaction was minimal in animals treated with Nanotrap/MSCs/Scaffold composites. Nanotraps enhanced MSC attachment and resulted in better tracheal regeneration in an animal model.
Polydopamine/hyaluronic acid‐based salivary gland tissue engineering platform inspired by roles of hyaluronic acid during salivary gland development
11Department of Physiology, School of Dentistry and Dental Research Institute, Seoul National
University, 2Department of Carbon Convergence Engineering, Wonkwang University, 3Department of Chemistry, Center for Nature‐inspired Technology (CNiT), Korea Advanced Institute of Science and Technology (KAIST)
Dry mouth, or xerostomia, caused by salivary gland dysfunction significantly impacts oral and systemic health and quality of life. Although in vitro‐generated artificial salivary glands have been considered as the fundamental solution, its structural complexity is difficult to reproduce using current biomaterials. Therefore, understanding and recapitulating the roles of biomacromolecules in salivary gland organogenesis is needed to solve these problems. Hyaluronic acid (HA) is a macromolecule abundant during salivary gland organogenesis, but its role remains unknown. Here, we verify the effects of HA on salivary gland organogenesis and artificial organ germ formation in solubilized and substrate‐immobilized forms. In embryonic submandibular glands (eSMG), we found dense HA layers encapsulating proliferative c‐Kit+ progenitor cells that were expressing CD44, an HA receptor. The blockage of HA synthesis, or degradation of HA, impaired eSMG growth by ablating the c‐Kit+ progenitor cell population. We also found that high‐molecular‐weight (HMW) HA has a significant role in eSMG growth. Based on these findings, we discovered that HA is also crucial for in vitro formation of salivary gland organ germs, one of the most promising candidates for salivary gland tissue regeneration. We significantly enhanced salivary gland organ germ formation by supplementing HMW HA in solution; this effect was further increased when the HMW HA was immobilized on the substrate by polydopamine/HA co‐immobilization. Our study suggests that the current use of HA in salivary gland tissue engineering can be further optimized.
Biomimetic 3D‐bioprinted skin scaffolds for wound healing and regeneration
1University of Oxford, 2Institue of Biomedical Engineering, University of Oxford, Oxford, UK
Chronic skin wounds, which fail to properly heal through normal healing stages, can have drastic and long‐lasting effects on patient health and wellbeing [1]. Full‐thickness wounds are of particular concern as they affect deeper tissues. Current cell‐based therapy is based on culturing autologous cells obtained by biopsy, which is insufficient area and painful to the patient, and are then applied on the wound, eventually with the help of a scaffold [2]. Tissue engineered skin can overcome these issues by using scaffolds especially designed to replicate the extracellular matrix (ECM) of natural skin. Bioprinting can further enhance these skin scaffolds, not only by incorporating cells but also due to the improved properties achieved over conventional methods.
This research aims to develop a novel bioink formulation based on carboxymethyl chitosan (CMC) and hyaluronic acid (HA). This bioink is used to bioprint scaffolds incorporating mesenchymal stem cells, which mimic the mechanical, structural and biological properties of natural skin. These scaffolds may lead to the development of a widespread clinical technique for treating burns, ulcers and other skin wounds more effectively. Moreover, by biomimicking the natural tissue, these scaffolds can overcome issues associated with implants, namely risk of rejection and/or infection.
References:
[1]. Patrulea et al., (2015). Eur. J. Pharm. Biopharm. [2]. P. Abdel‐Sayed et al., (2019). Adv. Wound Care
Microfluidic chips for modeling of outer blood‐retina barrier using iPSC derived RPE and endothelial cells
1NIH
The retinal pigmented epithelium (RPE) and choroid vasculature form a highly specialized tissue that provides critical support for retinal function and homeostasis. Here, we combined human iPSC‐derived RPE and endothelial cells (EC) with organ‐on‐a‐chip technology, to model the outer blood‐retinal barrier (oBRB). Using this platform, we aim to study patient‐specific mechanisms of retinal degenerative disorders such as macular edema.
The commercially available Emulate organ‐chip microfluidic system was used as the basis of the model. The apical channel of the chip was seeded with iPSC‐RPE cells that had been differentiated for 42 days. ECs were deposited in the basal channel and formed 3D monolayer to mimic the choroid capillaries. The 2 channels are separated by a permeable porous membrane. RPE cells seeded in the apical channel formed a monolayer on the membrane of the apical channel and displayed highly pigmented polygonal morphology with ZO‐1 expression. RPE tight junction integrity was confirmed using a fluorochrome‐ conjugated dextran permeability assay. In addition, a fluid transport assay was performed to examine RPE transcytosis and homeostasis function. When compared to control chips seeded with only RPE cells, the fluid transport activity in RPE‐EC chips was 2‐fold higher and in the range of physiological levels. These results support that both EC and RPE cells are critical components of fluid transport activity.
The combination of a microfluidic organ‐chip system with patient‐specific iPSC derivatives has provided us with a non‐invasive ex vivo model to study outer retinal physiology and a platform for drug discovery and toxicity screening.
Magnetic resonance relaxometry as a tool for tracking induced pluripotent stem cell variability
1School of Chemical and Biomedical Engineering, Nanyang Technological University, 2Critical Analytics for Manufacturing of Personalized Medicine, Singapore‐MIT Alliance for Research and Technology
In the development of cell therapy, iPSCs have significant advantages, specifically, their pluripotency, donor matching capabilities and self‐renewal. From a laboratory setting to a manufacturing setting, self‐ renewal, the ability to perpetually proliferate, passage after passage is especially useful. Yet, it is widely observed that iPSCs produced from culture can be subject to significant quality and phenotype variation, caused by known factors (thawing and handling protocols, passage numbers, contamination, etc.) as well as unattributable / uncontrollable factors. Currently, no method exists to quickly establish, in a label‐free manner, if one passage of iPSCs is the same as the previous passage, nor even the initial passage. In this study, we introduce Magnetic Resonance Relaxometry (MRR) 1 for the quick, label free measurement of the quality and variability in iPSC cultures. This method uses just 4 uL samples of suspended cells (<50k) for measurements that take 2 minutes with no chemical or biological processing of the cells. T2 relaxation time from MRR is strongly correlated with the intracellular iron (Fe3+) content. We demonstrated that several factors associated with iPSCs culture contribute to variations in resulting iPSCs, and that T2 measurement can be used to identify such variations. While it is desirable to control these variations by controlling cell production process more tightly, we believe that MRR is still a useful tool in quantifying iPSC quality and variability commonly observed in lab or manufacturing settings.
1. Fook Kong, T., et al. Enhancing malaria diagnosis through... Sci Rep
Natural scaffolds for cultured meat production
1 Biomanufacturing Technology, Bioprocessing Technology Institute (BTI), Agency for Science, Technology, and Research (A*STAR), 20 Biopolis Way, Singapore 138668 , *Correspondence: Deepak_Choudhury@bti.a‐star.edu.sg
Cultured meat production has gained aggressive momentum in the past few years [1, 2]. The technique involves isolating animal muscle cells and expanding them in in vitro settings to develop meat. To produce cultured meat with similar structural complexity as conventional meat, suitable animal‐free scaffolds must be generated. Scaffolds should possess appropriate thickness and mechanical properties and allow necessary cell attachment, proliferation and maturation [3]. We have developed proprietary highly macroporous plant‐based scaffolds as edible substrates. Our scaffolds comprise unique features that help align and orientate cells which is crucial for the differentiation and maturation of muscle cells. We have performed SEM to observe the scaffolds' surface micro‐topographies and pore sizes. In addition, we have evaluated thermal stability with TGA, identified functional moieties with FTIR and observed relevant mechanical rigidity. These scaffolds have been shown to support the growth and proliferation of a model rat muscle cell line.
References
1. Choudhury, D. et al. (2020) The Business of Cultured Meat. Trends in Biotechnology 38 (6), 573‐ 577.
2. Ng, ET et al. (2021) Cultured meat ‐ a patentometric analysis. Critical Reviews in Food Science and Nutrition, 1‐11.
3. Seah, JSH et al. (2021) Scaffolds for manufacturing cultured meat. Critical Reviews in Biotechnology, 1‐13.
Induced pluripotent stem cell‐derived corneal endothelial‐like cell therapy for corneal endothelial dysfunction
1Department of Ophthalmology, Asan Medical Center, University of Ulsan College of Medicine, 2Department of Biomedical Science, College of Life Science and Center for Embryo and Stem Cell Research, CHA Advanced Research Institute, CHA University
Corneal endothelial cells (CECs) do not proliferate or recover after illness or injury, resulting in decreased cell density and loss of pump/barrier function. Considering the donor cornea shortage, it is vital to establish robust methods to generate CECs from induced pluripotent stem cells (iPSCs). We investigated the efficacy and safety of transplantation of iPSC‐derived CECs into a corneal endothelial dysfunction (CED) rabbit model. Characteristics of iPSCs were analyzed using immunocytochemistry, teratoma assay, and quantitative real‐time PCR (qRT‐PCR). Differentiation of iPSCs into CECs was induced via neural crest cell (NCC) induction. CEC markers were detected by immunofluorescence and gene expression analyzed using qRT‐PCR. Cultured iPSC‐derived CECs were injected into the anterior chamber of a CED rabbit model. iPSCs expressed PSC markers OCT4, SOX2, TRA‐1‐60, and NANOG, formed teratomas, and differentiated into a three‐germ layer lineage. After culturing iPSC‐derived NCCs, zona occludens‐1 (ZO‐1) and Na+/K+ ATPase were expressed and cells exhibited a well‐preserved hexagonality. ATP1A1, COL8A1, and AQP1 mRNA expression was higher in iPSC‐derived CECs than in iPSCs and NCCs. Direct injection of iPSC‐derived CECs resulted in improved corneal transparency. PCR of DNA in the rabbit central corneas revealed a human mitochondrial DNA band. An increase in ZO‐1 and ATP1A1‐ positive cells was noted in rabbit corneal tissues transplanted with iPSC‐derived CECs. Successful transplantation of CECs, differentiated from iPSCs, into a CED animal model confirms the therapeutic efficacy of iPSC‐derived CECs, demonstrated by the restoration of corneal clarity. iPSC‐derived CECs represent a promising cell therapy approach in CED.
Improving anti‐oxidative resistance of mesenchymal stem cells (MSCs) by priming to quiescent state
1Basic Research Division, Rohto Pharmaceutical Co., Ltd., 2Regenerative Medicine Research Planning Division, Rohto Pharmaceutical Co., Ltd.
Mesenchymal stem cells (MSCs) have been the most commonly used in regenerative medicine with many clinical trials in different diseases. However, the efficacy in clinical trials has some limitations, partly due to the death of transplanted cells within a few hours after transplant. Combination of host factors could produce different stimuli, including oxidative stress, to transplanted MSCs, and affects their viability and functions. Therefore, in this study, we aimed to find a method to improve stress resistance of MSCs by inducing them into quiescent state by means of priming with abemaciclib, a CDK4/6 inhibitor. The results showed that with a moderate priming of abemaciclib, adipose‐derived (AD) MSCs could be significantly induced into quiescent state, which have more resistance to oxidative stress. Interestingly, after freezing, quiescent AD‐MSCs showed significantly higher resistance to oxidative stress, and could reverse to proliferation state. In conclusion, the results in this study suggest a simple and cost‐effective method which might enhance cell viability under oxidative stress, and could be applied to improve cell viability for cell transplantation in the future.
Label‐free detection of residual undifferentiated iPSCs from their differentiated progenitor cells by microfluidic raman spectroscopy
1Critical Analytics for Manufacturing of Personalised Medicine Interdisciplinary Research Group, Singapore‐MIT Alliance in Research and Technology (Singapore), 2Department of Electrical Engineering and Computer Science, Biological Engineering, Massachusetts Institute of Technology (USA); Critical Analytics for Manufacturing of Personalised Medicine Interdisciplinary Research Group, Singapore‐MIT Alliance in Research and Technology (Singapore), 3School of Chemical and Biomedical Engineering,
Nanyang Technological University (Singapore); Lee Kong Chian School of Medicine, Nanyang Technological University (Singapore); School of Materials Science and Engineering, Nanyang Technological University (Singapore), 4Xiamen University, 5School of Chemical and Biomedical
Engineering, Nanyang Technological University (Singapore) and Critical Analytics for Manufacturing of Personalized Medicine, Singapore‐MIT Alliance for Research and Technology (Singapore)
In spite of many advantages, iPSC‐derived cell therapy engineering still faces the issue of cellular heterogeneity. Especially, residual iPSCs amongst their differentiated progenitor cells, even at small percentages, could pose a significant risk for downstream neoplasticism. The current techniques to detect such residual iPSCs rely on undifferentiated residual's antigen labels (i.e., for flow cytometer analysis), marker genes (i.e., using qRT‐PCR analysis), or culture systems that enable iPSC growth. However, these assays still have the disadvantage of being destructive, costly, and time‐consuming in the cell manufacturing pipeline. Recently, Raman spectroscopy was shown to be able to detect iPSCs amongst differentiated progenitor cells [1], but this study was done on a fixed cell utilizing commercial, large‐scale Raman systems. In this study, iPSCs derived from Endothelial Cord Lining Epithelial cells (CLECs) were differentiated into Spinal‐cord Progenitor Cells (SCPCs) through a 10‐day differentiation process. A label‐free microfluidic Raman spectroscopy system was used to measure and detect the residual iPSCs spiked into the differentiated progenitor cells based on the glycogen and other markers, using a collection of live cells within a microfluidic channel (∼50 nL sample volume). The assay was done around 10 number of live SCPCs within the focal point, utilizing 200 mW laser on a home‐made optics, with 100s scanning time. The sensitivity and specificity of detecting residual iPSCs in differentiated SCPCs will be determined and presented. This system is useful for the integration into the in‐line quality assessment of impurity during the manufacturing process.
[1] Hsu et al., Proc Natl Acad Sci, 2020.
Measurement of critical motions in the medium change process in differentiation culture of retinal pigmented epithelial cells
1Joint Research Chair on Design for Advanced Medical System (Shibuya Corporation), Graduate School of Engineering, Osaka University, 2Department of Biotechnology, Graduate School of Engineering,
Osaka University, 3Department of Biotechnology, Joint Research Chair on Design for Advanced Medical System (Shibuya Corporation), Research Base for Cell Manufacturability, Graduate School of Engineering, Osaka University
Retinal pigmented epithelial (RPE) cells derived from hiPS cells are promising transplants for clinical application of age‐related macular degeneration. During the differentiation process of RPE cells, the culture forms the fragile layered structure where it is easy to detach the cells from the top of layer. This structural decay causes undesired fates. Therefore, the gentle and stable motions in the culture operation are required to preserve the structure, leading to the requirement of master‐hands by expert operators in the practical manufacturing. Culture vessel handling with motions such as lifting, placing, etc. is considered to one of the critical triggers that cause the extrinsic force to collapse the layered structure.
We have developed the motion calibrator to quantify the accelerations for three axes of the vessel motion during the culture operation and the analysis method using the parameters (high gravity force and its jerk) identifying the impulsive motion with high gravity force.
The detail protocol, which is set to be single motions step by step, for the manual operation of medium change in the quasi‐clean room is set. According to the protocol, the acceleration in manual operation was measured. It was difficult to detect each motion independently, showing the smooth operation. This means the sequential and parallel motions without any pause between motions were spontaneously performed in the manual operation. In addition, many impulsive motions are detected. These results exhibit the difficulty in maintenance and stability of the gentle motions, suggesting the necessity of master‐hands.
Outcomes and quality of pancreatic islet cells isolated from surgical specimens for research on diabetes mellitus
1Asan Medical Center
Isolating human islets of high quality and quantity is a prerequisite for research on diabetes. Human islet isolation is typically performed using pancreases from brain‐dead donors, making islet cell research difficult due to low availability. Pancreas tissue discarded after surgical resection may be a good alternative source of islet cells. To test this hypothesis, we isolated islets from discarded surgical specimens and evaluated islet yield and quality, as well as islet cell preparations. Eighty‐two segmental pancreases were processed using the Ricordi automated method, and islet yield and quality were investigated. The mean age of patients was 54.6, and the cohort included 32 diabetic patients. After purification, partial pancreases yielded an average of 59,593 ± 56,651 islet equivalents (IEQ) and 2,546 IEQ/g of digested pancreas with 71.5 ± 21% purity. Multivariate analysis revealed that diabetes (p = 0.0046), and the lobe used (p = 0.0156) significantly altered islet yield. Islets transplanted into diabetic mice displayed good viability and in vitro glucose responses, DNA/RNA quality, mitochondrial function, and glucose control, even though those are dependent on islet quality. Isolated cells also maintained high viability and function even after cryopreservation. Our findings indicate that partial pancreases discarded after surgery can be a valuable source of islets for diabetes research.
A method for mass production and characterization of human‐derived nasal septum cartilage chondrocytes (hNCs) for allogeneic cell therapy products
1Department of Biomedicine and Health Science, College of Medicine, The Catholic University of Korea, 2Department of Otolaryngology‐Head and Neck Surgery, College of Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea
Human‐derived nasal septum cartilage chondrocytes (hNCs) promise possibility in breakthroughs in arthritis therapies. Their potential, however, has been hurdled in a mostly laboratory setting due to expansion culture system challenges in the harvest of cells for clinical and manufacturing purposes. For example, several studies have investigated the production of hNCs in expansion culture systems, the use of conventional cultures has shown an unfavorable environment for hNCs growth and quality maintenance. Chondrocytes undergo dedifferentiation in the process of proliferation and culture, and such dedifferentiation loses the function of chondrocytes. Expansion culture manufacturing techniques commonly used for R&D and early clinical trials are unsuitable and expensive for the large‐scale production required for late‐stage clinical trials and commercial manufacturing. Disposable bioreactors are widely recognized as a feasible manufacturing solution, but must be optimized to meet hNCs unique process requirements. One of the key challenges for the future success of the commercialization of homeopathic remedies is the scalable ability to stably reproduce the yield and quality of hNC‐derived products produced in small‐scale R&D on a large scale sufficient for commercial manufacturing. It is to establish a good manufacturing technology. This study focused on using mass production and characterization to aid in optimizing the use of vertical‐wheel bioreactors for hNCs production.
Effect of ROCK inhibitor on aggregate growth of human induced pluripotent stem cells in suspension culture
1Department of Biotechnology, Graduate School of Engineering, Osaka University, 2Department of Biotechnology, Research Base for Cell Manufacturability, Graduate School of Engineering, Osaka University
Large numbers of human induced pluripotent stem cells (hiPSCs) are required, and the establishment of culture techniques is being studied as a powerful tool in clinical and industrial applications. It is well known that Rho‐associated protein kinase inhibitor (ROCK inhibitor) inhibits cell dissociation‐induced apoptosis in cultures of hiPSCs. Understanding the effects of ROCK inhibitor on the growth process of hiPSC aggregates after cell aggregation is important for stabilizing the aggregate culture.
In this study, two hiPSC lines (Tic and 1383D2) were cultured in suspension cultures without (control) or with ROCK inhibitor after aggregation. When aggregate morphology and size were compared, the aggregates of two lines treated with ROCK inhibitor formed larger, more compact aggregates and showed higher cell density (Tic: 5.79‐fold, 1383D2: 4.40‐fold), as compared to the control condition. Moreover, in 1383D2 cells treated with a ROCK inhibitor, the number of aggregates was decreased, indicating coalescence between cell aggregates. When the microstructure of the surface on cell aggregate was compared, the fibril structures were formed under the control condition and the particle structures were maintained with a treatment of ROCK inhibitor. In the cross‐sectional structure of the cell aggregate, the intercellular space was larger and particle structures existed with the treatment of ROCK inhibitor. Our results suggest that ROCK inhibitor enhances stable aggregate formation in association with promoting cell growth and coalescence between aggregates accompanied with structural alternation of surface on cell aggregate.
Spatio‐temporal analysis of phase transitions in corneal epithelial cell sheet using an agent‐based model
1Japan Tissue Engineering Co., Ltd., 2Department of Biotechnology, Graduate School of Engineering, Osaka University, 3Department of Biotechnology, Graduate School of Engineering, Research Base for
Cell Manufacturability, Osaka University, 4Department of Ophthalmology, Graduate School of Medicine, Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, 5Department of Biotechnology, Graduate School of Engineering, Global Center for Medical Engineering and Informatics, Osaka University, 6Division of Health Sciences, Graduate School of Medicine, Osaka University
Transplantation of autologous cultured corneal epithelial cell sheet is an approach for limbal stem cell deficiency. In culture of corneal epithelial cell sheet, time‐dependent change of cell sheet fluidity was observed, and it is considered that cell sheet fluidity relates to cell sheet structure. The transition from high cell fluidity (unjamming phase) to low cell fluidity (jamming phase) is called jamming transition or unjamming‐jamming transition (UJT) and has attracted attention in the field of biophysics as a phenomenon like the glass transition. We focused on unique pattern of phase transitions in the culture of corneal epithelial cell sheet which show jamming‐unjamming transition (JUT) after UJT within same culture.
It is important to understand the individual cell behaviors to clarify the mechanisms of emergent phenomena in cellular tissues. In this study, agent‐based model was used for simulation of the behavior and interactions of autonomous cells and their effects on the entire cell tissue. To represent the behaviors of corneal epithelial cells, kinetic model that consider the time‐dependent of cell‐cell connections, as well as models that represent asymmetric cell division, cell differentiation, and cell death have been constructed. Spatio‐temporal analysis about cell state transition between jamming and unjamming state revealed migration induced by surrounding cell migration (chain migration) contributes JUT in corneal epithelial cell sheet. Interestingly, chain migration disrupts cell‐cell connections and promotes further cell state transition in JUT. Our results are considered to be important for understanding mechanisms of phase transitions and time‐dependent changes of cell tissue structure.
Cell‐fiber culture system: A powerful platform for scalable expansion of adherent and non‐adherent cells
1CellFiber Co., Ltd., Tokyo, Japan.
Here, we investigated several culture scales (up to 100‐fold) using the “cell‐fiber culture system” and analyzed mesenchymal stem cells (MSCs) and T cells. The practical usage of cell and gene therapies (CGT) has been limited by many difficulties, such as scalability and shear stress. It is difficult to reliably move forward the cell culture from lab‐scale to experimental‐clinical‐scale to manufacture off‐the‐shelf products that can be provided to multiple patients. To solve these problems, we established the novel culture system using cell‐laden core‐shell hydrogel microfibers (Cell‐fiber). This system allows expansion of any cell type (adherent and non‐adherent). We used T cells as a non‐adherent cell culture model and MSCs as an adherent cell model. Based on several factors such as, the cell specific markers, differentiation potency, expansion rate, doubling time, viability, etc., we demonstrated that cell fiber culture system is a powerful tool to maintain constant cell quality at any scale. Cell‐fiber platform can be consistently used from standard small‐scale cell culture for initial test optimization (e.g., 6‐well plate) to large‐scale bioreactors for cell mass production. We propose that cell‐fiber technology will revolutionize cell manufacturing and directly benefit CGT.
Long‐term clinical efficacy and safety of three‐dimensionally printed biomaterial in patients with nasal septal deformities
1Catholic university of Korea
This is the first study to investigate the long‐term effect of safety and efficacy of the three‐ dimensionally(3D)‐printed bioresorbable implant (polycaprolactone (PCL)) for nasal septal reconstruction. Furthermore, to our knowledge, this study is the largest long term follow up clinical results undergoing the standardized operation protocol of the application of a 3D‐printed biomaterial.
This study included 14 patients who undergone clinical trial of the nasal septum reconstruction surgery with 3D‐printed PCL nasal septal implants. The change in total Nasal Obstruction Symptom Evaluation (NOSE) Scale scores between the postoperative 3 month and the current status (3.59 ± 0.51 years) was the primary outcome. Changes in bilateral nasal cavity minimum cross‐sectional area (MCA) and volume on acoustic rhinometry, nasal cavity cross‐sectional area at the osteomeatal unit and nasal septum angle in the paranasal sinus on computed tomography images, visual analog scale of the patients' subjective satisfaction were the secondary outcomes.
The results revealed no significant changes in the MCAs (Cohen's d:0.09; p = 0.711) and nasal volume (Cohen's d:0.26; p = 0.356) on acoustic rhinometry, area at the osteomeatal unit (Cohen's d:0.49; p = 0.064) and septum angles (Cohen's d:0.18; p = 0.831) on computed tomography, and NOSE scale (Cohen's d:0.14; p = 0.621) and patients' subjective satisfaction (Cohen's d:0.52; p = 0.076) at the follow up period.
This homogeneous composite microporous PCL nasal septal implant has demonstrated long‐term clinical efficacy and safety in human tissues that require maintenance of mechanical strength. Therefore, this implant could extend indication in the various craniofacial reconstructions in the future.
Use of intraluminal nitinol stent to prevent tracheal stenosis in tracheal anastomosis
1Department of Veterinary Surgery, Chungbuk National University, 2Department of Veterinary Surgery, Chonnam National University
The resulting injury and compromise of the airway can become lethal in a short time. Tracheal stenosis can cause respiratory problems in mature, small‐breed dogs. This study aimed to evaluate the placement of an intratracheal nitinol stent to prevent tracheal stenosis in canine tracheal anastomosis. Three client‐ owned toy and miniature dogs were recruited. The self‐expandable intratracheal stent was an alloy of nickel and titanium, at the same atomic ratio. The intratracheal stent diameter was the same size as the inner diameter of the trachea to be excised. Vital signs and respiratory patterns, C‐reactive protein, radiography, computed tomography, and endoscopy results after intraluminal stenting were assessed for 3‐5 months. Serum C‐reactive protein levels increased in all dogs up to 3 days after surgery but returned to normal level after 7 days. Lateral cervical radiographs showed that the intratracheal stent was properly placed without movement or stent fracture at the insertion site. No dogs showed evidence of intraluminal tracheal stenosis or tracheitis in the region of stent insertion on tracheoscopy and computed tomography after tracheal stent placement. After 1‐2 weeks of tracheal stent placement, all dogs resolved coughing and dyspnea signs and resumed normal activities. The intratracheal stent showed no movement or deformation in the trachea, and had flexibility and an appropriate radial force. Therefore, titanium alloy tracheal stents are useful in stenotic operations for tracheal reconstruction.
Acknowledgement: This work was carried out with the support of "Cooperative Research Program for Agriculture Science and Technology Development (PJ01620801)" Rural Development Administration, ROK
Therapeutic effect of combined mesenchymal stem cells and cartilage acellular matrix injection in a goats OA model
1Stem Cell and Regenerative Bioengineering Institute, Global R&D Center, Kangstem Biotech, 2kangstem biotech
Human umbilical cord blood‐derived MSCs (hUCB‐MSCs) have been studied in osteoarthritis (OA) and cartilage regeneration. Our previous study demonstrated that hUCB‐MSCs combined with cartilage acellular matrix injection (CAM Inj.) represent potential regenerative effect and anti‐inflammatory effects in the rabbit OA model.
This study has evaluated the safety and efficacy of hUCB‐MSCs combined with CAM Inj. in an anterior cruciate ligament transection (ACLT) with resection of medial menisci (MMx) in a goat model. In this study, 27 goats were divided into 5 group: normal (n = 3), OA (n = 6), OA+CAM Inj. (n = 6), OA+hUCB‐MSCs (n = 6), and OA+hUCB‐MSCs+CAM Inj. (n = 6). Lameness and radiographic parameters were assessed 6 months after administration, and macroscopic and histological evaluation of the goat articular cartilage was performed 6 months after intervention.
The results showed significant improvement in lameness score only in the OA+hUCB‐MSCs group at 5 months after treatment (*p < 0.05), whereas the K&L score showed significant improvement only in the OA+hUCB‐MSCs+CAM Inj. group 6 months after intervention (*p < 0.05). In addition, the gross findings showed significance in OA+CAM Inj. and OA+hUCB‐MSCs+CAM Inj. groups 6 months after treatment (*p < 0.05 and **p < 0.01).
In conclusion, treatment with a combination of hUCB‐MSCs and CAM Inj. reduced OA symptoms and induced effective cartilage tissue repair in a goat model. We suggest that the combination of hUCB‐MSCs and CAM Inj. can be suitable candidate for alternative OA therapeutics.
Effect of combined treatment with statins and ezetimibe in NASH model: Inhibition of macrophage
1Yonsei university wonju college of medicine, 2Wonju Severance Christian Hospital, Department of Internal Medicine, Division of Gastroenterology and Hepatology
Statin‐Ezetimibe combination therapy is widely used as a treatment of hyperlipidemia. There is limited evidence that statin and ezetimibe combination therapy have a beneficial effect in cirrhosis. In both vivo and vitro studies, we aimed to investigate the effects of statin and ezetimibe combination therapy on lipid‐ lowering effects and putative anti‐inflammation capacity in NASH‐related liver fibrosis in mouse NASH‐ cirrhosis model.
A High‐fat diet and Thioacetamide (300mg/kg, intraperitoneal injection twice a week for 8 weeks) was given to establish mice models with non‐alcoholic steatohepatitis (NASH) related histological change. 36 male mice were divided into six groups as follows: (1) regular diet (RD), (2) high‐fad fed (HFD) and not treated, (3) HFD and Thioacetamide (TAA) injected (HFD‐TAA), (4) HFD‐TAA, and ezetimibe treated (HFD‐TAA‐EZET), (5) HFD‐TAA and statin‐treated group (HFD‐TAA‐statin), and (6) HFD‐TAA and ezetimibe and statin combination‐treated group.
After treatment of combination therapy, hepatic fibrosis is ameliorated more than the control group. In vivo, Statin and ezetimibe combination therapy inhibit the migration of macrophages via lowering the expression of CCR5 and lowered inflammation markers such as IL‐1β, IL‐6, and TNF‐α. Also, it reduces the transcription mRNA level of α‐SMA, Collagen type 1, TGF‐β, and PDGF‐d. In vitro, statin and ezetimibe combination therapy inhibit macrophage activation resulting in inhibition of the MAPK pathway and lowering anti‐inflammatory cytokine (such as TNF a and IL‐6).
In the NASH‐cirrhosis mouse model, statin and ezetimibe combination therapy showed anti‐inflammatory effects through lowering the MAPK pathway and inhibiting macrophage activation.
Tissue integration patterns of noncrosslinked and crosslinked collagen membranes: An experimental in vivo study
1Department of Periodontology, Research Institute of Periodontal Regeneration, Yonsei University College of Dentistry
Background: The noncrosslinked and crosslinked collagen membranes are known to exhibit differential degradation characteristics resulting in contrasting orientation of the adjacent tissues and biological processes. The aim of this study was to conduct histomorphometric assessments of the noncrosslinked and crosslinked collagen membranes regarding neovascularization, tissue integration, tissue encapsulation and biodegradation.
Methods: Lateral bone augmentation was performed using both the noncrosslinked (BG) or the crosslinked collagen membrane (CM) in 15 beagle dogs, which were euthanized at 4, 8, and 16 weeks (n = 5 each) for histomorphometric analysis. The samples were assessed regarding neovascularization, tissue integration, encapsulation, remaining membrane area and pseudoperiosteum formation. The BG and CM groups were compared at different time periods using nonparametric statistical methods.
Results: The remaining membrane area of CM was significantly greater than BG at 16 weeks, however, there was no significant differences at 4 and 8 weeks. Conversely, the neovascularization score for CM was significantly less than BG at 16 weeks. BG exhibited significantly greater tissue integration and encapsulation scores than CM at all time periods apart from encapsulation at 16 weeks. Pseudoperiosteum formation was observed in the BG group at 16 weeks.
Conclusion: Although BG membranes were more rapidly biodegraded compared to CM, it gradually became replaced by connective tissue with complete integration and maturation of the surrounding tissues to form dense periosteum‐like connective tissue. Further studies need to be performed to validate the barrier effect of the pseudoperiosteum.
Long term alveolar bone critical sized defect confirmation for GBR membrane in beagle dogs
1KBIOHealth, 2HYUNDAI BIOLAND Co., Ltd
Anatomically standardized surgical periodontal defects have been largely used for bone graft or guided bone regeneration (GBR) membrane development, because they are similar to human class III furcation defects and do not heal spontaneously. The study was conducted over 4, 8 12, 24, and 48 weeks in beagle dogs to evaluate the critical sized bone (CSD) healing to the beagle dog's mandibular alveolar bone defect for GBR membrane. After 12 weeks healing from extraction of the 2 ‐ 4 premolar, 5 x 5 x 5 mm cubic shape CSD were formed. After operation, clinical signs were monitored, and radiographic examinations were performed. After the animal sacrificed, micro‐CT image analysis, histomorphometrical and histological examinations were performed. Bone formation results can be explained by the lack of bone graft and membrane that maintain space and prevent soft tissue invasion. However, adequate clot formation in the empty defect can still have bone formation ability. Even we made CSD, used in several studies, the defect showed continuous bone growth until 12 weeks. Theorically, critical sized defect does not heal spontaneously during the lifetime of the animal, but 50% to 70% spontaneous regeneration can be expected in acute defect models. In this study, unexpected high bone formation was observed 12 weeks after the defect creation, meaning that the alveolar defects in dogs have the potential for spontaneous healing. However, at 24 weeks, the bone healing decreased, and disuse atrophy appeared at 48 weeks.
Conditioned medium of human pluripotent stem cell‐derived neural precursor cells exerts neuroprotective and neuroregenerative effects against ischemic stroke model
1Yonsei university wonju college of medicine, 2Catholic Kwandong University College of Medical Convergence, 3Yonsei University College of Medicine
Increasing evidences support the notion that neural stem/progenitor cells (NPCs) having self‐renewal and multipotent capacities with strong anti‐inflammatory activity are an ideal cell type for repair of global cerebral damage, and a number of previous studies have shown that early therapeutic events upon transplantation of NPC to animal models of ischemic stroke readily protected neuronal cell damage and improved behavioral recovery through paracrine manners. Here, the regenerative potential of the conditioned medium from neural progenitor cells (NPC‐CM) in vivo and its potential mechanism of action were further explored. Adult male SD rats with permanent middle cerebral artery occlusion (pMCAo) were randomly assigned to 4 groups: control, vehicle, NPC‐CM single injection (NPC‐CM(S)) and NPC‐CM multi‐injection (NPC‐CM(M)) groups. Single intravenous injection of NPC‐CM exerted strong neuroregenerative potential with behavioral recovery and this was further enhanced by multi‐ injection by suppressing inflammatory damage and inducing endogenous neurogenesis leading to histopathological and functional recovery. Proteome analysis of NPC‐CM identified a number of proteins that are known to be associated with nervous system development, neurogenesis and angiogenesis. In addition, we assessed differential gene expression in NPC‐CM(S)‐ and NPC‐CM(M)‐treated rats and uncovered that the importance of the inflammatory response during stroke recovery. Some of the key hub acting genes in the interaction network were validated in the brain tissue of NPC‐CM‐treated rats by qRT‐ PCR. Thus, our finding demonstrated that NPC‐CM promoted functional recovery, reduced cerebral infract and inflammation with enhanced endogenous neurogenesis and highlights its potency in the stroke therapy.
In vivo high‐resolution flexible electrode array for multi‐site recording and stimulation
1KBIOHealth, 2Daegu Gyeongbuk Institute of Science & Technology (DGIST)
The Neural Interface systems for brain disease treatment, brain function research, and brain‐machine interface (BMI) implementation has focused on non‐invasive methods so far, but has a limitation in that it has a low resolution. The purpose of this study is to develop a flexible, high‐resolution Neural Interface systems with biocompatibility and mechanical properties to overcome the limitations of the conventional brain interface electrodes, and to improve long‐term use suitability for practical clinical use. In this study, we measured brain signals or stimulated neuronal cells to treat brain disorders and for brain research using non‐human primate. A minimal invasive hybrid electrocorticography (ECoG) electrode arrays was used without damaging the brain. These ECoG electrodes can be used to measure local field potential (LFP) across large areas of the brain. In this study, we present the results of in‐vivo recording and stimulation of the brain using the developed electrode array.
Age‐related macular degeneration model (AMD) simulated using a biomimetic nanofiber membrane
1KYUNGPOOK NATIONAL UNIVERSITY HOSPITAL, 2KYUNGPOOK NATIONAL UNIVERSITY
Age‐related macular degeneration (AMD), which has the highest risk of blindness in the global elderly population, is a serious disease that leads to blindness by destroying optical cells and loss of central vision. In this study, we constructed an AMD model that mimics the blood‐retinal barrier (BRB) using functional and structural black polycaprolactone (PCL) nanofibers that mimics Bruch's membrane. Bruch's membrane made in black makes it easy to observe the infiltrated cells. To simulate BRB, human retinal pigment epithelial cells (ARPE‐19) and human umbilical vein endothelial cells (HUVEC) were used. Lipofuscin and blue light were treated to induce AMD and the change was observed. As a result, it was confirmed that the mRNA expression of heme oxidase‐1 (HO‐1) increased and the mRNA expression of superoxide dismutase 2 (SOD2) and glutathione peroxidase (GPx) decreased. Due to the increase in oxidative stress production, the death of ARPE‐19 and the collapse of the BRB due to the collapse of the tight junction were observed, In addition, the increase of vascular endothelial growth factor (VEGF) expression in the AMD in vitro model induced the infiltration of HUVECs. Considering the change in hypoxia‐inducible factor 1 alpha (HIF1α) mRNA expression, it is thought that the increase in VEGF expression is due to hypoxia caused by photooxidation of A2E.
In conclusion, A2E and blue light‐induced AMD in vitro models fabricated with nanofiber membranes are also considered to be useful tools for future studies related to AMD.
Quantum dot‐loaded biopolymer‐based nanocomposite for skin infection treatment
1Pohang University of Science and Technology, 2Kyungpook National University
The emergence of multidrug resistant (MDR) bacteria has evolved in response to extensive use of antibiotics and represents a significant issue in the biomedical field and public health concern. However, a treatment of MDR bacteria has limitations due to time‐consuming for isolation and ineffective antibiotics. Photodynamic therapy using a quantum dot (QD) has been recently proposed as a promising solution for destructing MDR bacteria. The QDs can damage bacteria independently of the antibiotic resistance pattern by generating reactive oxygen species (ROS) responding to light exposure with great advantages of superior photostability and tunability compared with conventional organic photosensitizer. However, the essential challenges of QDs for application in the biomedical field are stability and dispersion. In this study, Mussel adhesive protein (MAP) as an organic material and QDs as an inorganic material were selected to construct an antibacterial and wearable patch that can be used for skin and soft tissue infections. This novel patch showed antibacterial activity against Gram‐negative and Gram‐positive bacteria as well as MDR bacteria in discs and absorbance tests, and cytotoxicity was not observed in mammalian cells. In this presentation, the innovated strategies for designing biopolymer‐quantum dot nanocomposite and their potential as photodynamic platforms are presented.
Effect of aronia extract on the regulation of collagen synthesis in skin cells and skin equivalents
1Department of Materials Science & Engineering, Pohang University of Science and Technology (POSTECH), 2Division of Cosmetic Science and Technology, Daegu Haany University, 3Department of Molecular Biology, Pusan National University, 4Department of Mechanical Engineering, Pohang
University of Science and Technology (POSTECH)
The collagen synthesis regulated by fibroblasts in the dermis is a key process in skin regeneration and reconstruction. Here, we investigated whether extracts of Aronia melanocarpa affect human skin via collagen synthesis. We focused on this process using two model systems: a 2D cell culture and a bioprinted 3D skin equivalent. The aronia extract was non‐cytotoxic and increased cell proliferation in neonatal human dermal fibroblasts. Aronia extract treatment did not lead to COL1A1 mRNA degradation. Instead, the transcription of COL1A1 increased in direct proportion to the aronia concentration. In addition, aronia extract affected collagen synthesis by inhibiting the expression of MMP1 and MMP3. We also generated skin equivalents from type 1 collagen and dermal fibroblasts via bioprinting. We analyzed the inhibition of collagen degradation mediated by aronia extracts in a 3D microenvironment. In the 3D dermis model, the compressive modulus, which is directly affected by collagen synthesis, increased in direct proportion to the concentration of the aronia extract, and the expression levels of MMP1 and MMP3 decreased in inverse proportion to the concentration. These results indicate that aronia extract is directly related to the expression of collagen synthesis‐related genes in the dermis of human skin, suggesting that this is a potential candidate for skin improvement.
Effect of oxytocin receptor inhibitor on hard tissue regeneration in dental pulp
1Department of Conservative Dentistry, Yonsei University College of Dentistry, 2Department of Oral Microbiology and Immunology, School of Dentistry and Dental Research Institute, Seoul National
University
Dental pulp‐derived stromal cells (DPSCs) are a crucial cell population for maintaining the tissue integrity of the pulp‐dentin complex. The oxytocin receptor (OXTR), a member of the G protein‐coupled receptor (GPCR) superfamily, plays versatile roles in diverse biological contexts. However, the role of OXTR in dental pulp has not yet been investigated. We demonstrate the biological functions and significance of OXTR in DPSCs through multidisciplinary approaches. Microarray data of 494 GPCR genes revealed high OXTR expression in human DPSCs (hDPSCs). Blocking OXTR activity increased the expression of osteogenic and odontogenic marker genes, promoting hDPSC differentiation. Additionally, preclinical experiment using a dog model demonstrated that the application of OXTR‐ inhibitor on dental pulp tissue induced significant hard tissue formation. These results provide a new insight into the role of the oxytocin‐OXTR system in the pulp‐dentin complex and new perspectives for the field of regenerative dentistry.
Risk of secondary nonhematologic malignancies after allogeneic stem cell transplantation: A nationwide case‐control cohort study
1Seoul St. Mary's hospital, Catholic university, Seoul Korea, 2 Department of Pharmacology, College of Medicine, The Catholic University of Korea,
Our study investigated the incidence of secondary nonhematologic malignancies in patients undergoing allogeneic hematopoietic stem cell transplantation (allo‐SCT) and explored its risk compared to the general population. A population‐based case cohorts with adult patients who received allo‐SCT between January 2002 and December 2018 and a control cohort with matched general population were extracted from the Korean National Health Insurance Service database. Each case and control cohort included 5177 patients. With a median follow‐up of 2374 days for the case cohort and 2269 days for the control cohort, the 10‐year cumulative incidence rate of nonhematologic malignancy was significantly higher in the case cohort compared to the control cohort (4.23% vs 2.3%, hazard ratio [HR] 1.73, 95% confidence interval [CI] 1.32‐2.25, P < .001). The sub‐class analysis according to cancer‐site revealed significantly higher risks of 10‐year cumulative incidence for cancers of head, neck and esophagus (HR 3.19, 95% CI 1.34‐ 7.59, P = .003); cancers involving upper gastrointestinal tract (HR 3.74, 95% CI 1.58‐8.85, P < .001), colorectal cancer (HR 2.02, 95% CI 1.04‐3.91, P = .029), thyroid cancer (HR 2.09, 95% CI 1.1‐ 3.97, P = .012), gynecological cancer (HR 2.69, 95% CI 1.04‐6.96, P = .048) in the case cohort compared to the control cohort. No significant differences were detected for cancers involving lung, mediastinum and heart, breast cancer in female, cancers of the hepatobiliary and pancreatic system and cancers associated with urological system. These findings suggest the need for enhanced screening for nonhematologic malignancies in allo‐SCT recipients compared to the general population.
Destroying multidrug‐resistant lung cancer by mitochondrial damage and ATP Inhibition using nanodrug
1Lee Gil Ya Cancer and Diabetes Institute, Gachon University, 2Department of Physiology, College of Medicine, Gachon University , 3Gachon Advanced Institute For Health Sciences and Technology,
GAIHST, Gachon University
Despite advances in various therapeutic modalities to treat cancer, multidrug resistance (MDR) and incomplete destruction of deeply buried lung tumors remain long‐standing problems those leading to tumor recurrence and low survival rates. Therefore, new strategies for the treatment of MDR tumors are urgently needed. In this study, it was confirmed that intracellular drug internalization of nano‐drugs was enhanced through the covalent bonding of specific nano‐sized carbon nanotubes and doxorubicin (DOX). In addition, carbon nanotube‐conjugated DOX (CNT‐DOX), which persists for a long time in the intracellular environment of multidrug‐resistant lung cells, induces mitochondrial damage and inhibits ATP production, thereby showing an effective therapeutic effect on drug‐resistant tumors. In conclusion, nanodrugs of a specific size induced greater apoptosis through selective accumulation of tumor cells, improved therapy for MDR lung cancer cells, and provided novel insight into strategies to overcome multi‐drug‐resistant lung cancer cells.
Polycaprolactone scaffold cell‐based nasal implant using 3D printing
1Soonchunhyang University Bucheon Hospital
Small molecule mediated intervertebral disc repair via regulation of proteoglycan metabolism
1The University of Hong Kong
Low back pain is associated with degeneration of intervertebral disc (IVD). Proteoglycan is a predominant extracellular matrix (ECM) component in the nucleus pulposus (NP) of IVD. A reduction of proteoglycan causes compromised disc biomechanics and dysfunction of spinal motion segment. We previously established a miniaturized dimethylmethylene blue assay‐based platform for high‐throughput proteoglycans measurement. In a follow‐up study of screening a library of over 50,000 small molecules, we revealed leads that could up‐ or down‐regulate proteoglycan production in primary chondrocytes. Subsequent screening identified a novel small molecule, termed TS17, which could promote deposition of proteoglycans in human NP cells in dose‐dependent manner. TS17 could inhibit aggrecan degradation along with reduced expression of proteoglycans‐degrading enzymes ADAMTS5 and MMP13. Moreover, TS17 showed a capacity in inhibiting interleukin‐1 mediated inflammation. In a mouse IVD injury model, both intradiscal and systematic delivery of TS17 could facilitate IVD repair, indicated by ECM preservation and inflammatory reduction in the NP. Pain behavioral study demonstrated that TS17 administration could alleviate cutaneous mechano‐hyperalgesia. Transcriptome analysis of TS17‐treated inflammatory NP cells suggested a role of iron transmembrane transport and CD36 being the top differentially expressed gene. Furthermore, TS17 could down‐regulate the phosphorylation of CD36 downstream effector NFkB. Altogether, our study implicated a potential of small molecule‐based IVD repair via a regulation of proteoglycan metabolism through CD36/NFkB axis.
Transverse tibial cortex transport surgery: A novel treatment strategy for diabetic ulcers and biological mechanisms
1The Chinese University of Hong Kong
Diabetic foot ulcers (DFU) are severe vascular and neurological complications in the in the lower limbs at later stage of diabetic patients. Severe DFU usually accompanies such as gangrene, deep and large ulcers and osteomyelitis. At present, 90% of DFU patients with severity at Wagner III or above end up with amputation. For those whose ulcers healed through conventional treatments, the recurrence rate is 40% within 1 year.
Since 2011, clinical scientists in China launched a novel strategy for the treatment of severe DFU by using transverse tibial cortex transport technique (TTT) with proven success. Following TTT, the wound healing become more responsive and effective and the wound healing rate and limb salvage rate was over 95% for Wagner, and 1‐year recurrence rate of DFU was less than 9%.
The possible underlying mechanisms for the TTL surgery are: (1) decompress the marrow cavity and promote microcirculation and angiogenesis in the distal limb. (2) systemic factors release to promote stem cells mobilization and wound healing. (3) regulate local inflammation such as macrophages transformation from M1 to M2 phase. (4) other possible mechanisms including improvement in the sympathetic never or lymphatic microcirculation functions. More studies are still needed to discover the underlying biological mechanisms of TTT treatment for DFU and other peripheral avascular diseases.
TTT surgery is a novel, simple and cost‐effective surgical method to promote DFU healing, especially for severe DUF cases, which has a high successful rate of limb salvage and low incidence of recurrence for severe DUF.
miR‐31‐3p functions as a tumor suppressor by directly targeting GABBR2 in prostate cancer
1CHA University, 2Ajou University, 3SOON CHUN HYANG University, 4Korea Institute of Radiological and Medical Sciences
MicroRNAs are key regulators of gene expression in tumorigenesis. In this study, we investigated the tumor‐suppressive function of miR‐31‐3p. Analysis of the Gene Expression Omnibus database revealed that the expression of miR‐31‐3p in prostate cancer tissues is lower than that in adjacent normal tissues from patients with prostate cancer. Moreover, miR‐31‐3p induces apoptosis in DU145, PC‐3, and LNCap prostate cancer cells, while those transfected with miR‐31‐3p exhibit significantly decreased cell proliferation, migration, invasiveness, and tumor sphere‐forming ability, as determined using the cell counting kit‐8, transwell, and sphere‐forming assays. Further analysis revealed that GABBR2 is a direct target of miR‐31‐3p. Within a DU145 xenograft murine model, intratumoral injection of a miR‐31‐3p mimic suppresses tumor growth. Taken together, the findings of this study suggest that miR‐31‐3p performs a novel tumor‐suppressive function in prostate cancer and may represent a novel target for anti‐ prostate cancer miRNA therapeutics.
Plasma and tissue proteomics to identify biomarkers for reduced bone healing capacities in patients comorbid with type 2 diabetes mellitus
1Department of Preclinical Development and Validation, Fraunhofer Institute for Cell Therapy and Immunology IZI, 2Julius Wolff Institute of Biomechanics and Musculoskeletal Regeneration, Charité Berlin, 3Institute for Computational Systems Biology, University of Hamburg
Bones have the remarkable property of healing without scarring. However, several diseases, including type 2 diabetes mellitus (T2DM), can lead to impaired fracture healing. However, the extent of reduction in bone healing capacity is patient‐specific and no indicative biomarker for patients at risk who require treatment beyond standard therapy through personalized scaffolds has yet been identified. In the SyMBoD project, we have acquired molecular profiles (plasma proteomics and metabolomics) of potential high‐risk patients as well as control patients with bone defects. For comparison, a rat model with a critical size femur defect treated with polycaprolactone (PCL)‐based scaffolds was established. Blood plasma from these rats were analyzed longitudinally by proteomics to monitor the initial fracture response and early phase of bone regeneration. This approach allowed the quantification of 667 plasma proteins, of which 242 were identified with altered profiles in terms of temporal response and/or with altered abundance between diabetic and healthy animals. AI‐assisted bioinformatics clustering and network analysis tools were applied to identify profiles of affected proteins including previously described acute‐ phase proteins, apolipoproteins, and fatty acid‐binding proteins. In addition to gain further mechanistically insights, plasma protein profiles were correlated with proteomic analysis of explanted regenerated tissues sampled 21 and 42 days after surgery and with clinical outcomes. In the future, the results obtained will be incorporated into the multi‐omics and multi‐tissue analysis of the rat model and human clinical samples to identify T2DM patients with risk of compromised bone healing and to offer personalized treatment with 3D‐printed, structurally optimized scaffolds.
3D microenvironment prevents simulated microgravity‐mediated changes in T cell transcriptome
1NYUAD
Human space travel and exploration are of interest to both the industrial and scientific community. However, there are many adverse effects of spaceflight on human physiology. In particular, there is a lack of understanding to the extent by which microgravity affects the immune system. T cells, key players of the adaptive immune system and long term immunity, are present not only in blood circulation, but also reside within tissue. As of yet, studies investigating the effects of microgravity on T cells are limited to peripheral blood or traditional 2D cell culture that recapitulates circulating blood. To better mimic interstitial tissue, 3D cell culture has been well established for physiologically and pathologically relevant models. In this work, we utilize 2D and 3D cell culture to gain an understanding of how simulated microgravity affects both circulating and tissue resident T cells. T cells were studied in both resting and activated stages. We found that 3D cell culture attenuates the effects of simulated microgravity on the T cells transcriptome and nuclear irregularities compared to 2D cell culture. Interestingly, simulated microgravity appears to have less effect on activated T cells compared to those in the resting stage. Overall, our work provides novel insights into the effects of simulated microgravity on circulating and tissue resident T cells which could provide benefits for the health of space travelers.
Hyper O‐GlcNAcylation facilitates regenerative dentin formation via inflammation modulation
1Department of Biochemistry, School of Dentistry, IHBR, Kyungpook National University, 2Department of Oral and Maxillofacial Surgery, School of Dentistry, IHBR, Kyungpook National University,
3Department of Conservative Dentistry, School of Dentistry, IHBR, Kyungpook National University,
4Department of Pharmacology, School of Dentistry, IHBR, Kyungpook National University, 5Department of Oral Medicine, School of Dentistry, IHBR, Kyungpook National University
O‐GlcNAcylation is the posttranslational modification of the proteins catalyzed by two enzymes; OGT and OGA. O‐GlcNAc modification of protein on serine or threonine residue is crucial for tissue specification, cell viability, and embryonic development. Moreover, the hyper O‐GlcNAcylation of the cellular proteins has shown preventive effects during inflammation in the heart and vascular dysfunction. However, its role during hard tissue especially dentin formation and regeneration have not been explored yet. In this study, the pharmacological elevation of O‐GlcNAcylation by OGA inhibitor drug following up pulp exposer in mouse molars resulted in reparative dentin formation via inflammation prevention. Altered morphological changes and cellular physiology were examined with histology and immunohistochemistry. After OGA inhibitor local delivery in the mice pulpal cavity, histology, and cellular physiology such as modulation of differentiation and inflammation were examined using histology and immunohistochemistry. OGA inhibited specimens showed altered localization patterns of Nestin, NF‐κB, MPO, Runx2, TGF‐β1, and TNF‐∂ after 3 and 5 days. Furthermore, OGA inhibited specimens showed facilitated dentin‐bridge formation after 42 days of local drug delivery when compared with the control. Micro‐CT images further confirmed the dentin‐bridge structure in the OGA inhibited specimens when compared to the control. From these results, we concluded that hyper O‐GlcNAcylation with OGA inhibition would facilitate reparative dentin formation via inflammation prevention and activation of signaling regulations. Therefore, the elevated O‐GlcNAcylation of the cellular proteins might be crucial for inflammation prevention and patterned formation of dentin regeneration.
Anti‐biofouling, waterproof and insulating encapsulation for bioelectronics with improved longevity and robustness
1YONSEI UNIVERSITY
Bioelectronics including biosensor applications have made significant progresses in biomedical research. Accurate signal acquirement and electrical stimulation therapy were realized by attaching the device onto the skin or inserting the device into one's body, The major challenge of the bioelectronics is the need of encapsulation which enable stable performance in contact with biological environment for long‐term. In the case of conventional bioelectronic devices, as the moisture is not completely blocked, the device failure became inevitable that hindered practical application as an implantable electronics. Also, the side effect of immune response including inflammatory response can occur, which prevents from integration between the device and the tissue. Therefore, development of the encapsulation with longevity is essential for long‐term operatable bioelectronics.
Here, we overcome the limitations by developing biocompatible organic materials‐based encapsulation coating with anti‐biofouling coating. This coating consists of an adhesive polydopamine layer, a perfluoropolymer layer and a lubricant swelling layer. The substrate was swollen in fluorinated oil which exhibit anti‐biofouling, water‐repellant and insulating properties. The insulation property was demonstrated by the maintenance of the leakage current at 10‐9∼10‐10 for more than a month. In addition, in vitro experiment was performed by immersing the touch panel with the coating in the PBS solution at 37.5 degrees. The resultant data indicates that the device performance lasted even after 3 weeks of incubation. Lastly, the encapsulation layer can be easily coated on flexible bioelectronics due to the inherent flexibility of organic materials.
Development of in‐situ evaluation system for the surface lubrication function of tissue‐engineered cartilage by using surface plasmon resonance
1Department of Biomedical Engineering, Doshisha University, 2Graduate School of Life and Medical Sciences, Doshisha University
Hydration lubrication has been proposed as one of the mechanisms to achieve excellent low friction at the cartilage surface. In this mechanism, phospholipids, hyaluronic acid, and lubricin act synergistically, and the hydration capacity of the hydrophilic head group of phospholipids mainly contributes to a decrease of friction. Furthermore, the interaction between lubricin and hyaluronic acid is also known to affect lubrication function by changing viscoelastic properties at the frictional interface. Hence, we focused on the maturation process of the surface lubrication based on the molecular expression to regenerate a physically functional tissue.
In order to evaluate the molecular interactions involved in construction of hydration layer, we developed a surface plasmon resonance microscope installed with a friction measuring apparatus enabling to acquire the information of those interactions specific to the contact interface. This system equips a variable laser incidence angle mechanism to detect changes in various molecular interactions and their expression profiles at the frictional interface. In this study, chondrocyte‐agarose complex tissues were cultured and the interaction between hyaluronic acid grafted on the gold film, the detector part of the microscope, and molecules expressed on the tissue surface was analyzed under friction test. The friction coefficient of tissue‐engineered cartilage decreased with the increase of culture day, and changes in area and intensity of the molecular interaction at the tissue surface could be in situ detected during friction. This system would provide further insight into the interactions which contribute to hydration lubrication.
Concurrence of cartilage degeneration, vessel formation and subchondral bone remodelling in osteoarthritic hip joints
1University College London
Osteoarthritis (OA) is a degenerative joint disease that affects both cartilage and subchondral bone (SCB). With the progression of OA, as results of changes in the loading pattern and possible vascular pathology, bone remodelling and resorption occur 1‐2, which affect the physical environment supporting the overlying cartilage. Understanding the co‐localisation of these factors can lead to the development of possible prognostic factors and can assist us in creating more effective biological treatments. In this study, we report the changes in local distribution of volumetric bone mineral density (vBMD), tidemark morphology and vascular changes with OA to evaluate any concurrence of cartilage degeneration and SCB remodelling.
In this study, human femoral heads (n = 4) were collected during a total hip replacement operation due to OA. Cartilage was graded using ICRS classification3. To determine the remodelling of the subchondral bone, a peripheral quantitative CT (pQCT) was used to assess the vBMD distribution within the SCB. Histology/immunohistochemistry were used to evaluate vascular changes.
The examination of retrieved tissues revealed cartilage in different stages of degeneration, from normal to severely abnormal. Subchondral vBMD decreased with cartilage ICRS grade confirming bone remodelling in all samples. CD31 positive cells were identified in different regions of the femoral head confirming the concurrence of changes in blood vessels, cartilage degeneration and SCB remodelling.
1 Bhatia et al., Journal of Pharmacy & Bioallied Sciences
2 Findlay, Rheumatology
Sustained drug delivery of doxorubicin and enhanced anti‐cancer efficacy via intra‐tumoral injection of in‐situ forming click‐cross‐linked hydrogel
1Ajou University
The primary clinical treatment for solid tumors is a surgery, which is the cancer tissue is directly incised and removed. Following the surgery, a drug treatment or radiotherapy methods are used to remove cancer tissues that have not been completely removed after dissecting the cancer tissues, or to prevent cancer metastasis by remaining cancer cells and cancer tissues. Among numerous drugs, FDA approved Doxorubicin (DXR) have been used as an anticancer drug. DXR has hilarious anticancer effects, but it is very toxic in high plasma concentration and has a short lifetime in human body. For these reasons, we have prepared a click cross‐linkable hyaluronic acid hydrogel that can be directly injected into cancer to improve the efficacy of DXR and reduce DXR toxicity. Since the biorthogonal reagents, trans‐ cyclooctene and methyl tetrazine are immediately reacted in bodies and known for low toxicity, we individually introduced trans‐cyclooctene and methyl tetrazine into hyaluronic acid (HA) so that the trans‐cyclooctene conjugated HA and methyl tetrazine conjugated HA immediately formed DXR‐ containing hydrogel after being injected in the tumor and the drug could be sustainably released. Click cross‐linked HA(Cc‐HA) hydrogel consistently suppressed tumor growth, whereas DXR loaded non‐ crosslinked HA and DXR solution inhibited the tumor growth for very short period and tumors were dramatically increased. In conclusion, we successfully prepared Cc‐HA hydrogel capable of forming a drug depot within tumor and the anticancer effect of DXR loaded hydrogel was successfully increased.
Treatment of acne vulgaris through electrostatic interaction between adapalene and cationic polycaprolactone emulsifier
1Ajou university
Adapalene (ADP) is a widely used for the treatment of acne vulgaris due to its significant inflammatory inhibition and low side effects. However, ADP has limitation that it's negative charge potential reduces the permeability to negatively charged skin. This study is conducted to increase the stability and treatment effect of ADP using electrostatic interaction. Emulsion using methoxy polyethylene glycol‐b‐poly(ɛ‐ caprolactone) (PC) and PC with amine pendant group (PC‐NH2) as negative charge emulsifiers and positive charge emulsifiers, respectively, were prepared to evaluate the effects of acne treatment through electrostatic interaction between emulsifier and ADP. As the number of amine pendant group of PC‐NH2 increases, the positive charge potential becomes stronger and the negative charge potential caused by ADP decreases. The stronger the positive charge of PC‐NH2 emulsion, the better the stability of the emulsion and the therapeutic effect of acne than ADP alone and PC emulsion. This indicates that PC‐NH2 emulsifier helps effectively penetrate ADP into the skin. Through histological analysis, it is confirmed that PC‐NH2 emulsion with a high positive charge can effectively suppress the expression of macrophages and inflammation. Therefore, the positively charged PC‐NH2 emulsifier can be used as an effective acne treatment strategy through the electrostatic interaction between the negatively charged ADP and the skin.
Computational modeling approach to measure cell nucleus elasticity using traction force microscopy
1 Korea University
Finite Element Modelling (FEM) approach has been proved to be key technique to estimate elasticity of cell by comparing with AFM technique and compare force‐indentation depth response of cell and predict intercellular force transduction and distribution. We aim to develop a novel TFM‐based technique to compute the intracellular forces generated by a migrating doxycycline‐induced progerin expressing HeLa cells using image analysis and the finite element model (FEM). We first culture cells over the ECM‐ mimicking hydrogels where fluorescent bead microspheres embedded and the dishes were mounted on fluorescence microscope. We next captured live cell imaging of an in‐focus phase contrast image of cell and bead. In order to quantify bead displacements between the relaxed and stressed states of the substrate, we used newly developed image processing algorithm and later used bead displacement values to estimate the traction forces generated by subcellular organelles. Later we use these TFM based results for nucleus modeling, by applying control magnitude of stress to the cell model at the point of interest using substrate stain technique. Later, we measured the stain at the nucleus location to evaluate young modulus of cell nucleus.
Application of surface plasmon resonance in detection of rectal cancer
1University of Chinese Academy of Science
Rectal cancer is one of the most common malignant tumor. With the incidence rate increasing, it shows younger and malignant trend. Due to poor surgical results, early detection of rectal cancer is necessary. The commonly used clinical detection methods are Enteroscope, MRI and CT, which are complex and costly, and the acceptability of invasive detection methods is low. Therefore, they usually found advanced cancer.
Due to intestinal bleeding, fecal occult blood (FOB) detection can be used for early preventive detection of rectal cancer. FOB detection is the qualitative detection of trace hemoglobin (Hb) in fecal samples. The clinical common combined immune method and monoclonal antibody method have high sensitivity, but it is complex and costly.
Surface plasmon resonance (SPR) is a real‐time, label‐free and highly sensitive optical detection technology. At present, it has been widely used in biotechnology, medicine and other fields. This technology can directly reflect the change of physical and chemical properties on the interface through the change of optical signal.
In this study, we will use a homemade SPR detector to detect ultra‐low concentration Hb to simulate the detection of FOB. We use self‐assembled monolayer technology to modify SPR chip by aptamer. Compared with antibody, aptamer has shorter screening cycle; lower working concentration; smaller batch difference; simple amplification process; lower cost and better stability. The LOD of the self‐made SPR detector is 2.55nM, compared with original methods increased 125%. Therefore, this technology can be used as a new method for clinical detection of rectal cancer.
Fabrication of photothermal film for deicing process based on gold nano‐ aggregate encapsulated yolk‐shell structure
1Sungkyunkwan University
Ice accumulation on vessels, airplanes, or on the off‐shore plants surfaces causes major accidents and difficulties in operation. Therefore, highly efficient and environmentally friendly materials for use as deicing surfaces are in continuous demand. Accordingly, photothermal materials have gained enormous attention owing to their outstanding performance in the removal of ice. Herein, a gold nano‐aggregate yolk‐shell structure (GNA‐YS) was introduced as a deicing material. GNA‐YS was homogeneously dispersed in photocurable polyurethane acrylate and used to fabricate a highly efficient and sunlight‐ responsive GNA‐YS film. Upon irradiation with an 810 nm light‐emitting diode (LED) (135 mW cm−2), the temperature of the GNA‐YS film increased by approximately 70 °C within 2 min, which rapidly (within 6 min) melted the accumulated ice. Moreover, the GNA‐YS film exhibited a temperature increase of 15 °C in a refrigerator under LED illumination for 1 min. Additionally excellent stability was confirmed through repeated experiments. The newly developed deicing GNA‐YS film has prospective applicability in vessels or airplanes.
Development of hybrid skin model to evaluate UV‐protective effects
1Department of Biomedical Engineering, Doshisha University, 2Graduate School of Life and Medical Sciences, Doshisha University
In this study, we developed the hybrid skin model that enables to evaluate skin damage caused by UV with the reproduced UV absorption properties similar to skin. The in vitro model is needed to evaluate UV protection effects of skin care products while avoiding the burden on the subject and individual differences in screening. The hybrid skin model had the PDMS layer consisted of the melanin‐containing and the hemoglobin‐containing layers for reproduction of UV absorption properties, and the collagen nanofiber and the fibroblast layers for evaluation of biological damage by UV. Concentration of melanin and hemoglobin, and thickness of the melanin‐containing and the hemoglobin‐containing PDMS layers were optimized using UV absorption properties of mouse skin. The optimized concentration of melanin and hemoglobin were 0.09 and 0.27 w/v%, and the optimized thickness of the melanin‐containing and the hemoglobin‐containing PDMS layers were 1080 and 1960 μm. Collagen nanofiber layer fabricated by an electrospinning method was crosslinked with glutaraldehyde to improve water resistance, and then laminated on the hemoglobin‐side of the PDMS layer. Fibroblasts were cultured on the collagen nanofiber layer of the skin model. UV damage can be evaluated by measuring changes in collagen structure and cell activity due to UV light irradiated through skin care products located on the melanin‐side of the PDMS layer.
The anti‐tumor effect and mechanism of PDK4 in bladder cancer.
1Kyungpook National University, 2Kyungpook National University Hospital
Bladder cancer is the second most common urinary tract cancer in the world. Over 430,000 men and women are diagnosed as bladder cancer every year. Most bladder cancer patients are diagnosed as non‐ muscle invasive bladder cancer (NMIBC), however, 10‐15% of NMIBC progress to muscle invasive bladder cancer (MIBC) by metastasis. Metastasis plays a key role in increased cancer related death rate. Pyruvate dehydrogenase kinase 4 (PDK4) is a member of PDK4 family which is located in the mitochondrial matrix of cells. Recently PDK4 is emerged as tumorigenesis related protein. Therefore, we explored the relationship of TRPM7 and bladder cancer.
In siRNA treated cells, we were able to detect increased p‐AKT and p‐ERK1/2 level while total forms stay equal in mock and negative control treated cells. In addition, the expression of e‐cadherin was increased, and n‐cadherin was decreased compared to mock and negative control treated cells. In vivo analysis, we performed xenograft model and observed the suppressed tumor progression rate in PDK4 knockout J82 cell injected mice.
Taken together, TRPM7 plays a key role in migration and invasion in bladder cancer cell line via mediation of p‐Akt, p‐ERK and p‐JNK signaling pathway. In addition, the present study suggests the possibility of TRPM7 as a bladder cancer predicator.
Time‐series correlation multivariate algorithm model to improve the accuracy of continuous glucose monitoring
1University of Chinese Academy of Sciences
The key to treat diabetes and reduce its harm is to scientifically monitor the blood glucose (BG) level and take a series of cost‐effective interventions to improve the health status of patients. Continuous glucose monitor (CGM) sensors are medical devices currently used for real‐time monitoring of blood glucose level. However, the current CGM devices are often accompanied by the problems of limited measurement accuracy, weak stability and small scope of application. Therefore, it needs patients to measure the fingertip blood glucose for calibration frequently.
This work uses the time‐series correlation multivariate algorithm model to improve the accuracy of blood glucose concentration and prediction. Considering CGM signal, insulin amount, carbohydrate intake and other influencing factors, combined with statistical learning method Principal Component Analysis (PCA) to determine the calibration principal component to improve the robustness of the model.
In this study, the algorithm will be pretrained and tested on the public dataset to preliminarily verify the effectiveness of the model. Then the pretraining model will be fine‐tuned on our local dataset to compare with our previous work, and the clinical applicability of the algorithm will be verified by Clark grid analysis.
Suppression of cancer cell migration by TGFβ activated fibroblasts in a co culture model of the tumor microenvironment
1Korea University
Tumor microenvironment (TME), which consists of various cell types embedded in an altered extracellular matrix (ECM), plays an important role in metastasis through cell‐cell and cell‐ECM interactions. Within the TME, fibroblasts are key cellular components of tumors as they play a role in cancer metastasis by being involved in mechanisms such as matrix deposition and remodeling, which can be dependent on their quiescent or activated state. However, the role of fibroblasts in the origin and initiation of cancer cell invasion is yet to be fully understood.
In this study, cancer cell progression, based on the activation and inactivation of fibroblasts by TGFβ treatment, was tested in a 2D coculture model. Traction Force Microscopy (TFM) was utilized to measure traction and velocity forces regarding cellular migration. Our results show higher traction and localized vimentin expression at the boundary of the patterned activated fibroblast coculture group. This is opposite to other findings where activated fibroblasts assist tumor progression. While co culture of cancer cells with normal fibroblast facilitate the migration of cancer cells, co culture of cancer cells with activated fibroblasts prevented migration.
Investigating and targeting activated fibroblasts in the cancer microenvironment will help us better comprehend the mechanisms of cancer cell progression.
Submicron‐structure surface chip apply in early cancer screening by surface plasmon resonance
1University of Chinese Academy of Science
Due to advances in surface submicron‐structure fabrication technology, surface‐enhanced sensor chips based on localized surface plasmon resonance (LSPR) effect have been widely used in chemical and biological analysis. The submicron‐structure surface fabrication has enhanced sensitivity and quantification of surface refractive index change. Traditional photolithography has significant advantages in fabricating long‐range ordered, high‐fidelity nanostructure arrays, but its complex and time‐consuming process leads to high economic costs, therefore, it is necessary to use an easy and high‐throughput photolithography process and micro‐nano patterning process to fabricate surface‐enhanced sensor chips. This research will use nanoimprint lithography, plasma etching, and metal film deposition to fabricate surface‐enhanced SPR sensor chips with periodically distributed arrays of gold nanorod structures. the results showed the submicron‐structure surface not only can enhance the sensing sensitivity, but also has the lower detection limit. Due to the high sensitivity and lower detection limit, the submicron‐structure surface SPR chip was very suitable apply in early cancer screening.
Effect of vaginal sildenafil on the regeneration of refractory thin endometrium in thawed embryo transfer cycles: A case series
1Seoul National University Hospital
This study aims to evaluate the effect of vaginal (sildenafil VS) on patients who experienced the cancellation of thawed embryo transfer cycles due to refractory thin endometrium.
The medical records of patients who visited the fertility clinic of Seoul National University Hospital for thawed embryo transfer cycles were retrospectively reviewed. Six patients and 15 cycles were included in this case series study. Patients received oral estradiol (valerate EV) starting on day 3 of their cycles from 4 to 10mg. The cycle was cancelled if endometrial (thickness EMT) measured by transvaginal ultrasound failed to reach more than 7 mm. Patients started EV and VS in the next cycle on day 3 of their cycles. The EMT was measured on the day of luteal phase (support LPS) initiation, and VS was stopped on the same day. The EMT was compared between cancelled cycles and VS cycles using the Mann‐Whitney test.
The mean age was higher in VS cycles (38.11 vs. 36.5 years, p = 0.388), and mean BMI, AMH and dosage of EV was similar between VS cycles and cancelled cycles (BMI: 27.12 vs. 27.11 kg/m2, p = 1.000; AMH: 4.67 vs. 5.33 ng/mL, p = 0.776; EV dose: 9.25 vs. 10 mg, p = 0.368). The mean EMT on the day of LPS initiation was 5.45mm in cancelled cycles and 6.86mm in VS cycles, which showed a 25.9% increase (p = 0.026). However, none of these cycles resulted in clinical pregnancy.
Vaginal sildenafil can be one of treatment options for refractory thin endometrium when previous thawed embryo transfer cycles were cancelled.
Multifunctional synthetic nanoenzyme embedded colorimetric paper biosensor for rapid detection of hydrogen peroxide
1Korea National University of Transportation, 2Hanyang University, 3Temple University
Here, we present the fabrication of novel molybdenum based synthetic nanozymes (Synz) and their applications as a paper‐based colorimetric biosensor for the detection of hydrogen peroxide (H2O2) and L‐cysteine. Physiochemical characterizations indicate a flower shape architecture with a negative surface potential of ‐26 eV and presence of carboxyl and hydroxyl groups in Synz. These blue colored Synz has demonstrated photothermal activity under near‐infrared region (NIR) laser irradiations (808 nm; 2W/cm2) and possess computerized tomography (CT) contrasting and H2O2 mediated oxygenic properties. Furthermore, Synz are selective and sensitive to H2O2, where the color of the Synz solution has changed from blue to white as well as change in the absorbance spectra in the NIR region (750 nm) when incubated with H2O2. We later coated Synz over whatmann filter paper and monitored change in the color of Synz paper from blue to white. A substantial change in the color of Synz has been observed until H2O2 conc of 60μM. Collective our results manifests Synz as a promising multifunctional nanomaterial and a low‐cost paper based colorimetric biosensor for diverse biomedical applications.
The influence of fine dust in DEPs –inhalated diabetic wound models
1SoonChunHyang University , 2SoonChunHyang University Bucheon Hospital, 3SoonChunHyang University
Exposure to diesel engine exhaust particles (DEPs) has been associated with several adverse health outcomes in which inflammation seems to play a key role. But, it is un‐known the effect of exposure to DEPs on infection wounds in vivo. This study investigated pro‐ inflammatory enzymes, cyclooxygenase‐ 2 (COX‐2) and pro‐inflammatory cytokines, tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 for the evaluation of the influence of Diesel exhaust particles on wounds.
The effect of wound healing lead material using a streptozotocin‐induced diabetic rat model exposed to fine dust
1SoonChunHyang University, 2SoonChunHyang University Bucheon Hospital
Due to ultrafine dust, immunity is lowered and the probability of being exposed to various diseases is increasing. Among them, diabetes is a problem caused by complications, which can get worse. In this study, the degree of healing was confirmed using histology and molecular biology by applying a therapeutic substance after exposing diabetic wound‐induced mice to fine dust. The concentration of ultrafine dust was determined to be 80 μg/m3.
Effect of electric muscle stimulation on the improvement of deltoid muscle atrophy
1Konkuk University Medical Center, 2Konkuk University
Immobilization after trauma or surgery induces skeletal muscle atrophy, and the improvement of muscle atrophy is critical for the successful clinical outcomes. The purpose of this study was to evaluate the effect of the electric muscle stimulation (EMS) on the improvement of deltoid muscle atrophy after establishing deltoid muscle atrophy model in rats. The deltoid muscle atrophy was the most prominent at 3 weeks after shoulder immobilization, then the atrophy was gradually recovered but not to the normal level. The Murf1 and Atrogin (muscle atrophy‐related factors) were strongly induced at initial phase and gradually decreased at around 3 weeks, and the expression of MyoD (myogenesis‐related factor) showed inverse relationship compared to the Murf1 and Atrogin. The expression of IL‐1b and IL6 (proinflammatory cytokine) were prominent at 1 week but decreased afterwards, and that of Fabp4 and C/EBPa (adipogenesis‐related factors) were increased after 3 weeks. Thus, the 3 weeks, when the deltoid atrophy was the most prominent with the change of relevant gene expressions, were selected as the time point for the evaluation of the EMS effect. The deltoid muscle cell size of the immobilized shoulder was significantly smaller than the intact shoulder in all rats. The cell size of the LE‐EMS group was similar with the control group, but that of the HE‐EMS group were bigger than other groups. The HE‐EMS might be one of the solutions for strengthening atrophied skeletal muscles and facilitated rehabilitation after trauma or surgery.
Targeted delivery of apoptotic cell‐derived nanovesicles prevents cardiac remodeling and attenuates cardiac function exacerbation
1Korea Institute of Science and Technology, 2The Catholic University of Korea
Inflammation plays an important role in the myocardial infarction (MI) pathophysiology. Therefore, the modulation of macrophages phenotype in early stage of MI is crucial in MI treatment. Herein, we developed apoptotic bodies‐mimetic nanovesicles derived from apoptotic fibroblasts (ApoNVs) conjugated with dextran and ischemic cardiac homing peptide (ApoNV‐DCs) for ischemia‐reperfusion injured heart treatment. Intravenously injected ApoNV‐DCs actively targeted ischemic myocardium via the conjugation with ischemic cardiac homing peptide and were selectively phagocytosed by macrophages in infarcted myocardium via the conjugation with dextran. ApoNV‐DCs polarized macrophages from M1 to M2 phenotype, resulting in the attenuation of inflammation. Four weeks after injection, ApoNV‐DCs attenuated cardiac remodeling, preserved blood vessels, and prevented cardiac function exacerbation of IR‐injured heart. Together, our findings may open new avenues for the immunomodulation by targeted delivery of anti‐inflammatory nanovesicles that can be universally applied for various inflammatory diseases.
Effective delivery of osteoinductive composite‐spheroids laden hydrogel for bone tissue engineering
1Department of bioengineering, Hanyang University, 222 Wangsimni‐ro, seongdong‐gu, seoul, 04763, Republic of Korea, 2Department of Bioengineering, Hanyang University, 222 Wangsimni‐ro, Seongdong‐ gu, Seoul, 04763, Republic of Korea
Hydrogels incorporating stem cell spheroids and osteoinductive biomolecules have been investigated for bone reconstruction due to convenience of delivering molecules and similarity in cellular microenvironment with natural extracellular matrix. However, the limited diffusion and interactions between biomolecules and cells hindered the differentiation capacity of stem cells, and the heterogeneous distribution and limited cell‐spreading induced abnormal and islet‐type in vivo bone formation.
In this study, we developed the human adipose‐derived stem cell spheroids laden gelatin (methacrylate GelMA) hydrogel engineered with the functional nanofibers to address the current issues. The spheroids were homogeneously distributed within a hydrogel by modulating the size of spheroids and the mechanical property of hydrogels. Briefly, the small spheroids (167 cells and 0.02 ug fibers) showed even distribution within the hydrogel than the larger spheroids, and the hydrogel with lower mechanical property (400 Pa) revealed the more proliferation of cells than the stiffer gel. Furthermore, the fibers were coated with bone morphogenetic protein 2 by polydopamine mediated adhesion to effectively give osteogenic signals from the inside of spheroids. The stem cells in spheroids with BMP‐2 immobilized fibers showed the greater osteogenic differentiation than the groups where BMP‐2 was in media or hydrogel; the BMP‐2 in a spheroid group showed 14.29 ug of calcium deposition while the other groups were 7.46 ug and 7.43 ug, respectively. In conclusion, spheroids‐laden hydrogels with effective delivery of inductive factors using engineered fibers dramatically enhanced proliferation and osteogenic differentiation of the encapsulated spheroids, and would be used as a potential treatment of bone injuries.
Inhibitory effect of ginseng derived extracellular nanovesicles on osteoclast differentiation
1Department of Biomedical Science and Technology, Graduate School, Kyung Hee University, 26 Kyugheedae‐ro, Dongdaemum‐gu, Seoul 02447, Republic of Korea, 2Department of Dentistry, Graduate School, Kyung Hee University, 26 Kyugheedae‐ro, Dongdaemum‐gu, Seoul 02447, Republic of Korea, 3Department of Dental Materials, School of Dentistry, Kyung Hee University, 26 Kyugheedae‐ro, Dongdaemum‐gu, Seoul 02447, Republic of Korea, 4Biofriends, 23 Kyugheedae‐ro, Dongdaemum‐gu, Seoul 02447, Republic of Korea
Plant derived extracellular nanovesicles have a great potentiality with their complexed components, which can make difference from normal plant extract by having similarity with exosomes in round, lipid bilayer morphology and inner components. Plant derived extracellular nanovesicles have different efficacy with their own proteins, RNAs, which constitute that plant. In particular, ginseng is known for their anti‐inflammation ability, anti‐cancer ability, improving immunity, and various ginsenoside components showed the ability to inhibit bone resorption for preventing osteoporosis. In this study, we confirmed the ability to inhibit osteoclast formation with ginseng derived extracellular nanovesicles (GDN). GDN was obtained by centrifugation with sucrose gradient method. The average size and zeta potential was measured 71.42nm and ‐18.8mV, respectively, using DLS, Zeta potential. Also GDN showed specific round morphology in TEM observations, which can be concerned as extracellular nanovesicles. Bone marrow‐derived macrophages were used to demonstrate cytotoxicity and inhibitory effect of osteoclast differentiation of GDN. The results showed that high viability and proliferation with 3, 5, 7μg/ml concentration of GDN as evaluated by Live/Dead staining and CCK‐8 assay. TRAP and F‐ actin staining were carried out to estimate inhibitory effect of osteoclast differentiation and then GDN has a great inhibitory effect from 1μg/ml to 5μg/ml concentration. Moreover, in‐vivo analysis (micro‐CT, bone volume/total volume, bone mineral density) also demonstrated GDN's inhibitory effect of osteoclast differentiation in LPS‐induced bone resorption mouse model. Consequently, our results suggest that GDN have anti‐osteoporosis ability by inhibit differentiation of osteoclast, and therefore can be a promising approach for bone loss diseases.
Fabrication of spheroids using hydrogels with self‐assembly system and bioreactor
1Department of Biomedical Science and Technology, Graduate School, Kyung Hee University, 26
Kyugheedae‐ro, Dongdaemum‐gu, Seoul 02447, Republic of Korea, 2Department of Dentistry, Graduate School, Kyung Hee University, 26 Kyugheedae‐ro, Dongdaemum‐gu, Seoul 02447, Republic of
Korea, 3Department of Dental Materials, School of Dentistry, Kyung Hee University, 26 Kyugheedae‐ro, Dongdaemum‐gu, Seoul 02447, Republic of Korea
Tissue engineering aims to create large tissues that are close to the organ in the human body. However, it is difficult to increase the size of the tissue, and even if the size is increased there are restrictions such as apoptosis occurring in the tissue. To overcome these limitations, we use self‐assembly hydrogels with bioreactors to create spheroid that mimic relatively large tissues. In this study, we introduced different two types of cells encapsulated in a hydrogel to maintain the characteristics of each cell type for as long as possible, and a self‐assembly system was used to integrate the two parts. The fabricated spheroid has various advantages that providing enough space for cell growth and promoting cell proliferation due to the inherent porous structure of the hydrogel by supplying nutrients and oxygen. We found that the spheroid formation can be adjusted by controlling the degradation rate by the concentration of hydrogel and the density of cells. In addition, we investigated the optimal time that the spheroid was transferred to the bioreactor, supplying sufficient medium for enhancing the spheroid growth. As a result, several spheroids were combined to make a large organoid under the influence of self‐assembly hydrogels surrounding spheroid. On basis of these advantages, we suggest that the fabricated spheroid using hydrogels with self‐assembly, and bioreactor has potential application in field of tissue engineering by transplantation at the defect site.
Multi‐functional membrane with bioactive layer and anti‐adhesion layer for tendon regeneration
1Dankook University, 2Hannam University
Although the development of surgical techniques for tendon injury are rapidly growing, insufficient regeneration of tendon and inevitably occurred tissue adhesion during the healing process continues to be a challenge for orthopedic surgeons. To overcome the limitations, delivery of cells/growth factors (for tendon regeneration); drug therapy, physical barrier, physical rehabilitation training (for prevention of tissue adhesion) are intensively investigated. In this study, we developed a bi‐layered Janus membrane which can enhance tendon regeneration (by sustained release of PDGF‐BB from unique pore structure; tissue regeneration layer) and prevent tissue adhesion (by physical separation of alginate between defect and surrounding tissues; anti‐adhesion layer). The morphology, growth factor release behavior, tenogenic differentiation (on bioactive layer), and cell adhesiveness (on anti‐ adhesion layer) were investigated.
Development of PCL‐based 3D printing scaffold with unique morphology for bone regeneration
1Dankook University, 2Hannam University, 3Gyeongsang National University School of Medicine
Bone defects which can occur in patients suffering from trauma, infection or tumor resection need intervention therapy to achieve sufficient restoration. However, traditional bone grafts cannot completely meet the clinical requirements. In recent years, 3D printing scaffolds have gained increasing interest as an alternative for bone reconstruction. However, their insufficient biological properties for bone regeneration still remained as a critical problem. In this study, we fabricated a growth factor‐loaded PCL 3D printing scaffold with leaf‐stacked structure which can allow sustained release of the growth factor as well as improved cell adhesiveness. The morphology, mechanical properties, growth factor release behavior, cell adhesiveness/proliferation/differentiation (in vitro); and new bone formation using beagle dog animal model (in vivo) of 3D printing scaffold with leaf‐stacked structure were investigated.
Development of blood plasma‐immobilized porous film with leaf‐stacked structure as a hemostatic agent
1Dankook University, 2Hannam University, 3Kyungpook National University Hospital
Bleeding is one of the major causes of death in trauma patients. It is known that death occurs when the amount of bleeding exceeds 40% of the total blood volume. To prevent excessive life‐threatening bleeding, various hemostatic agents have been developed. Recently, many hemostatic agents based on polymeric biomaterials are commonly adapted in clinical fields to manage bleeding. Platelet‐rich plasma (PRP) containing a lot of blood clotting factors is also considered as a hemostatic agent. We expected that if the PRP can be adsorbed on surface with large surface area, it will maximize their hemostatic effect. Therefore, the main aim of this study was to develop a PRP‐adsorbed porous film with leaf‐stacked structure (PRP‐FLSS) and to confirm the feasibility of the film as a hemostatic agent. The morphology, cytotoxicity, hemolysis analysis, blood coagulation (in vitro); prevention of hemorrhage, and histological evaluation (in vivo) of PRP‐FLSS were investigated.
Fabrication and characterization of cell spheroid system containing porous microparticles
1Dankook University, 2Hannam University
In recent years, a cell spheroid system is considered as one of the most common and versatile way for 3D cell culture. However, limited size, weak structural stability and heterogeneous cell differentiation of cell spheroid due to the insufficient supply of oxygen/nutrients into the central region, relatively weak cell‐ cell interaction, and different concentration of signaling molecules along the cell depth of the spheroid are still remained as critical challenges. In this study, we fabricated porous microparticles with leaf‐stacked structure throughout entire matrix (LSS particles) which can provide space for sufficient supply of oxygen/nutrients in cell spheroids, act as a filler for improved structural stability of cell spheroids, and allow sustained release of signaling molecules for effective induction of cell differentiation throughout whole cell spheroids. A cell spheroid system was prepared using the LSS particles and human bone marrow‐derived mesenchymal stem cells (hBMSCs). The morphology, release pattern of bone morphogenetic protein‐2 (BMP‐2) from LSS particles, cell survival/structural stability/osteogenic differentiation of hBMSCs in cell/LSS spheroid system were observed. We also evaluated whether the cell/LSS spheroid system can promote bone regeneration using a SD rat (calvarial defect model).
Development of bi‐layer GBR membrane for simultaneous regeneration of bone and epithelium
1Dankook University, 2Hannam University, 3Gyeongsang National University School of Medicine
Recent studies related with guided bone regeneration (GBR) membrane have focused on exclusion of rapidly growing non‐osteogenic cells into the bone defect for effective bone regeneration. Although it is well‐established that the GBR membrane can accelerate new bone formation, a high risk of infection caused by loosening between membrane and epithelium remains as a clinical challenge. Many surgeons continue to raise the need for development of GBR membrane which can induce simultaneous reconstruction of bone & epithelium. In this study, we developed a membrane with leaf‐stacked structure on both faces. It was expected that the FLSS allows sustained release of BMP‐2 and PDGF‐BB from each side, and thus achieves effective regeneration of bone (by BMP‐2) and epithelium (by PDGF‐BB) respectively. The morphology, physical properties, growth factor release pattern and cell behaviors (adhesiveness/migration/differentiation) of growth factors‐loaded FLSS were investigated.
PMMA‐based bone cement to prevent adjacent vertebral fractures after vertebroplasty
1Dankook university, 2Gyeongsang National University Hospital, 3Hannam University, 4Korea Institute of Industrial Technology
Vertebroplasty is commonly used technique to treat osteoporotic vertebral compression fractures. In this method, bone cement is injected into the defected vertebral body. Although the vertebroplasty using poly(methylmethacrylate) (PMMA)‐based bone cement has been adapted in clinical practices as a gold standard to treat vertebral fracture, adjacent vertebral fractures caused by high modulus of injected bone cement is remained as a challenge of orthopedic surgeons. To solve the limitation of vertebroplasty using PMMA bone cement, we developed a PMMA bone cement containing polydimethylsiloxane PDMS (i.e., Sylgard) as a plasticizer to reduce modulus. The PMMA/PDMS mixture was prepared by simple mixing, and PDMS in the bone cement was rapidly and homogeneously solidified by exothermic heat during PMMA polymerization. The SEM‐EDS mapping, XRF analysis, mechanical property, injectability and cytotoxicity of PMMA/PDMS mixture were investigated. And, biomechanical test using osteoporotic porcine bone was also conducted.
Temperature sensitive polymer hydrogel patch with controllable release system for skin tissue regeneration
1Department of Dentistry, Graduate School, Kyung Hee University, 26 Kyugheedae–ro, Dongdaemum–gu, Seoul 02447, Republic of Korea, 2Department of Dental, Materials, School of Dentistry, Kyung Hee University, 26 Kyugheedae–ro, Dongdaemum–gu, Seoul 02447, Republic of Korea, 3Biofriends, 23 Kyugheedae–ro, Dongdaemum–gu, Seoul 02447, Republic of Korea, 4Department of Dental, Education, School of Dentistry, Kyung Hee University, 26 Kyugheedae–ro, Dongdaemum–gu, Seoul 02447, Republic of Korea
Damaged skin caused by injury or illness can significant complications and disability. Therefore, it is essential to prevent infection and regenerate wounds through rapid regeneration. In an effort to restore damaged skin tissue, a lot of research has been conducted in tissue engineering fields. Among these, wound dressing foam is commonly used at initial treatment of skin defect via rapidly drug releasement. However, it has side effect of causing re‐damage to injury sites during detachment due to adhesive bond contained in the dressing foam. To overcome these drawbacks, we aimed to prepared biomedical patch using hydrogel which has its inherent capacity of biocompatibility, mechanical properties, and drug delivery. In this study, we were fabricated N‐isopropylacrylamide‐Polyvinyl alcohol (N‐P hydrogel) hydrogel with N‐isopropylacrylamide (NIPAAm), most used temperature sensitive polymer, which can enhance porosity of hydrogel and drug releasement to skin at body temperature. In addition, Polyvinyl alcohol (PVA) was added to rapid drug release. In this result, N‐P hydrogel had increased swelling ratio, porosity, and degradability unlike decreased mechanical strength as PVA was increased. Additionally, PVA added hydrogel showed increased drug release profile than NIPAAm hydrogel only. Furthermore, it was found that all hydrogels showed no significant cytotoxic effects up to 48 h. In conclusion, N‐P hydrogel show rapid and sustained drug release behavior in controlled manner through concentration of PVA. Our findings suggest that N‐P hydrogel can be a useful strategy as biomedical hydrogel patch owing to non‐cytotoxicity and controllable drug releasing behavior.
Vascular perfusion enabled by microchannel network in ischemic disease model using hydrogel with three‐dimensional microtubular structures
1Chung‐Ang University, 2Yonsei University College of Medicine, 3Sookmyung Women's University
While angiogenesis is the key factor to treat ischemic disease, angiogenesis induction such as local treatment with pro‐angiogenic molecules has critical side effects including inflammatory coupling, tumorous vascular activation, and off‐target circulation. Here, a structural angiogenic induction is applied to physically engineer three‐dimensional channel networks in ischemic region. Microchannel networks are generated in a gelatin hydrogel to overcome the diffusion limit of nutrients and oxygen three‐ dimensionally. Hydrogel implantation in mouse and porcine models of hindlimb ischemia rescues severely damaged tissues by the ingrowth of neighboring host vessels with microchannel perfusion, mediated by size‐specific regenerative macrophage polarization.
Tailoring the bioactivity of a cell‐derived extracellular matrix (ECM)‐ based material to exhibit superior pro‐angiogenic and osteogenic properties
1Institute for Tissue Engineering and Regenerative Medicine, The Chinese University of Hong Kong
A bone defect is a serious condition that often results in limited recovery due to defect size, co‐ morbidities, and limited grafting sources. Although the therapeutic potential of mesenchymal stem cells (MSCs) for bone healing has been demonstrated, MSC‐based therapy still faces various challenges such as inefficient cell engraftment and limited survival. MSCs are also excellent producers of extracellular matrix (ECM), a native acellular network that provides structural support, as well as preserves, stabilizes and presents bioactive factors. We hypothesized that ECM derived from osteogenically induced MSCs can promote osteogenesis and angiogenesis and thus serve as a potential biologic to promote bone regeneration.
An ECM‐based biomaterial was synthesized based on the co‐precipitation of dextran sulfate (DxS) and ECM during culture of osteogenically induced MSCs, which resulted in an enhanced ECM deposition and an enrichment of osteogenic factors within the decellularized material. The proliferation and osteogenic differentiation of reseeded naïve MSCs were strongly enhanced especially on this material. Moreover, the hybrid material also promoted endothelial cell proliferation and sprouting most strongly, indicating it to exhibit superior pro‐angiogenic properties. Experiments involving the disruption of ECM topography suggested that the superior bioactivity of the hybrid material might be partially mediated by the ECM micro‐ and nanostructure, which was preserved during material processing.
The strategy of DxS‐driven co‐precipitation and enrichment of bioactive factors results in the production of ECM‐based materials with superior pro‐angiogenic and osteogenic properties. Future work will focus on investigating the therapeutic potential of this biologic in a bone defect model.
Mesenchymal stromal cell exosomes modulate macrophage activities to promote joint repair in osteoarthritis
1Faculty of Dentistry, National University of Singapore, Singapore, 2Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, 3Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 4Department of
Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 5Tissue Engineering Program, Life Sciences Institute, National University of Singapore,
Singapore, 6Integrative Sciences and Engineering Program, NUS Graduate School, National University of Singapore, Singapore
Despite several reports of mesenchymal stromal/stem cell (MSC) exosomes for joint repair in osteoarthritis (OA), the immunomodulatory activities of MSC exosomes in alleviating OA remain to be elucidated. Here, we investigated the immunomodulatory activities of MSC exosomes in a rat model of temporomandibular joint osteoarthritis (TMJOA). Forty‐seven rats were randomly assigned to OA+Exo, OA+PBS, sham, and naïve groups. Following OA induction, OA+Exo rats received three intra‐articular injections of exosomes (1.3 × 1010 particles/injection), whereas OA+PBS rats received equivalent phosphate‐buffered saline (PBS) injections. Sham rats received needle pricks while naïve rats were age‐ matched control. At 1 week, OA+Exo rats had preferential synovial infiltration of anti‐inflammatory CD206+ M2 over pro‐inflammatory CD86+ M1 macrophages, and elevated gene expression of M2 over M1 associated markers in the synovium that culminated in an improved synovium inflammation score. Compared to OA+PBS rats, OA+Exo rats showed early suppression of systemic inflammation, with significantly reduced circulating SSCHiCD43HiRP1Hi neutrophils and CD43LoHis48Hi classical monocytes, accompanied by suppression of pro‐inflammatory plasma cytokines such as IL6, IL12, IL17A, IFNγ and TNFα. By 8 weeks, OA+Exo rats showed pain recovery with increased head withdrawal threshold, enhanced cartilage and subchondral bone restoration with improved Mankin score and augmented bone structural parameters compared to OA+PBS rats, and were comparable to that of sham and naïve rats. Using primary rat macrophages, we could attribute the preferential M2 over M1 macrophage infiltration observed during TMJ repair to the enhanced M2 over M1 macrophage polarization mediated by MSC exosomes.
Enhancement of muscle tissue regeneration supplemented with bioactive components
1Department of Biomechatronic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan University (SKKU), 16419, Suwon, South Korea., 2Sungkyunkwan university
Insufficient oxygen delivery to the tissue can lead to hypoxia and tissue death. In tissue engineering, a three‐dimensional (3D) scaffold ladened with cells can have a similar effect at the core, preventing cell proliferation. To overcome this issue, we have utilized a photosynthetic cyanobacterium (Synechococcus elongatus) to produce oxygen inside a 3D bioconstruct ladened with C2C12 myoblasts. Various concentration of cyanobacteria has been cultured with C2C12 to optimize for the highest cell viability and growth. As result, the C2C12 growth was significantly higher for the co‐cultured groups compared to the C2C12 culture alone. In addition, to determine that photosynthesis of the cyanobacteria is the cause of the higher cell proliferation, we have examined co‐culture with and without a light source. Furthermore, to induce myoblast alignment, we have utilized shear‐induced alignment at the nozzle. The cellular morphology has been analyzed with DAPI/Phalloidin staining, and the degree of myogenic differentiation has been analyzed with DAPI/MHC (Myosin Heavy Chain) staining, as well as various myogenic related gene expressions (Myog, Myod1, Myh2, TnT). Based on the results, the inclusion of Cyanobacteria to the aligned C2C12 myoblast can promote efficient multinucleated myotubes at the core and surface of the muscle construct.
The bone regenerative potential of RANTES/CCL5 in the calvarial defects of rat
11.Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, 2 Department of Periodontology, Oral science research center, College of Dentistry, Yonsei University, 2Department of Periodontology, Research Institute for Periodontal Regeneration,
College of Dentistry, Yonsei University
The objective of the present study is to compare the bone regenerative potential of chemokine ligand 5 (RANTES/CCL5) soaked to the collagen matrix according to the various concentration in the calvarial defects of rats. A total of 53 rats were prepared for the experiment. Circular defects of 6 mm in diameter were made using trephine bur in calvarium of the rat. The defects were randomly allocated to the five groups: (i) negative control (no grafting) group; (ii) positive control (collagen matrix only) group; (iii) low‐CCL5 (0.1μg/ml) group; (iv) moderate‐CCL5 (1μg/ml) group; and (v) high‐CCL5 (10μg/ml) group. The samples were collected at 2 weeks and 8 weeks postoperative. Histometric analysis was performed to measure the % of newly bone formation (%NB) within each defect. Kruskal‐Wallis test was conducted to determine the differences between the groups and Mann‐Whitney test between each two groups. There were no adverse reactions in any of the specimens. Histomorphometry at 2 weeks revealed that the low‐ CCL5 (18.69 ± 5.04%) and moderate‐CCL5 (21.63 ± 4.86%) groups demonstrated significantly higher
%NB than the negative control group (8.33 ± 3.35%; p < 0.005), whereas the positive control and high‐ CCL5 groups did not show significant difference compared to the negative control group. At 8 weeks, however, there were no statistical difference in %NB among the five groups. It was found that CCL5 showed a higher regenerative potential in the early healing stage when it was soaked to the collagen matrix with the concentation of 0.1μg/ml and 1μg/ml.
Aligned alginate based cell‐laden nanofibrous produced by cell electrospinning for corneal stromal regeneration
1Sungkyunkwan University
Recent studies reflect the importance of using natural biopolymers as three‐dimensional structure that has always been of particular interest to regenerate/reconstruct damaged tissues. It is noteworthy that mimicking tissue‐like structures can play an important role to induce the physico‐chemical and biological properties. In accordance to corneal stromal tissue, it is composed of aligned collagen fibers. To mimic the biophysical cue, cell‐electrospinning (CE) showed excellent potential to produce arranged cell‐laden fibers. In this study, we fabricated corneal stromal layer by a CE‐process to achieve not only homogeneous cell distribution with a high cell viability, but also highly aligned cells, which are guided by aligned alginate fibers. Briefly, the bioink for CE process was composed of poly (ethylene oxide) (PEO) and alginate. To fabricate homogenously aligned fibers, various parameters of the process were appropriately selected. The processing condition was considered to produce electrospun fibers containing bovine corneal keratocyte cells (BCKCs) to ensure suitable condition and guidance for cell alignment and elongation. We successfully produced aligned cell laden microfiber composed of aligned nano‐scale fibers and micropores. The fabricated cell‐laden fibers revealed enhanced cell alignment compared to conventional structure. Based on these results, the cell‐laden fibers showed improved biological properties making those a new and potential strategy for further studies towards corneal stromal regeneration.
Fabrication of mechanically reinforced alginate/PCL scaffolds for hard tissue engineering
1Sungkyunkwan University
Alginate bioink has been regularly used for bioprinting process to fabricate 3D scaffolds for tissue engineering applications due to its excellent biocompatibility, rapid crosslinking ability, and low toxicity characteristics. However, due to low mechanical strength, the application of alginate in tissue engineering is limited. Herein, PCL particles have been incorporated into alginate hydrogel to overcome the mechanical limitations. Briefly, pulverized PCL powder was mixed with alginate hydrogel and printed on the cryogenic printing stage (‐20°C). Then, the printed structures were immersed in CaCl2 solution to induce ionic crosslinking. To enhance interactions between PCL powder and alginate hydrogel, the printed structures were thermally treated. Various parameters such as alginate concentration and PCL concentration, have been optimized. The biological evaluation including cell viability, proliferation, and osteogenic ability of the proposed structure was evaluated with MG‐63 osteoblast‐like cells. As result, the alginate/PCL hybrid structures showed significant mechanical and biological enhancement. Based on the results, the proposed scaffold shows promising potential in bone tissue engineering.
Novel concept of guided bone (regeneration GBR) using collagen membrane with rhBMP‐2
1Yonsei Univ
The purpose of this study was to suggest novel concept of guided bone regeneration(GBR) using collagen membrane with rhBMP‐2 by confirming that osteogenic ability is enhanced when collagen membrane with rhBMP‐2 is applied to CSD.
Four bone defects with a diameter of 8 mm were formed in the calvaria of New zealand white rabbits. The experimental groups were set as follows, and the control group was left empty.
Group 1(M): only collagen membrane, group 2(MB): collagen membrane+BCP, group 3(BMP1‐MB): collagen membrane with rhBMP‐2(1.0mg/ml)+BCP, group 4(BMP0.5‐MB): collagen membrane with rhBMP‐2(0.5mg/ml)+BCP. After 2, 4, and 8 weeks of healing, the rabbits were sacrificed for histological and histomorphological analysis. In the histomorphological analysis, BMP0.5‐MB showed statistically significantly higher new bone formation (32.56 ± 12.62%, p < 0.05). For each group, the osteogenic rate was highest in the order of C, M, MB, BMP1‐MB, and BMP0.5‐MB. The longer the healing period, the higher the bone formation rate(%). In histological analysis, continuous new bone was observed in the upper part of the bone defect in BMP1‐MB and BMP0.5‐MB, whereas no continuous forms were observed in the other groups.
In conclusion, the application of collagen membrane with rhBMP‐2 and BCP together when performing GBR procedures is more effective for new bone formation. A novel GBR method with collagen membrane with rhBMP‐2, which can rapidly form a natural bone wall in the early stage, is proposed to induce quantitatively and qualitatively excellent bone regeneration in CSD.
Therapeutic potential of multiple cycles collection of conditioned medium from different cell sources on wound healing model: In vitro study
1Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Cheras, Kuala Lumpur, 56000, Malaysia. , 2Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Cheras, Kuala Lumpur, 56000, Malaysia.
A biphasic construct for osteochondral complex using modified transwell model
1National University of Singapore
The generation of osteochondral tissue has been a challenge due to its hierarchical structure composed of articular cartilage and subchondral bone region. In this study, a dual microenvironmental biphasic construct was developed. The chondral layer was hydrogel composed of methacrylated chondroitin sulfate (CSMA) and gelatin methacryloyl (GelMA), and the subchondral region was porous silk fibroin (SF) scaffold prepared by salt leaching method. The insert membrane of traswell plate was firstly coated with a thin layer of polydimethylsiloxane (PDMS) to seal the pores and then punched to make a hole, through which the chondral layer and osteogenic layer could be connected. Such a design allowed separate medium for chondrogenic and osteogenic differentiation in the upper and lower chamber. Mesenchymal stem cells (MSCs) were encapsulated in the CSMA/GelMa hydrogel of the chondral layer and seeded on the SF scaffold in the osteogenic layer. MSCs differentiation in each layer will be investigated in terms of gene expression and histology.
Effects of (glycosaminoglycan GAG) content in donor cartilage extracellular matrix on the functional properties of osteochondral allograft evaluated by μCT non‐destructive analysis
1Department of Orthopedic Surgery, School of Medicine, Ajou University, 2Department of Molecular Science and Technology and Department of Orthopedic Surgery, School of Medicine, Ajou University, , 3Cell Therapy Center, Ajou Medical Center, 4Department of Molecular Science and Technology, Ajou University, 5Department of Biomedical Sciences, Graduate School of Ajou University
A transplantable pre‐vascularized tissue platform by using a multi‐ material microfluidic 3D bioprinting method
1POSTECH, Pohang, Gyeongbuk, 37666, Republic of Korea
3D bioprinting is promise technology in tissue regeneration. By controlling a combination of biomaterials and cells and positioning of each bioink, 3D bioprinting can recapitulate the complexity of organs. Additionally, research about decellularized extracellular matrix (dECM) is also actively conducted. Tissue‐specific dECM can provide target tissue‐friendly microenvironment. However, as each tissue requires different mechanical properties, a different printing strategy is required regarding the viscosity of target tissue‐specific dECM. This study presents a microfluidic 3D bioprinting technique to fabricate core‐shell strut structures. Due to alginate ionic crosslinking of hybrid bioink (a mixture of vascular‐ tissue‐derived decellularized extracellular matrix (VdECM) and alginate) at the shell, target tissue‐ specific dECM can be stably printed at the core regardless of viscosity by the same printing strategy. A pre‐vascularized shell, which is consist of a hybrid bioink with Human Umbilical Vein Endothelial Cells (HUVECs), can support neovascularization after transplantation under the subcutaneous site. In addition, we investigated the versatility of this platform by fabricating both pre‐vascularized cardiac patch and liver tissue having human induced pluripotent stem cell‐derived (hiPSC) cardiomyocytes and hepatocytes with each tissue‐specific dECM, respectively. The pre‐vascularized cardiac patch showed higher maturation of cardiomyocytes demonstrated by gene expression and electrophysiology, and pre‐vascularized liver tissue showed upregulated albumin secretion and urea production than each tissue without HUVECs. After transplantation, the pre‐vascularized tissues showed neovascularization and migration of HUVECs patch to host and did not show any severe immune reaction. This platform will provide versatile defected organ‐ specific transplantable pre‐vascularized tissue that can support neovascularization and suppress immune reaction.
Exosome‐encapsulating tissue‐adhesive patch for diabetic wound regeneration
1Department of Biotechnology, Yonsei University, 03722 Seodaemun‐gu, Seoul, Republic of Korea
Diabetes, one of the most prevalent health problems affecting approximately half a billion people worldwide, is well‐known to be associated with serious complications such as diabetic ulcer and limb amputation. Therefore, various effective therapies to accelerate diabetic wound regeneration have been developed. As one example, cell‐derived exosomes containing endogenous therapeutic molecules have been of great interest as a potential candidate for cell‐free therapy for tissue regeneration. This study proposes a tissue‐adhesive hyaluronic acid (HA) hydrogel scaffold to improve the stability and in vivo retention of exosomes. The HA hydrogel scaffold was biocompatible and supported sustained release of exosomes. Further in vitro analysis indicated that HA‐hydrogel scaffold containing exosomes promoted vascularization and cell growth. Finally, the therapeutic efficacy of the exosome‐incorporating HA hydrogel patch was confirmed to enhance wound healing and skin regeneration in skin wound model of diabetic mice.
This work was supported by the Korea Evaluation Institute of Industrial (Technology KEIT) grant funded by the Korea government (MSIT) (No. 20009125).
Fabrication of a cell‐aggregates loaded hepatic tissuevia cell‐printing system
1Sungkyunkwan University
In the human body, liver tissue plays crucial role in the xenobiotic metabolism and protein synthesis. In that sense, a biomedical scaffold should mimic the 3D architecture of a functional hepatic lobule to provide the liver‐specific functions. The hepatic lobule is composed of anatomically complex structure including densely linked hepatocytes (hepatic cordon) and vessels. Recently, various researchers reported about fabrication of hepatic tissues consisting of hepatocyte‐aggregates. However, most reports are limited to formation of 2D functional units, while only few studies have introduced the development of hepatic constructs in a 3D environment. Moreover, the vascularization for the 3D lobule constructs should be considered to improve hepatic functionalities and cellular microenvironments. In the present study, we have focused on the fabrication of vascularized 3D hepatic constructs consisting of hepatocyte‐cordons and vessels using hepatocyte‐ and endothelial cell‐laden bionks and 3D cell‐printing system. To obtain the hepatocyte‐aggregates loaded 3D constructs, we have applied the in situ stimulation during the cell‐ printing process, which can induce cellular movement including migration, aggregation, and alignment in the hydrogel. As result, the 3D vascularized hepatic constructs containing hepatocyte‐aggregates exhibited improved liver‐specific gene expressions and secretion of albumin compared to the conventionally printed hepatocyte‐laden construct.
Gelatin incorporation in VEGF‐loaded PVA‐Tyramine hydrogels to enhance cellular interaction and vascular infiltration
1University of Otago, 2UNSW Sydney
Stimulation of angiogenesis via the delivery of growth factors (GFs) like vascular endothelial growth factor (VEGF) is a promising strategy for the treatment of avascular necrosis. Tyraminated poly(vinyl alcohol) hydrogels (PVA‐Tyr), which covalently bind GFs, were proposed as a platform for the controlled delivery of therapeutic levels of VEGF to the necrotic areas. Nevertheless, PVA hydrophilicity and bioinertn nature limits its integration with host tissues. The aim of this study was to investigate the suitability of incorporating gelatin, an FDA‐approved, non‐immunogenic biomaterial with biological recognition sites, as a strategy to facilitate cell migration and blood vessels invasion of PVA‐ Tyr hydrogels.
We systematically evaluated the effect of incorporating progressively higher gelatin concentrations (0.01‐ 5wt%) on PVA‐Tyr hydrogels physico‐chemical properties and cell adhesion capability. Then, the biofunctionality of the released VEGF to promote vascularization was evaluated via chorioallantoic membrane (CAM) assay. Finally, VEGF‐loaded PVA‐Tyr hydrogels with or without gelatin were implanted in a subcutaneous mouse model.
The incorporation of 1% gelatin significantly enhanced endothelial cell attachment without compromising hydrogels physico‐chemical properties, degradation time and VEGF release profile. The CAM assay results showed that covalently binding VEGF to the PVA‐Tyr network does not hamper its native bioactivity. Furthermore, incorporating gelatin in the polymer network promoted neo‐angiogenesis. In vivo, 3D reconstruction of the vessel network and histological analyses highlighted higher vascular infiltration in the hydrogels where either gelatin or VEGF were included compared to PVA‐Tyr control. These results indicate the suitability of PVA‐Tyr/gelatin hydrogels as VEGF delivery tool to promote vascularization.
Microfluidic chip development for vascularized bone marrow niche
1Seoul National University, Korea National University of Transportation, 2Korea National University of Transportation
Bone marrow is essential for producing hematopoietic cells such as red and white blood cells and the formation of trabecular bone tissues. However, it is very difficult to study physiological and pathological processes related to bone marrow and its reaction to diverse therapeutic agents. The bone marrow is located deep inside the bone, making it challenging to observe physiological processes in vivo. In addition, the bone marrow has a highly complex system involving multiple cell types and processes, so it is difficult to mimic bone marrow microenvironment in 2D in vitro environment. Under these circumstances, niche‐on‐a‐chip can be an alternative approach to studying cellular behavior and function in bone marrow by mimicking the intrinsic fluidic microenvironment with high fidelity. Also, niche‐on‐a‐ chip presents a unique opportunity to recreate bone marrow‐related disease models that can be developed as a next‐generation platform for drug delivery systems. This study recapitulated the complex bone marrow fluidic microenvironment by controlling osteoclastogenesis, osteogenesis, and angiogenesis on a chip. This study introduces an appropriate biochip design considering the type of scaffold, the concentration of bone minerals such as whitlockite nanoparticles, and the pore size. The formation of bone marrow like 3D structures and blood vessels, and the induction of osteogenic markers demonstrated the successful development of niche‐on‐a‐chip.
Polycaprolactone/gelatin/polydeoxyribonucleotides nanofiber for wound healing application
1Pukyong National University
In the present study, we extracted polydeoxyribonucleotides (PDRN) from Patiria pectinifera and evaluated their cytotoxicity and wound healing effect by MTT assay, FDA/PI staining, Picro‐Sirius red staining, and western blot analysis. After then, we fabricated polycaprolactone (PCL)/gelatin (Gel)/PDRN nanofibrous membranes by an electrospinning technique for wound dressing application. The average fiber diameters of PCL, PCL/Gel, and PCL/Gel/PDRN nanofibrous membrane were 0.58 ± 0.20, 0.44 ± 0.15, and 0.51 ± 0.18 μm, respectively. Based on universal tensile machine (UTM) analysis, the PCL/Gel and PCL/Gel/PDRN nanofibrous membranes significantly increased tensile properties compared with the PCL nanofibrous membrane. Additionally, the biocompatibility of fabricated nanofibrous membranes was examined using MTT assay and FDA/PI staining on human dermal fibroblast (HDF) and human keratinocytes (HaCaT). The fabricated fibrous membranes show proper biocompatibility. In vivo experiments, we confirmed that the PCL/Gel/PDRN nanofibrous membrane accelerated the initial wound healing process by full‐thickness skin defect model and histological analysis. Based on these results, the PCL/Gel/PDRN nanofibrous membrane can be used as a candidate biomaterial for wound dressing applications and skin tissue engineering.
Triple cross‐linked methacrylate kappa‐carrageenan/poly(vinyl alcohol)/chitooligosaccharide wound dressing hydrogel for wound healing application
1Pukyong national university, 2Korea Conformity Laboratories, 3Korea Institute of Ocean Science & Technology, 4National Marine Biodiversity Institute of Korea
A variety of physical and biological features of hydrogel‐based wound dressings stimulate early tissue regeneration and wound healing. A poly (vinyl alcohol) (PVA)/methacrylate kappa‐carrageenan (‐CaMA) composite hydrogel encapsulated with chitooligosaccharide (COS) was prepared in a cassette using repeated freeze/thaw cycles, photo‐crosslinking, and chemical crosslinking to create an effective and mechanically robust wound dressing. Following that, the proposed triple‐crosslinked hydrogel's chemical, physical, mechanical, in vitro biocompatibility, and antimicrobial properties were investigated. An excisional wound‐healing mouse model was used for in vivo examination of full‐thickness wound‐healing properties, as well as histomorphological evaluation of sectioned tissue samples. The PVA/‐CaMA/COS (P‐CaC) hydrogel had a consistently thick, porous three‐dimensional architecture with homogeneously distributed pores, a high fluid absorption and retention capacity without compromising mechanical stability, and good in vitro biocompatibility, according to the findings. The wounds dressed with the proposed P‐CaC hydrogel were entirely healed by day 14, according to macroscopic photographs from the full‐thickness skin wound model, while histomorphological data confirmed full re‐epithelization and rapid skin‐tissue remodeling. As a result of our research, the composite P‐CaC hydrogel has a high potential for usage as a wound dressing.
Topographical regulation for local bone regeneration in type 1 diabetes mellitus: In‐vivo
1Department of Dental Biomaterials, School of Dentistry, Kyungpook National University, 2Department of Pharmacology, School of Dentistry, Kyungpook National University
Objectives
Diabetes mellitus (DM) has been associated with oxidative stress‐related disease and the complication of ROS production, which affects macrophage activities for osteoimmunomodulation in musculoskeletal system. Based on topographic strategies to promote proliferation and differentiation of mesenchymal cells, this study investigated that characterized scaffold topographies showed to regulate macrophage polarizations for bone regeneration at critical defects of calvaria in DM rats.
Methods
A single streptozotocin (STZ) was intraperitoneally injected to induce DM and both body weights and blood glucose levels were measured for 2 weeks in Sprague‐Dawley rats. Mesoporous scaffolds with two characterized surfaces (rough and smooth surfaces; Rs and Ss) were created using poly‐ɛ‐ caprolactone solutions and salt‐leaching technique was used for macropores. Two types of scaffolds were implanted into critical defects on calvaria. After 3 and 6 weeks, all groups were analyzed using histomorphometry, immunohistochemistry, and micro‐CT.
Results
In 2 weeks, DM rats showed significantly higher ROS, blood glucose levels, and lower body‐weights after STZ‐injection than the healthy. At 3‐ and 6‐weeks after scaffold transplantations, Rs group showed significantly higher expression levels of anti‐inflammatory phenotype (M2) of macrophage using IL‐10 and CD163. Although ALP had no expressions, Ss group had higher OCN expression than Rs. Based on the quantification of bone formation by histomorphometry and micro‐CT, Rs group had statistically higher bone parameters.
Conclusion
Typical topographies could promote macrophage polarization for anti‐inflammatory phenotype and enhance bone regeneration in T1DM. Therefore, surface characterization could be the promising strategy for bone regeneration to regulate immunological interaction, topography‐macrophage.
The effect of nanohydroxyapatite incorporated with micro RNA 21 in regulating osteogenesis
1Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, 2University Kebangsaan Malaysia
Anti‐senescence ion‐delivering nanocarrier for recovering therapeutic properties of long‐term‐cultured human adipose‐derived stem cells
1Sungkyunkwan University
Human adipose‐derived stem cells (hADSCs) have been used in various fields of tissue engineering because of their promising therapeutic efficacy. However, the stemness of hADSCs cannot be maintained for long durations, and their therapeutic cellular functions, such as paracrine factor secretion decrease during long‐term cell culture. To facilitate the use of long‐term‐cultured hADSCs (L‐ADSCs), we designed a novel therapeutic anti‐senescence ion‐delivering nanocarrier (AIN) that is capable of recovering the therapeutic properties of L‐ADSCs. In the present study, we introduced a low‐pH‐ responsive ion nanocarrier capable of delivering transition metal ions that can enhance angiogenic paracrine factor secretion from L‐ADSCs. The AINs were delivered to L‐ADSCs in an intracellular manner through endocytosis. Low pH conditions within the endosomes induced the release of transition metal ions (Fe) into the L‐ADSCs that in turn caused a mild elevation in the levels of reactive oxygen species (ROS). This mild elevation in ROS levels induced a downregulation of senescence‐related gene expression and an upregulation of stemness‐related gene expression. The angiogenic paracrine factor secretion from L‐ADSCs was significantly enhanced, and this was evidenced by the observed therapeutic efficacy in response to treatment of a wound‐closing mouse model with conditioned medium obtained from AIN‐treated L‐ADSCs that was similar to that observed in response to treatment with short‐term‐ cultured adipose‐derived stem cells. This study suggests a novel method and strategy for cell‐based tissue regeneration that can overcome the limitations of the low stemness and therapeutic efficacy of stem cells that occurs during long‐term cell culture.
Mesenchymal stem cell and hydrogel treatment of oral ulcer
1Dankook institute of medicine and optics, Dankook university, 2Interdisciplinary Program for Medical Laser, College of Medicine, Dankook University, 3Department of Otorhinolaryngolog‐Head and Neck Surgery, Dankook University College of Medicine
The study investigated the effects of tonsil‐derived mesenchymal stem cells embedded in thermo‐ responsive injectable hydrogel to treat a chemically‐induced oral ulcer in vivo. The thermo‐responsive hydrogel used was biocompatible with anti‐inflammatory properties. Mesenchymal stem cells have been known to possess features for fast wound healing. The wound healing effects of treatment using stem cell and hydrogel alone and in combination was determined. Animal models of oral ulcerations were induced by acetic acid injection in buccal mucosa after anesthesia. Oral ulcers were confirmed 3 days after induction. Each treatment was performed once after confirmation of the ulcer in the animal model. Changes in the dimensions of the ulcers after 1, 3, and 7 days of treatment were determined. Wound healing and inflammatory response were evaluated by histological and immunohistochemical analysis. Significant dimensional changes in the size of the ulcers were observed after treatment. The hydrogel allowed for longer retention of the delivered stem cells in the wound area with no observable inflammatory response. The combination appears to synergize early wound healing of the ulcers based on the histological data. This preliminary study stands as proof of concept regarding the potential use of MSCs, hydrogel, and combination therapy for oral ulcers that have not been previously investigated. Each treatment and combination therapy enhances wound healing. Further experimentation will be conducted to expand the understanding of how each treatment affects the healing mechanism of ulcers and combination therapy.
Salivary gland stem cell‐derived exosomes produced by a Wnt‐loaded microwell culture accelerates the recovery from salivary gland dysfunction in murine salivary gland damage models
1Department of Otorhinolaryngology, Gangnam Severance Hospital, Yonsei University College of Medicine, 2Department of Chemical and Biomolecular Engineering, Yonsei University
Purpose: This study aims to investigate the regenerative effects of salivary gland stem cell‐derived exosomes produced in a 3D microwell scaffold with sustained Wnt release on salivary gland dysfunction in salivary duct obstruction‐induced salivary gland damage models of mice.
Methods: Wnt3a protein (WNT)‐loaded PLGA electrospun nanofiber was fabricated to enable sustained WNT release over seven days. This WNT‐loaded nanofiber was incorporated with a PCL microwell scaffold by using a custom‐made insert. Human salivary gland‐derived stem cells (hSGSCs) were cultured on either 2D plastic dishes or 3D bare PLGA nanofiber‐PCL microwells (Mock‐Microwell) and WNT‐loaded PLGA nanofiber‐PCL microwells (WNT‐Microwell). We retrogradely infused exosomes isolated by ultracentrifugation from the culture media into the salivary ducts of mice. We investigated the regenerative potentials of hSGSC‐derived exosomes on the recovery from the salivary gland dysfunction in murine damage models.
Results: hSGSCs cultured on both microwell scaffolds formed 3D spheroids, spheroids on WNT‐ Microwell showed significantly higher stem cell‐associated gene and protein expression and produced more abundant exosomes with enhanced paracrine activity compared to those on PLGA‐Microwell. The mice treated with exosomes from SGSCs spheroids in WNT‐Microwell (exosomeWNT) showed significant improvement in gland damage compared to those isolated from SGSCs spheroids in Mock‐ Microwell (exosomeMock) and 2D plastic culture (exosome2D).
Conclusion: We manufactured a sustained WNT‐releasing microwell scaffold via electrospinning and photolithography. The resultant scaffold culture system facilitates hSGSCs spheroids formation and exosome production. The exosome isolated from hSGSC spheroids in WNT‐Microwell showed enhanced regenerative potential in obstruction‐induced salivary damage mice models.
Secretome of human fetal cartilage progenitor cells as potential treatment agent for testosterone‐induced hair loss
1Cell Therapy Center, Ajou Medical Center, Suwon, 2Department of Molecular Science and Technology, Ajou University, Suwon, 3Department of Orthopedic Surgery, School of Medicine, Ajou University, Suwon, Korea, 4Department of Biomedical Sciences, Inha University College of Medicine, Incheon
Matrilin3/TGFβ3 gelatin microparticles promote chondrogenesis, prevent hypertrophy, and induce paracrine release in MSC spheroid for disc regeneration
1Dongguk University, 2Chung Ang University, 3CHA University
Degenerative disc disease (DDD) is the major cause of severe lower back pain and disability in the aging population worldwide. Among the current treatments for this disease include cell based‐therapies such as the injection of both disc‐ and non‐disc‐derived chondrocytes. These strategies, indeed, have shown significant improvements in the patients' condition. However, further development of these therapies is required to not only provide healthy chondrocytes but also to promote regeneration of the defective cells in the injury site. Here, we report that the incorporation of gelatin microparticles conjugated with transforming growth factor‐beta 3 and matrilin 3 promoted chondrogenic differentiation of adipose‐ derived mesenchymal stem cell spheroids while preventing hypertrophy and terminal differentiation of cells. ASC‐spheroids bearing the composite microparticles showed increased expression of several chondrogenesis markers such as SOX9, ACAN, and COL2A as indicated by quantitative PCR, western blot, and immunostaining analyses. Interestingly, these spheroids also induced the release of chondrogenic cytokines such as TGF‐β proteins that promoted the regeneration of degenerative chondrocytes in vitro. Finally, injections of these composite spheroids in a rat model of intervertebral disc disease promoted the restoration of the chondrogenic properties of the cells, thereby allowing regeneration of the chondrogenic tissue in vivo. This study suggests that the combinatorial use of biofunctionalized microparticles and 3D cell culture system is an effective and efficient way of inducing directed differentiation of cells that can be utilized for various clinical applications.
Alginate patch containing extracellular matrix effectively delivers mesenchymal stem cell‐derived secretomes for advanced skin wound healing
1Korea Institute of Science and Technology
Human mesenchymal stem cell (hMSC) secretomes contain various therapeutic factors, such as growth factors (GFs), cytokines and other extracellular molecules. Extracellular matrix (ECM) is a complex of diverse macromolecules and it has many functions, including GFs reserve. In this study, we have developed a novel alginate patch, where it incorporates hMSC secretomes and cell‐derived ECM together. The secretomes were obtained from concentrated conditioned media (CCM) of hMSC and ECM was obtained from in vitro cultured human lung fibroblast via decellularization. Alginate hydrogels were constructed with or without ECM and rehydrated with CCM or serum‐free media (SFM) after air‐dried. We prepared four different groups: alginate/SFM (AS), alginate/ECM/SFM (AES), alginate/CCM (AC), and alginate/ECM/CCM (AEC). The content of GFs and cytokines profiles in the CCMs was determined by proteome microarray. BCA assay and ELISA showed that AEC could hold much larger amount of secretomes. We confirmed faster wound closure and increased collagen synthesis of human dermal fibroblast (hDFB) in vitro when treated with AEC patch. Moreover, murine full‐thickness wounds were administered with those alginate patches. Evaluations were carried out through wound closure rate, H&E, immunofluorescence and herovici staining, respectively. On day 7, AEC patch showed significantly improved re‐epithelization, neovascularization and cellular recruitment. We also noticed at 14 days, significantly promoted mature vessel formation and increased mature collagen deposition, along with normalized epidermis thickness when treated with AEC patch. Our study demonstrates that AEC patch can hold and deliver secretomes effectively and lead to enhanced skin wound healing by their therapeutic effect.
Thermosensitive copolymer coated and redox‐induced dissolvable microsphere for efficient cell harvesting during 3D cell culturing
1National Taiwan University of Science and Technology
The use of microspheres for culturing adherent cells has been proven as an important method, allowing for obtaining adequate number of cells in limited space and volume of medium for the intended cell‐based medical applications. However, the use of proteolytic enzymes for cell harvesting from the microsphere resulted in cell damage and loss of functionality which could affect subsequent cell‐based applications. Therefore, nonenzymatic cell detachment using thermosensitive polymer matrix is necessary for maintaining cell quality after harvesting. In this study, first, we prepared thermosensitive PNIPAm‐co‐ AAc‐b‐PS and PNIPAm‐co‐AAm‐b‐PS copolymers and low critical solution temperature (LCST) was tuned near to body temperature. Then, spin coated polymer films were prepared for cell adhesion and thermal‐induced cell detachment. Furthermore, the polymer was used to coat cytodex 3 for 3D cell culturing and interestingly enhanced cell detachment with preserved potential of recovery was observed at a temperature of below LCST. Secondly, we developed a disulfide crosslinked gelatin‐based microsphere that disintegrated upon exposure to redox agents. The microsphere was then coated with PNIPAm‐ALA copolymer, resulting in a positively charged thermosensitive surface and improved cell attachment. The addition of GSH, DTT and L‐cysteine resulted in further cleavage of the disulfide bond in the microsphere and subsequent dissolution of the microsphere for complete cell detachment. Therefore, surface modification of dissolvable microcarriers with dissolvable thermosensitive copolymer was worthwhile for non‐enzymatic cell detachment and has the potential to be used for cell expansion and harvesting adequate live cells of high quality and functionality for tissue engineering or cell therapy.
Engineered silk protein‐based core‐shell electrospun immunomodulatory fibrous scaffold for tissue regeneration with angiogenesis
1CSIR ‐ Central Leather Research Institute, Academy of Scientific and Innovative Research (AcSIR), Ghaziabad‐201002, India, 2CSIR ‐ Central Leather Research Institute, Chennai, India
Diabetic wound healing remains challenging due to persistent inflammation, increased matrix destruction, and improper activation of soluble components. Fabrication of tissue‐engineered scaffold that simultaneously suppress chronic inflammation and achieve highly ordered healing has gained attention for treating chronic diabetic wound. In this study, a multifunctional fibrous scaffold has been developed via core‐shell electrospinning of synthetic polycaprolactone (PCL) as core and Dextran‐Silk as shell. Coaxial electrospun fibers with uniform morphology of diameter 800nm ±300nm, with core (285 nm) were developed, showing stability for four weeks. Silk protein in shell were incorporated with a catechol group containing Dihydroxy phenylalanine (DOPA) via genetic code expansion by residue‐specific incorporation to enhance the vasculature. The alpha‐helical structure of silk and its functional groups were evaluated by CD and FT‐IR spectroscopy. Dual drugs, antioxidant eugenol and anti‐inflammatory crisaborole were loaded in the core and shell respectively. Drug release kinetics were monitored and pH
5.5 responsive release of crisaborole was confirmed by NMR analysis. The water contact angle and mechanical property characterization revealed hydrophilicity and tensile strength (1 ± 0.2MPa) of fiber respectively, which favors cellular interaction of matrix. The developed core‐shell scaffold enhanced adhesion, migration and angiogenesis in vitro, evaluated in fibroblast (NIH 3T3) and endothelial (EA.hy926) cells. Further, the scaffold demonstrated anti‐inflammatory activity by reduced proinflammatory cytokines production in LPS‐induced monocyte (THP‐1) cell lines. Reprograming macrophage status, considered a promising strategy to combat inflammatory condition in wounds was adopted in this study. The dual drug‐loaded multi‐faceted electrospun matrices could emerge as potential candidate to treat chronic diabetic wounds.
The effect of macromolecular crowding on decellularized graft mediated mesenchymal stromal cell delivery for treatment of wounds
1Science Foundation Ireland (SFI) Centre for Research in Medical Devices (CURAM), Biomedical Sciences Building, National University of Ireland Galway (NUI Galway), Galway, Ireland, 2Regenerative Medicine Institute (REMEDI), Biomedical Sciences Building, National University of Ireland Galway (NUI Galway), Galway, Ireland, 3Orbsen Therapeutics Ltd, IDA Business Park, Dangan, Galway,
Ireland, 4Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Charles Institute of Dermatology, Conway Institute of Biomolecular & Biomedical Research and School of Mechanical & Materials Engineering, University College Dublin (UCD), Dublin, Ireland
Decellularized grafts (DGs) retain the native extracellular matrix (ECM) composition and structure of the tissues from which they have derived, providing a favourable microenvironment for cell migration and proliferation. As mesenchymal stromal cell (MSC) delivery system, unfortunately, DGs still do not achieve prolonged transplanted cell localization and viability at the wound bed, imposing the need for further development of either their processing or their cell culture approach. Herein, we ventured to assess whether macromolecular crowding (MMC), a biophysical phenomenon that enhances and accelerates ECM deposition in culture, can enhance the therapeutic potential of MSC‐DGs constructs.
To validate this, human umbilical cord derived MSCs (hUC‐MSCs) were cultured on the basement membrane (BM) or connective tissue (CT) side of XenoMEM™ membrane, a decellularized porcine peritoneal DG for three days without and with MMC. Histological analysis showed that the structure of BM side was dense with small pore size and the CT side was porous with large pore size. When hUC‐ MSCs were seeded on the BM side, they were aggregated on the surface and when they were seeded on the CT side, they exhibited dispersed distribution. LIVE/DEAD™ analysis revealed that neither XenoMEM™ nor MMC affected cell morphology and viability. Cell metabolic activity and ECM deposition analysis are ongoing. Current data indicate that XenoMEM™ is suitable DG for hUC‐MSC attachment and growth. Further analysis is needed to determine whether MMC will result in augmented therapeutic potential of this MSC‐XenoMEM™ construct.
Novel implantable, wireless electricity auto‐generating patch accelerates the wound healing process by modulating mechanosensitive ion channels
1Institute of Tissue Regeneration Engineering (ITREN), Dankook University, South Korea; Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine,
Dankook University, South Korea, 2Institute of Tissue Regeneration Engineering (ITREN), Dankook
University, South Korea; Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, South Korea; College of Natural Science.
Dankook University, Cheonan, 31116, Republic of Korea, 3Institute of Tissue Regeneration Engineering (ITREN), Dankook University, South Korea; Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, South Korea; College of
Natural Science. Dankook University, Cheonan, 31116, Republic of Korea; Department of Biomaterials Science, School of Dentistry, Dankook University, South Korea; UCL Eastman‐Korea Dental Medicine Innovation Centre, Dankook University, South Korea
Therapeutic nanoglass paste as a drug‐free platform for the regeneration of bacteria‐infected hard tissues
1Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan, 31116, Republic of Korea, Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan, 31116, Republic of Korea, Department of Biomaterials Science, College of Dentistry, Dankook University, Cheonan, 31116, Republic of Korea,
UCL Eastman‐Korea Dental Medicine Innovation Centre, Dankook University, Cheonan, 31116, Republic of Korea, Cell & Matter Institute, Dankook University, Cheonan, 31116, South Korea, 2Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan, 31116, Republic of Korea,
Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan, 31116, Republic of Korea, 3Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan, 31116, Republic of Korea , Department of
Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine,
Dankook University, Cheonan, 31116, Republic of Korea, 4Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan, 31116, Republic of Korea, Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan, 31116, Republic of Korea, UCL Eastman‐Korea Dental Medicine Innovation Centre, Dankook University, Cheonan, 31116, Republic of Korea
Cobalt doped silica microcarrier with action of promoting angiogenesis and bactericidal potential through dual‐ion delivery
11 Institute of Tissue Regeneration Engineering (ITREN), Dankook University, South Korea. 2
Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, South Korea, 21 Institute of Tissue Regeneration Engineering (ITREN),
Dankook University, South Korea. 2 Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, South Korea. 3 Department of Biomaterials Science, School of Dentistry, Dankook University, South Korea. 4 UCL Eastman‐Korea
Dental Medicine Innovation Centre, Dankook University, South Korea
Floating electrode‐dielectric barrier discharge‐based plasma can accelerate skin regeneration in a full‐thickness skin defect mouse model
1Soonchunhyang Institute of Medi‐bio Science (SIMS), Soonchunhyang University, Republic of Korea, 2HK‐MnS Co. Ltd., Republic of Korea, 3Department of Plastic and Reconstructive Surgery, Soonchunhyang University Bucheon Hospital, Soonchunhyang University College of Medicine, Republic of Korea
The incidence of various wounds such as critical‐sized full‐thickness skin wounds, diabetic ulcers, and atopic dermatitis is rapidly increasing with the growth of an aging population. Although non‐thermal plasma‐induced active species have shown to promote hemostatic effects and wound healing processes, little is known about its action mechanisms in wound healing processes. Here, we aim to investigate the safety and efficacy of custom‐made non‐thermal plasma devices using a full‐thickness skin defect mouse model. Our initial findings revealed that our floating electrode‐dielectric barrier discharge (FE‐DBD)‐ based atmospheric pressure plasma could promote significantly faster wound healing in the plasma‐ treated experimental group, compared to un‐treated wounds (control group). Furthermore, we observed the increased thickness of dermis and the suppressed hyperplasia at the epidermis in plasma‐treated skin tissues, which are equivalent to the normal skin, and the inflammation‐related markers, including IL‐1β, IL‐6 and Cox2 genes, were significantly down‐regulated by the plasma treatment. Taken together, plasma treatment could accelerate the wound healing processes without noticeable side effects, whereas it could suppress the pro‐inflammatory genes, suggesting that our FE‐DBD‐based plasma can be a potential therapeutic option for treating various wounds.
Elucidating the role of cell surface free thiol groups in myogenic differentiation of skeletal muscle progenitor cells by mild reduction of cell surface
1Soonchunhyang Institute of Medi‐bio Science (SIMS)
Skeletal muscle consists of 40% of the weight of a healthy human body and severe muscle damage or muscular dystrophy has less shown to regenerate damaged myofibers through the proliferation and differentiation of skeletal muscle stem cells into functionally multinucleated myotubes. Here, we aim to elucidate the role of cell surface free thiol groups in myogenic differentiation of skeletal muscle progenitor cells through changes in the formation of focal adhesion (FA) complexes and its subsequent contribution to the fusion of mononucleated myoblasts into multinucleated myotubes. Our finding revealed that mild reduction of cell surface proteins using a tris(2‐carboxyethyl) phosphine hydrochloride (TCEP) could generate free thiol groups on cell surfaces and lead to the increased FA expression of skeletal muscle progenitor cells. Interestingly, cell surface thiol groups generated by TCEP could dynamically regulate myogenic differentiation of skeletal muscle progenitor cells. For examples, TCEP‐ treated cells, which were initially seeded with low cell density (∼40k cells/cm2), exhibited the inhibited myogenic differentiation potential by limiting the fusion events, whereas TCEP‐treated cells, which were initially seeded with relatively high cell density (∼70k cells/cm2), underwent robust myogenic differentiation, which were evident by myogenic marker expression such as myosin heavy chain and desmin, in both mRNA and protein levels. The results described in this study provide a proof‐of‐principle that cell surface free thiol groups can be a novel therapeutic target to treat devastating muscle wasting diseases.
The multiple deliveries of bioactive ions and growth factor with antibacterial/angiogenic and osteogenic/odontogenic capacity of nano‐ therapeutic particles for regeneration of degenerated/infected tissue by bacteria
1Dankook University, ITREN, 2UCL Eastman Dental Institute, England, 3National Research Centre, Cairo, Egypt
Therapeutic options are relatively limited in clinics to successfully repair infected/degenerated tissues. However, the prevalent treatment is the complete removal of the whole infected tissue, which leads to a loss of tissue functions. The dental pulp infection, one of the most common dental problems, was selected as a clinically relevant case to regenerate using a multifunctional nano‐therapeutic approach. For this, a mesoporous bioactive glass nano‐delivery system incorporating silicate, calcium, copper, and loading epidermal growth factor (EGF) was designed to provide antibacterial/pro‐angiogenic and osteo/odontogenic multiple therapeutic effects. The Cu‐BGn treatment to human umbilical vein endothelial cells (HUVEC) led to significant enhancement of the migration, tubule formation and expression of the angiogenic gene (e.g. vascular endothelial growth factor, VEGF). Furthermore, the EGF‐loaded Cu‐BGn (EGF@Cu‐BGn) showed pro‐angiogenic effects with antibacterial activity against
E. faecalis, a pathogen commonly involved in pulp infection. Of note, under the co‐culture condition of HUVEC with E. faecalis, the secretion of VEGF was up‐regulated. In addition, the osteo/odontogenic stimulation of the EGF@Cu‐BGn was proven with human dental pulp stem cells. The local administration of the EGF@Cu‐BGn in a rat molar tooth defect infected with E. faecalis revealed significant in vivo regenerative capacity, highlighting the nano‐therapeutic uses of the multifunctional nanoparticles for regenerating infected/ damaged hard tissues.
Effects of enamel matrix derivative on the cellular viability and differentiation potential of cell spheroids composed of gingiva‐derived stem cells
1Department of Periodontics, College of Medicine, The Catholic University of Korea
Enamel matrix derivative is an extract of enamel matrix which mainly contains amelogenins. It is reported to promote the formation of enamel and periodontal ligaments. This study aimed to evaluate the effects of enamel matrix derivative on the cellular viability, osteogenic differentiation and mineralization of spheroids made of human gingiva derived stem cells.
Spheroids were prepared using microwells and were cultured in enamel matrix derivative at dilutions of 0, 0.001, 0.01, 0.1 and 1. The morphological evaluation was done on Days 1, 3, 5, and 7. Determination of the qualitative cellular viability was performed using Live/Dead Kit assay on Days 3 and 7. The quantitative cellular viability was evaluated with Cell Counting Kit‐8 on Days 1, 3, 5, and 7. The Alkaline phosphatase activity assays were performed on Days 7 and 14. The Alizarin Red S staining was performed on Days 7 and 14. Real‐time polymerase chain reaction was done on Days 7 and 14 to analyze expression of the targeted mRNAs.
The applied stem cells produced well‐formed spheroids regardless of enamel matrix. The shape and their viability were maintained throughout the entire period. Most of the cells emitted green fluorescence during the cultured period. The addition of enamel matrix derivative did not show significant disparities (P > 0.05). Alkaline phosphatase activity and Alizarin Red S results provided insights into the effects of enamel matrix derivatives on osteogenic differentiation.
Based on findings, we conclude that enamel matrix derivative can be applied for the stem cell spheroids for regeneration.
Enhanced wound healing with decellularized amniotic membrane hydrogels by supercritical CO2 process
1Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea, 2Research Institute for Convergence Science, Seoul National University
The amniotic membrane (AM) which is the most inside membrane for protecting the fetus has excellent bioactive characteristics because of the various factors it has like abundant cytokines, growth factors, and anti‐ inflammatory and good angiogenic factors. In the wound healing process, the levels of transforming growth factor (TGF)‐β present a critical factor; they are elevated during the inflammation phase and remain high in chronic wounds. Interestingly, epidermal growth factors (EGF), one of the main components of an AM, make a fine‐tuning and good suppression of the TGF‐ β pathway. In this study, we fabricated bioactive hydrogels having low immunogenic and proangiogenic properties for wound healing via decellularization of the AM using supercritical fluid carbon dioxide (scCO2). First, we confirmed that decellularized AM hydrogels fabricated by the scCO2 process had low DNA contents, while collagen and the glycosaminoglycan (GAG) almost remained similar to the level of native AM. And, from the in vitro and in vivo experiments, we confirmed that angiogenesis and cell growth were enhanced onto dAM hydrogels by the scCO2 process. Also pro‐angiogenesis, tissue regeneration, and wound healing were enhanced in the dAM hydrogels treated group in the skin defect models. In conclusion, it is considered that the amniotic membrane‐based hydrogels have a high potential as therapeutic material for wound healing.
The effect of hFC‐MSCs on the induces bone formation by notch signaling
1Department of Orthopaedic Surgery, CHA Bundang Medical Center, CHA University, Gyeonggi‐do 13496, 2Fetal Stem Cell Research Center, CHA Advanced Research Institute, Gyeonggi‐do 13488, 3Department of Biomedical Science, CHA University, Gyeonggi‐do 13488
Fetal mesenchymal stem cells arise as a novel strategy with diverse advantages such as lower immunogenicity, higher potential for expansion, and also possessing a broader possibility to differentiate into various cell lineages, compared with adult stem cells. This study evaluated the ability of human fetal mesenchymal stem cells (hFC‐MSCs) in osteogenic differentiation and bone regeneration, comparing with adult bone marrow mesenchymal stem cells (BM‐MSCs) in vitro and in vivo. We isolated and characterized MSCs from human fetal calvaria. Time‐course research was conducted to assess cell proliferation, bone marker gene expression, alkaline phosphatase (ALP) activity, and mineralization. hFC‐ MSCs were implanted to the healthy rat with calvarial defects compared with adult BM‐MSCs implantation, evaluating the bone healing effect of hFC‐MSCs with micro‐CT evaluation and histology analysis. Additionally, RNA‐sequencing analysis and heatmap study were done to identify the different level of gene expression between hFC‐MSCs and BM‐MSCs. Among the genes related to osteoblast development showing a different gene expression between hFC‐MSCs and BM‐MSCs, HEY1 and HES1 showed significantly high expression levels which are the key target genes of Notch signaling pathway. We examined the Notch signaling marker gene expression by qRT‐PCR and western blot analysis in hFC‐ MSCs. we also used DAPT which inhibits Notch signaling pathway to confirm that Notch signaling is related to the osteogenic differentiation effect of hFC‐MSCs. This study shows for the first time that human fetal calvarial MSCs could be a great strategy for bone regeneration as a better alternative to adult derived stem cells.
Thiolated mesoporous silica nanoparticles for the treatment of oxidative stress‐associated osteoporosis
1Indian Institute of Technology Ropar, 2Department of chemistry, Indian Institute of Technology, Ropar
Osteoporosis (OP) is a metabolic bone disorder, characterized by the decreased bone mass, which leads to brittle bone and bone fractures. Conventional treatment for OP includes use of bisphosphonates, growth factors, and hormonal therapy, which are effective in stabilizing bone mass but fail to control the oxidative stress. Therefore, we have developed thiolated mesoporous silica nanoparticles (MSN‐SH) to exploit the intrinsic osteogenic potential of MSNs and impart antioxidant properties to it by thiolation to improve cell adhesion and facilitate proliferation of osteoblast cells, resulting in an increased bone mass. MSNs were fabricated by sol–gel method using TEOS as a precursor and surface functionalized with thiol (‐SH) groups using 3‐mercaptopropyl trimethoxysilane as a donor group to obtain MSN‐SH. The nanomaterials were characterized by FTIR, TGA, XRD, DLS, and BET analysis, and their antioxidant properties, cell viability, and cell differentiating potential was investigated. The nanomaterial exhibited 90% antioxidant activity, and cell viability assay in pre‐osteoblast (MC3T3‐E1) cells for 72 h demonstrated that MSN‐SH are cell proliferative in nature. Accelerated bone differentiation, evident from increased alkaline phosphatase (ALP) activity and calcium deposition, was observed in pre‐osteoblast cell line. MSN‐SH was able to neutralize free radicals and protect cells against ROS‐induced cell death. More than 90% cells were found viable in MTT assay using HUVEC cells. Thus, we have developed thiolated mesoporous silica nanoparticles with improved antioxidant properties and osteogenic activities for controlling the oxidative stress to prevent the disease progression and treatment of osteoporosis.
Wound healing effects of extremely low‐frequency electromagnetic fields through activation and differentiation of stem cells
1Department of Medical Biotechnology, Dongguk University
Electromagnetic fields (EMFs) have been widely used in the stimulation of wound healing and for relieving pain. EMFs affect cell activity, proliferation, and differentiation by affecting cell membranes or intracellular proteins, such as ion channels.
EMFs did not induce toxicity in the hair bulb (organoids HBO), and EMF promoted the expression of ALP, versican, β‐catenin, and several cytokines in HBOs. Our results demonstrated that EMF promoted hair shaft growth in HFs due to the effect of cytokines and adhesion molecules via the Wnt/β‐catenin pathway.
Also, EMF treatment can promote differentiation of human dental pulp stem cells(hDPSCs). The hDPSCs showed frequency‐dependent differences in protein and gene expression, and odontoblast‐related markers, particularly β‐catenin, p‐GSK‐3β, and p‐p38, were increased.
Additionally, in cerebral ischemia mice model, neural‐related proteins were upregulated in the EMF groups compared with the control and cell group. Upon conducting rotarod tests, the cell/EMF group exhibited significant differences in motor coordination at 13 days post‐treatment when compared with the control and stem‐cell‐treated group. And, the cell and cell/EMF group exhibited a significant reduction in the expression of MMP‐9, TNF‐α and IFN‐γ in the induced ischemic area compared with the control.
Acknowledgement: This study was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (HI19C0757). Also, this study was also supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF‐ 2019R1I1A2A01063305).
Dendritic cell‐derived nanovesicles for targeted delivery of immune checkpoint inhibitors to improve therapeutic efficacy and prevent side effects
1Seoul National University
Despite the anti‐cytotoxic T lymphocyte‐associated protein 4 antibody (αCTLA‐4)'s clinically demonstrated efficacy, the therapy's low response rate and immune‐related adverse events (irAEs) in cancer patients are major drawbacks. The non‐specific stimulation of T cells and the poor activation of tumor‐specific cytotoxic T lymphocytes (CTLs) are the main downsides of αCTLA‐4 treatment. To resolve these concerns, αCTLA‐4 was delivered using dendritic cell‐derived nanovesicles carrying tumor antigens (DCNV‐TAs), which only engage with tumor‐specific T cells, resulting in tumor‐specific CTL activation. In syngeneic tumor‐bearing mice, treatment with αCTLA‐4‐conjugated DCNV‐TAs dramatically suppressed tumor growth and reduced irAEs when compared to traditional αCTLA‐4 therapy. This study reveals that presenting αCTLA‐4 and tumor antigens in a spatiotemporal manner allows for targeted activation of tumor‐specific T cells and enhances αCTLA‐4's anticancer activity without causing systemic irAEs.
Human hair keratin gradient hydrogels for skin regeneration
1School of Materials Science and Engineering, Nanyang Technological University, Singapore, 2Institute for Health Technologies, Interdisciplinary Graduate Programme, Nanyang Technological University, Singapore, 3Environmental Chemistry and Materials Centre, Nanyang Environment and Water Research Institution, Singapore, 4Center for Nanotechnology and Nanotoxicology, Harvard T.H. Chan School of Public Health, Harvard University, United States
Gradient hydrogels have received attention due to the possibility to incorporate physical and chemical gradients to mimic those present in vivo with higher relevance. Human hair keratins (HHK) are an established biomaterial in forming various templates for biomedical applications essentially due to the presence of versatile functional groups and the cell adhesion motif LDV (leucine‐aspartic‐valine). By exploiting the high cysteine content in HHK and their affinity to silver ions, we have developed a novel gradient HHK hydrogel through a single‐step fabrication process. The gradient gelation is facilitated by the diffusion of silver ions into solubilized HHK through a thin membrane first formed upon a dropwise addition of HHK into the ion bath. This gradient was confirmed through Turbiscan analysis where transmission decreased as light travelled across the hydrogel. Also, rheological studies highlighted a gradient breakdown of the hydrogel where the storage modulus remained greater than the loss modulus at high strain despite an initial decrease. This gradient profile rendered the hydrogel with improved toughness. Moreover, scanning electron microscopy unveiled the presence of a dual microstructure within a single construct: a porous spongy layer and a non‐porous compact layer, which mimics the dermis and epidermis of native skin, respectively. Unconstrained by silver, a HHK gradient hydrogel could also be formed with other biologically relevant metal ions. Alongside HHK, gelatin methacryloyl was found suitable to form a composite gradient hydrogel, displaying potential for skin regeneration. Beyond the skin, this tunable HHK gradient hydrogel system is potentially tailorable towards other biomedical applications.
Challenges in mixing of multiple components in nanocomposite bioink for 3D bioprinting
1Department of Chemical and Biomolecular Engineering, Seoul National University of Science and
Technology, Seoul 01811, 21. Convergence Institute of Biomedical Engineering and Biomaterials, Seoul National University of Science and Technology, Seoul 01811/ 2. Department of Chemical and Biomolecular Engineering, Seoul National University of Science and Technology, Seoul 01811,
3Convergence Institute of Biomedical Engineering and Biomaterials, Seoul National University of Science and Technology, Seoul 01811, 41. Convergence Institute of Biomedical Engineering and Biomaterials, Seoul National University of Science and Technology, Seoul 01811/ 2. Department of
Chemical and Biomolecular Engineering, Seoul National University of Science and Technology, Seoul 01811/ 3. Functional, Innovative and Smart Textiles, PSG Institute of Advanced Studies, Coimbatore 641004, India
Most of the advanced bioinks are multicomponent to ensure the structural stability of 3D bioprinted hydrogels. More than one gels, and bioactive nanomaterials are often used to improve the shape fidelity after printing. The homogeneous mixing of such components in a bioink is essential to achieve uniform biomechanical properties of the construct. Our designed twin screw extrusion (TSE) head is capable to synchronously perform systematic mixing and 3D bioprinting. In this study, an ionic gel (alginate) and alpha‐tricalcium phosphate (α‐TCP) micro/nanoparticles were used to optimize the extrusion and mixing of the TSE head. The uniform dispersion of micro/nanoparticles is observed with the TSE‐processed bioink samples within 60 s of screw mixing. The extrusion flow rates follow linear correlation with screw rpm over 15 – 30 rpm. The homogeneous distribution of micro/nanomaterials is observed in real time mixing and bioink printing using the TSE extruder. This process has higher repeatability and increased batch consistency than the conventional method of bioink component mixing and its subsequent 3D bioprinting. The requirement for controlled mixing of bioink components in tissue engineering can be successfully addressed using this technology.
Even tissue formation by uniform cell distribution during 3D bioprinting
1Department of Chemical and Biomolecular Engineering, Seoul National University of Science and Technology, Seoul 01811, 21. Department of Chemical and Biomolecular Engineering, Seoul National
University of Science and Technology, Seoul 01811/ 2. Convergence Institute of Biomedical Engineering and Biomaterials, Seoul National University of Science and Technology, Seoul 01811, 31. Department of Chemical and Biomolecular Engineering, Seoul National University of Science and Technology, Seoul 01811/ 2. Convergence Institute of Biomedical Engineering and Biomaterials, Seoul National University of Science and Technology, Seoul 01811/ 3. Functional, Innovative and Smart Textiles, PSG Institute of Advanced Studies, Coimbatore 641004, India
The progress in three‐dimensional printing (3D) technologies attracts the medical practitioners to adopt patient specific prosthesis and implants. Cell embedded hydrogel (known as bioink) printing or 3 D bioprinting offers new dimension in reconstructive surgery for regeneration of the targeted tissue or organ. Though cell encapsulated polymer gels are printed using three dimensionally computer‐controlled machine in this process, the cell distribution in the hydrogel matrix often inhomogeneous due to difficulty in mixing and cell sedimentation problem during large construct printing.
Our designed and patented twin screw extrusion head enables low shear uniform cell mixing with the hydrogels along with the synchronous 3D bioprinting. The cell mixing and extrusion with moderately crosslinked biocompatible, nontoxic polymer sodium alginate gel was carried out. After printing the cell encapsulated gels are further cross‐linked with calcium chloride solution.
3D bioprinter was used to create different shapes and multilayered structures. Homogeneous distribution of cells inside the gel matrix with good cell viability was observed. The cell immobilized gel structures were cultured with cell culture medium in CO2 incubator under controlled conditions. The even tissue formation was recorded after 7 days of cell culture study. This technique can be used for reconstruction of skin, bone, or other tissues.
Kombucha‐cultured nanocellulose for 3D bioprinting
1Department of Chemical and Biomolecular Engineering, Seoul National University of Science and Technology, Seoul 01811, 21 Department of Chemical and Biomolecular Engineering, Seoul National
University of Science and Technology, Seoul 01811/ 2. Convergence Institute of Biomedical Engineering and Biomaterials, Seoul National University of Science and Technology, Seoul 01811/ 3. Functional, Innovative and Smart Textiles, PSG Institute of Advanced Studies, Coimbatore 641004, India, 31
Department of Chemical and Biomolecular Engineering, Seoul National University of Science and
Technology, Seoul 01811/ 2. Convergence Institute of Biomedical Engineering and Biomaterials, Seoul National University of Science and Technology, Seoul 01811
Bacterial cellulose has gained researchers attention for the tissue engineering scaffolding purpose due to its pure and nontoxic form. The symbiotic culture of bacteria and yeast (SCOBY) resulted in the kombucha tea which is traditionally used as herbal medicine. During the culture process, a byproduct of nanocellulose pellicle formed which has good mechanical strength, biocompatibility, and hydrophilicity. However, it lacks processability and shape formation ability due to highly entangled nanofibrous natural structure formation during its growth. Thus, it has limited use in tissue engineering. In this study, the kombucha sheet is prepared and it is partially hydrolyzed in a controlled manner through acid treatment. After this treatment, its extrusion ability and shape formation ability are increased, and it is possible to give various shapes. This material is studied for its biomechanical properties after 3D printing. Both human dermal fibroblast cells and mouse osteoblast cells show high cell viability with this gel material. The scaffold is found to be cell supportive in nature providing good attachment, growth, and proliferation for both the cells. The green process of synthesis of this material offers a new possibility for its successful use as a bioink for 3D bioprinting. The controlled partial hydrolysis process preserves most of the gel strength and cytocompatibility while making it suitable for 3D printing/bioprinting.
Isolation of colon cancer cells using membrane filtration method
1National Central University
Colon carcinoma is one of the most fatal and common gastrointestinal cancers in the world. Cancer‐ initiation cells (CICs), or cancer stem cells (CSCs), are responsible for tumor initiation, growth, and metastasis. The purification and isolation of tumorigenic colon CSCs (CICs) from primary colon tumor tissues should be valuable for the development of novel diagnostic and personal therapeutic treatments in the future. In this study, we investigated the purification of CSCs (CICs) of colon carcinoma cell line CoLo205 and SW480 from human fibroblasts (CG1639) (model of primary colon carcinoma cells) as well as primary colon carcinoma cells from colon tumor tissues of patients utilizing the filtration method via nylon mesh filters (pore size = 11 um) and poly(lactide‐co‐glycolic acid)/silk screen (PLGA/SK) filters with pore size = 28–37 μm. In the separation of colon carcinoma cells from fibroblasts, both cells were stained with Cell Tracker Red and Blue, respectively for their identification using flow cytometry. The isolation efficiency was characterized using (i) CD44 and CD133 marker expression, (ii) colony‐forming unit assay, and (iii) carcinoembryonic antigen (CEA) production. Colon cancer cells preferentially permeated through the membranes compared to the fibroblasts. Furthermore, colon CSCs (CICs) were purified in the migrated cells compared to the cells in permeation solution and recovery solution. We expect to establish a purification method for a primary colon carcinoma cell line with a high proportion of colon CSCs (CICs) from the patient's tumor tissue using the filtration method developed in this study.
Photo‐crosslinked gelatin‐methacryloyl hydrogel strengthened with in situ formed nanoparticles for regeneration of rabbit calvarial defects
1Department of Periodontology, Research Institute for Periodontal Regeneration, Yonsei University
College of Dentistry, 2Seoul National University of Science and Technology, Department of Chemical and Biomolecular Engineering
Fabrication of cell scaffold capable of sustained oxygen release by hydroxyapatite formation on calcium peroxide
1Osaka University
In tissue engineering field, in vitro construction of thick three‐dimensional tissues is still a major challenge because limited diffusion of oxygen inside these tissues causes cell necrosis. Although many researchers reported oxygen releasing materials using calcium peroxide (CaO2) since it generates oxygen by the reaction with water, the initial burst release of oxygen is issue [1]. Accordingly, the sustainable release of oxygen from biomaterials are strongly desired.
To achieve this end, we suppressed the reaction between CaO2 and water through the formation of hydroxyapatite (HAp) on the surface of CaO2 using phosphate buffer (PB). This HAp‐CaO2 showed sustained oxygen release compared to unmodified CaO2. Furthermore, oxygen releasable gelatin hydrogel enzymatically crosslinked by transglutaminase were successfully fabricated with HAp‐CaO2 and oxygen supply from this biomaterial improved cell viability under hypoxia condition.
Dissolved oxygen amount in PB and HEPES were measured after the addition of 1 mg/mL CaO2. In HEPES, CaO2 showed the initial burst increase of dissolved oxygen, suggesting that the reaction between CaO2 and water was proceeded quickly. By contrast, sustained oxygen release was observed in PB. SEM‐ EDX and XRD measurements of CaO2 immersed in PB confirmed the formation of HAp on CaO2. According to these results, initial burst release of oxygen from CaO2 is suppressed in PB because the formation of HAp prevents the reaction between CaO2 and water.
[1] K. Park et al., Biomaterials
Three‐dimensionally printed biphasic calcium phosphate blocks with different pore diameters for regeneration in rabbit calvarial defects
1Department of periodontology, Research institute of periodontal regeneration, Yonsei University College of Dentistry, Seoul, Korea
Design and fabrication of bone scaffolds with the auxetic structure
1 Industry 4.0 Convergence Bionics Engineering, Pukyong National University, 2Major of Biomedical Engineering, Division of Smart Healthcare, College of Information Technology and Convergence, Pukyong National University
Repeating three‐dimensional (3D)‐architected metamaterial scaffolds can be useful in bone tissue engineering applications due to exceptional and tunable mechanical properties, which can improve the load‐bearing behavior of the scaffolds. However, they need a precise fabrication technique to make 3D interconnected porous architectures. In this respect, 3D printing is a promising technique to fabricate intricate predesigned 3D structures. Therefore, this study aims to propose a novel 3D metamaterial using auxetic repetitive patterns with simple and gradient architectures and diverse Poisson's ratios. The geometries are fabricated by fused deposition modeling (FDM) and a polymer filament. Additionally, the mechanical properties of the scaffolds are investigated by the experimental and finite element method (FEM) and compared with each other. Correlation between pore architectures and the mechanical properties of scaffolds under tensile and compression loads are investigated, and the architectures are optimized by FEM. The study indicates that the feasibility of using auxetic metamaterial scaffolds as an alternative for an effective strategy in bone tissue engineering.
Cell laden gelatin hydrogel with carbodiimide or genipin‐crosslinked for glottic insufficiency: An in vitro study
1Department of Otorhinolaryngology‐Head and Neck Surgery, Universiti Kebangsaan Malaysia, 2Centre of Tissue Engineering and Regenerative Medicine, Universiti Kebangsaan Malaysia
Application of bacterial cellulose membrane in cancer cell isolation
1Department of Biomedical Engineering, National Taiwan University
Circulating tumor cells (CTCs) have huge potential in prognosis prediction, assessment of the efficacy of chemotherapy drugs, survival duration of patients, early diagnosis of cancer disease, and so on. Separation and subculture of CTCs are difficult tasks due to their extreme rarity and vulnerability in the bloodstream. There are certain drawbacks with current technologies, including high cost, high heterogeneity, low protein expression, and low sample purity. To overcome those limitations, we demonstrated a label‐free and distinctive cancer cell capture and expansion system based on the specific affinity difference between bacterial cellulose (BC) for WBCs and cancer cells. The system utilized regioselective oxidation of primary (C6) hydroxyl groups to create BC containing an imparted carbonyl group. The membrane becomes a cellphilic environment ideal for cell quick attachment. Afterward, the selective culture was used to deplete WBCs and expand cancer cells. Conclusively, more than 90% of cancer cells could attach to the BC efficiently within 1 hr and then could proliferate with WBCs being depleted through days of selective culture. It is expected that this system has a tremendous application in isolation and expansion of CTCs for oncotic liquid biopsy.
Cytocompatibility of corneal cells towards ovine collagen type 1 hydrogel
1Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000, Cheras, Kuala Lumpur, Malaysia, 2Department of Ophthalmology, Faculty of Medicine, National University Malaysia, 56000, Cheras, Kuala Lumpur, Malaysia., 3Department of
Ophthalmology, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000, Cheras, Kuala Lumpur, Malaysia., 4Institute of Microengineering and Nanoelectronics, Universiti Kebangsaan Malaysia, 43600, Cheras, Kuala Lumpur, Malaysia, 5Department of Pathology, Faculty of Medicine, Universiti
Kebangsaan Malaysia, 56000, Cheras, Kuala Lumpur, Malaysia., 6Department of Anatomy, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000, Cheras, Kuala Lumpur, Malaysia.
Corneal transplantation is a gold standard for corneal disorders treatment, but the shortage of tissue donors remained a critical problem. The presence of ovine tendon collagen type‐1 (OTC‐1) hydrogel can overcome the shortage of donated corneas. The aim is to evaluate the physicochemical and biocompatibility of the OTC‐1 hydrogel with corneal cells. Collagen fibril of OTC‐1 hydrogel was aligned by using a rocker and double crosslinked by using genipin and quercetin. The physicochemical are evaluated (microstructure, porosity, biodegradation rate, energy‐dispersive X‐ray spectroscopy (EDX), Fourier transform infrared spectrophotometry (FTIR), and X‐ray diffraction (XRD) and mechanical testing). In vitro biocompatibility is assessed by culturing the epithelial and fibroblast on the scaffold. Results showed the scaffold has a uniform interconnected porous structure with acceptable porosity (>70%). The biodegradation rate was 1 month. EDX identified the main elements of the scaffold, (carbon (C) 50.28%, nitrogen (N) 18.78%, and oxygen (O) 30.94%) based on the atomic percentage. FTIR confirmed the presence of collagen type 1 with functional groups (amide A: 3302 cm−1, amide B: 2926 cm−1, amide I: 1631 cm−1, amide II: 1547 cm−1, and amide III: 1237 cm−1). The XRD reported an amorphous phase instead of crystallinity on the fabricated OTC‐1 hydrogel. The ultimate tensile strength was 2.21 ± 0.70 MPa at a strain rate of 0.5%/s. Confluence epithelial cells migrated over the scaffold and keratocytes inside the scaffold. Thus, these properties suggest its potential be developed into a corneal tissue substitute.
Design, fabrication, and assessment of a robust modified‐honeycomb‐ structure scaffold with enhanced interconnectivity for bone tissue engineering
1Department of Mechanical Engineering, Wonkwang University, 2Mechabiogroup, Wonkwang University,
3Department of Mechanical and Design Engineering, Wonkwang University
To date, the fabrication of PCL (polycaprolactone) scaffolds with mechanical properties similar to the bone tissue is still a challenge. To enhance the mechanical properties of PCL scaffolds, previous studies have proved that the variation or manipulation of structures or material improve its mechanical properties. For example, geometrically speaking, Kagome‐structure scaffold is a structure with high compressive modulus and well in‐vitro response for bone tissue engineered scaffolds. However, Kagome‐structure is not the best way to optimize the mechanical properties of scaffolds from the viewpoint of geometry. On the other hand, honeycomb‐structure, which is a stronger structure with respect to its weight could be a promising structure for bone tissue engineering if its three‐dimensional interconnectivity is enhanced. The proposed modified‐honeycomb‐structure scaffold enhances its interconnectivity after adding pores at its walls and have excellent structural integrity. The compressive stiffness of the modified‐honeycomb‐ structure scaffold was investigated by numerical analysis and validated using a universal testing machine. The effective compressive modulus of the modified‐honeycomb‐structure scaffold was significantly superior to those of Kagome‐structure and grid‐structure scaffold having similar pore size of 500 μm and porosity of 50%. In addition, the modified‐honeycomb‐structure scaffold was successfully fabricated by a 3D bioprinter with a PED head, and cell proliferation was evaluated. The modified‐honeycomb‐structured scaffold was more favorable for cell proliferation than the Kagome‐structure and grid‐structure scaffolds. In conclusion, the present study proposes a modified‐honeycomb‐structure scaffolds with both, improvement of mechanical properties and has well three‐dimensional interconnectivity that enhanced cell proliferation showing a strong potential for bone tissue engineering.
Bioinspired peptide hydrogels for controlled delivery of viral vectors to reprogram endogenous reactive astrocytes to neurons in acquired brain injury
1ACRF Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, ANU College of Health & Medicine, Australia, 2The Graeme Clark Institute, The University of Melbourne, Melbourne, Australia, 3iMPACT, School of Medicine, Deakin University, Waurn Ponds, VIC 3216, Australia, Federation Uni, 4The Florey Institute of Neuroscience and Mental Health, The
University of Melbourne, Parkville, Melbourne, VIC 3010, Australia, 5School of Human Sciences, The University of Western Australia, and Perron Institute for Neurological and Translational Science, Perth, WA 6009, Australia
The incapacity of the central nervous system (CNS) to regenerate is a barrier to the effective treatment of neurodegenerative diseases and traumatic injuries. Following CNS injury, astrocytes undergo reactive gliosis and create a glial scar that cordons off the injury that restricts the extension of regenerating axons through the injury site. Reversing this natural biological response to CNS injury is challenging using existing treatment approaches. Therefore, converting glial cells into functional neurons in situ provides an efficient way to obtain desirable endogenous neurons from a large cellular pool for ‘‘on‐site’’ brain repair. Here, we examined the ability of adeno‐associated viral vector (AAV) encoded with genes relevant for reprogramming astrocytes into functional neurons. Our in vitro results demonstrated the successful conversion of 25% and 65% reactive astrocytes to neurons through direct and indirect reprogramming, respectively. The in vivo generation of induced neurons represents the next generation of treatment options for patients with brain injuries or ND diseases. However, challenges remain with their uncontrolled dissemination and neutralization.
To address this, we utilize a library of Fmoc Self‐Assembling (Fmoc‐SAPs) hydrogels to provide an extracellular matrix (ECM)‐like structure to preserve damaged cells and facilitate their survival, proliferation, and differentiation. These Fmoc‐SAPs are able to provide structural support to damaged tissue and act as a local transport system for the delivery of AAV and neurotrophic factors in a controlled manner. These injectable hydrogels addressed multiple difficulties with viral vector delivery by shielding viral vectors, confining them to the site of therapeutic need and minimizing immunogenicity.
Development and evaluation of freeze dried and electrospun scaffolds from chitosan, gelatin and nano ceramic phosphate for bone tissue engineering
1National Institute of Technology Rourkela, India
Gelatin, chitosan and dihydrogen calcium phosphate anhydrous (DCPA) based composite scaffolds with tailored architectures and properties has great potential for bone regeneration. Herein, we aimed to improve the physicochemical, mechanical and osteogenic properties of 3D scaffold by incorporation of DCPA nanoparticles into biopolymer matrix with variation in composition in the prepared scaffolds. Scaffolds were prepared from the slurry containing gelatin, chitosan and synthesized DCPA nanoparticle using lyophilisation technique. The prepared scaffold showed interconnected porosity with pore size varying between 110‐ 200 micrometer. With increase in DCPA content from 5 wt% to 10 wt%, the compressive strength of the scaffold increased from 1 to 2.36 MPa. Higher cellular activities were observed in DCPA containing scaffold as compared to pure gelatin chitosan scaffold suggesting the fact that DCPA addition into the scaffold promoted better osteoblast adhesion and proliferation as evident from MTT assay and SEM investigation of osteoblast cultured scaffolds. The results demonstrated that both mechanical strength and osteogenic properties of gelatin/chitosan scaffold could be improved by addition of DCPA nanoparticles into it. Separately, scaffolds were prepared from a mixture of gelatin, chitosan solution in acetic acid and aqueous suspension of DCPA nanoparticles using electrospinning technique. The electrospun scaffold with average fiber diameter of nearly 120 nm were successfully prepared at applied voltage 20 kV, 15 cm of distance between collector and needle tip and flow rate at 8 mL/min. The results demonstrated that both physicochemical and mechanical properties of gelatin/chitosan scaffold could be improved by addition of DCPA nanoparticles into it.
Investigation of the potential for osteochondral tissue regeneration via a novel biphasic 3D printed silk reinforced scaffold
1Biomaterials and Tissue Engineering Group, Dept. of Oral Biology, University of Leeds, 2CReaTE
Group, Dept. of Orthopaedic Surgery, University of Otago Christchurch, 3Graduate School of Biomedical Engineering, UNSW Sydney, 4School of mechanical engineering, University of Leeds, 5School of Biomedical Sciences, University of Leeds
Osteochondral tissue damage remains a major clinical challenge due to its prevalence in the young, active population. There is a need for a unique tissue engineering approach which is able to regenerate both articular cartilage and subchondral bone. 3D printing is an exciting scaffold development method for tissue regeneration, especially within personalised medicine. However, many 3D printing techniques rely on creating a lattice structure, which often demonstrates poor cell bridging between filaments due to its large pore size; reducing regenerative capacity as cells are unable to efficiently remodel the scaffold. We tackled this issue by developing a novel silk reinforced 3D printed scaffold. These biphasic scaffolds consist of 3D printed poly(ethylene glycol)‐terephthalate‐poly(butylene‐terephthalate) (PEGT/PBT) lattice, infilled with a cast and freeze dried porous silk scaffold, connected to a seamless silk top layer. Compression testing showed that scaffolds had a compressive modulus of 12.7 ± 0.9 MPa and ultimate compressive strength of 1.56 ± 0.1 MPa, theoretically allowing for their survival during implantation and joint articulation without stress‐shielding mechanosensitive cells. Fluorescence microscopy showed biphasic scaffolds can support human bone marrow stromal cell (HBMSC) attachment and spreading after
24 hours of seeding. Histological analysis also demonstrated scaffolds support osteogenic or chondrogenic differentiation of seeded HBMSCs. By combining two different and unique materials, our biphasic scaffold possesses the mechanical and structural advantages of PEGT/PBT with the biocompatibility and cell supporting characteristics of silk, overcoming the disadvantages of the individual materials. Our work demonstrates that this biphasic scaffold has suitable properties for osteochondral tissue regeneration.
A novel biofabrication process to generate vascularised 3D bioprinted constructs to support islet transplantation for the treatment of type 1 diabetes
1University of Wollongong, 2University of Adelaide
Pancreatic islet transplantation has been a promising life‐changing treatment for individuals with severe type 1 diabetes; however, post‐transplant islet survival has limited the broader application of this treatment. At present, revascularisation of transplanted islets remains a critical step to ensure islet survival and maximal function The intra‐islet vasculature is highly tuned to respond to changes in blood glucose levels to achieve euglycemia. During islet isolation, there is complete disruption of the intra‐islet vasculature. Post‐transplantation the rate of intra‐islet revascularisation and intra‐islet vascular density will determine the survival rate and functional capacity of the transplanted islets. Therefore, we investigated an alternative biofabrication approach creating vascularised islet‐ladened constructs via 3D bioprinting. 3D bioprinting utilises biomimicry to create tissue constructs that mimic the cellular architectures found in nature, in this case pancreatic like insulin‐secreting tissue (PLIST). In this study, a novel approach for the vascularisation of cell‐ladened constructs utilising the Janus nozzle through a 3D bioprinting approach is investigated. The Janus nozzle simultaneously deposits two bioinks side‐by‐side, with an alginate/gelatin based bioink and a cell‐ladened gelatin/fibrinogen based bioink. This study demonstrates utility of the Janus nozzle to facilitate vascularisation in a 3D bioprinted construct through specific deposition of human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs), the first step towards creating PLIST. In addition, this biofabrication method exhibits a supportive environment for cell viability and structural integrity of the construct. These results demonstrate the potential for the inclusion of islets within this vascularised 3D construct to treat type 1 diabetes.
3D printed natural hydroxyapatite‐embedded titanium implants promoting osseointegration
1Sunchon National Univ
Biomaterials play a vital role in the modulation of physicochemical properties of dental or orthopedic implants as well as its biological responses in human body. Here, we report a novel strategy to develop Ti6Al4V (Ti) implants embedded with natural hydroxyapatite derived from equine bones (EB) which overcome the limitation of conventional hydroxyapatite‐coated titanium implants. Due to its flexibility in fusing feedstock, we employed a selective laser sintering technique for the facile fabrication of Ti/EB implants and also confirmed distribution of EB particle on the surface via a FE‐SEM observation. The developed Ti/EB implants not only exhibited higher hydrophilicity and protein adsorption but also enhanced crystalline structure suitable for implantation. We found that our platform greatly influences the attachment, proliferation, and osteogenic differentiation capabilities of MC3T3‐E1 cells through the in vitro study. Furthermore, Ti/EB implants alleviated the inflammatory responses of macrophage which can contribute to promotion of osseointegration when they are implanted. Consequently, our developed functional platform would be a promising candidate replacing conventional dental or orthopedic implants and our methodology could be utilized to design advanced tissue engineering implants.
Development of 3D printable calcium phosphate cement based on cockle shell powder/β‐TCP
1Sunchon National University
Calcium Phosphate (Cement CPC) scaffolds fabricated by 3D printing are increasingly used in the field of bone tissue repair. Generally, production rate of CPCs is low since the amount of calcium phosphate compounds is small in conventional raw materials, such as bovine bone and porcine bone. On the other hand, cockle shell contains more than 99% of CaCO3, one of the main constituents of CPC, in the form of amorphous aragonite which has excellent bioavailability. Therefore, in this study, cockle shell powder and β‐TCP based CPC scaffolds were fabricated through an extrusion method using nozzles which are narrower than conventional one for CPC 3D printing. Their morphology and biocompatibility were investigated. With this method, high‐added value can be created for fishing villages due to the recycling of natural materials which is thrown away as marine waste.
Natural killer cell membrane coated gold nanoparticles for cell membrane immunotherapy
1Dongguk university
Here, we developed natural killer (NK) cell membrane coated gold nanoparticles (AuNPs) as a versatile platform for a cancer immunotherapy to eliminate primary tumors and inhibit metastasis. NK cells are the first line of defense against tumors in innate immunity via surface‐ligand recognition processes, even without prior activation. NK cells recognize tumors via interactions between tumor‐cell‐expressed ligands and NK cell‐membrane receptors such as NKG2D and DNAX accessory molecule 1 (DNAM‐1). NK cell membrane coated nanoparticles (CMC NPs) were developed using AuNPs as biocompatible inorganic templates. In addition to augmented surface‐ligand interaction via the surface‐presented components, AuNP‐mediated photothermal efficacy could be also utilized for enhanced anticancer treatment. To fabricate CMC NPs, a solution comprising NK cell membrane vesicles and AuNP templates was repeatedly passed through a porous polycarbonate membrane using a mini extruder. NK CMC NPs could induce cancer cell death through synergistic effects of (1) the recognition of surface ligands in target cancer cells and (2) photothermal efficacy under near‐infrared irradiation. Therefore, the incorporation of NK cellular membrane components onto AuNPs and the administration of NK CMC NPs toward cancer cell lines could be a potential strategy for effective cancer immunotherapy.
The fabrication of highly porous cell‐laden structure
1Sungkyunkwan university
Scaffold‐based tissue engineering aims to develop biocompatible scaffolds for the recovery of damaged tissues and organs. Therefore, appropriate mechanical strength, biocompatibility, biodegradability, and porosity are key characteristics of scaffolds for tissue engineering. Amongst them, the high porosity can be effective in transferring oxygen and nutrients to the host cell. Previous examples of fabricating porous structures such as the gas foam method and salt leaching method show that high porosity can be achieved. During these processes high temperature and pressure or solvent are needed, therefore it is difficult to manufacture a structure containing cells using these conventional methods. To overcome this issue, we propose a simple process using mesh filter in obtaining porous cell‐laden structure using gelatin methacrylate (Gel‐ma) bio‐ink bearing with human adipose derived stem cells (hASCs). The amphiphilic behavior (containing both hydrophobic and hydrophilic amino acids) of Gel‐ma would contribute to capturing of the air molecules, forming porous hydrogel. We have considered various of parameters such as the Gel‐ma concentration, flow rate, the size of mesh, and manufacturing temperature, and UV crosslinking conditions (UV intensity and crosslinking time) for optimal structural maintenance and cell survival. The cells encapsulated deep inside the porous hydrogels can survive due to the nutrient and oxygen transportation. Moreover, cellular activities (cell stretching, proliferation) were significantly higher in the porous cell‐laden structure compared to the conventional cell‐laden hydrogel.
Collagen‐based bioink for 3D bioprinting to obtain mechanically enhanced porous 3D cell‐laden structure
1Sungkyunkwan University
According to the increase of the global demand for tissue replacement or regeneration, 3D bioprinting technology has been emerging with an incessant series of revolutions to regenerate such damaged tissues or organs into functioning tissues or organs. Bioink is a main part of the 3D bioprinting, which contains cells and biomaterials that play key roles for the tissue regeneration. It requires sufficient mechanical properties to print and sustain the 3D structure and biological properties to induce cell proliferation and tissue repair. Therefore, various materials, techniques and combinations were used to enhance the properties of bioink and the printing procedure. In this study, collagen, a main component of the extracellular matrix, was used as the base material of bioink. However, natural hydrogels like collagen have shown serious limitation of deficient mechanical properties. Therefore, an innovative method to enhance the properties of the collage‐based bioink was developed using collagen fibrillation in this study. In addition, the developed biomaterial has shown hierarchically porous structure, which is also required for active regeneration. In conclusion, the collagen‐based newly developed bioink can be used in the 3D bioprinting of tissue structure with upregulated performances for tissue engineering.
Fabrication of 3D cell‐constructs using photocrosslinkable bioin
1Sungkyunkwan University
Recently, bioprinting strategies to fabricate cell‐laden structures have been regarded as an effective tool in regenerating and restoring functionalities of damaged tissues. However, the insufficient cell‐to‐cell interactions in the current bioconstructs are a substantial challenge for successful tissue engineering. To overcome this issue, we have treated cells with photocrosslinkable natural proteins to fabricate 3D structures with elevated cell‐to‐cell interactions. To attain optimal printability and cell‐to‐cell interactions, various parameters have been investigated. Then, the bioink containing the cells was extruded into an alginate‐based printing bath in order to fabricate 3D structures. Printing parameters such as nozzle moving speed, the volumetric flowrate of bioink, and UV crosslinking conditions were investigated to achieve high cell viability (> 90%). To evaluate the biological effects of the described system, conventionally fabricated bioconstructs were compared. As a result, the protein‐treated cells exhibited a higher positive area of cadherin, demonstrating elevated cell‐to‐cell interactions.
Development of a novel hemostatic biomaterial using keratin‐conjugated fibrinogen for oral tissue regeneration
1School of Dentistry, Kyung Hee University, 2KERAMEDIX, 3College of Health & Medical Sciences, Cheongju University
Traumatic injury is often accompanied by uncontrolled bleeding, and various kinds of hemostatic biomaterial have been applied to cure such traumatic injury. Among hemostatic biomaterial, fibrinogen is widely used for blood clotting and wound healing. Recently, human hair keratin has been considered as a potent biomaterial for wound healing and hemorrhage control due to its favorable biocompatibility. Hence, I developed keratin‐conjugated fibrinogen (KRT‐FIB). In this study, rheological property, swelling behavior and surface morphology of the KRT‐FIB based hemostatic biomaterial were evaluated, and cellular interaction of the biomaterial inducing cell viability, proliferation and migration was investigated. The KFS‐6 showed the highest hemostatic ability from in vitro whole blood hemostatic test. In addition, KRT‐FIB based hemostatic biomaterial reduced bleeding time and bleeding amounts compared to the current hemostats as assessed via in vivo rat liver punching model. KRT‐FIB is a promising hemostatic biomaterial for the application to oral tissue regeneration.
This work was supported by the Technology Innovation Program (20008650, Development of Recombinant Keratin Protein‐based Biomaterials for Soft Tissue Regeneration) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No.2022R1C1C1010472).
Topical bioadhesive hemostatic agents for bleeding site care at visceral surgeries
1Department of Plastic and Reconstructive Surgery, The Catholic University of Korea, 2Department of Chemical Engineering, Pohang University of Science and Engineering, 3Division of Chemical Engineering and Materials Science, Ewha Womans University
Appropriate and timely application of topical hemostatic agents, which can reduce blood loss, is able to not only reducing the operation time and cost at visceral surgeries, but also improving the recovery prognosis of the patients. Among major commercial hemostatic materials, fibrinogen, thrombin, and collagen are effective but they have risks of infection and immune response depending on original sources. Other materials like synthetic polymers or oxidized polysaccharides may acidify the surrounding tissue during degradation. In this study, therefore, we introduced bioengineered mussel adhesive protein (MAP) as novel hemostatic material due to its accomplished underwater adhesiveness and biocompatibility. MAP is rich in L‐3,4‐dioxyphenylalanine (DOPA) to participate in multiple adhesion mechanisms and L‐lysine to contact with negatively charged blood cells or plasma proteins. We investigated the platelet aggregation and tissue adhesion ability of MAP. Then, we also used silk proteins for mechanical supporting and fabricated them into two formulations: hydrogel patch and electrospun nanofiber mat. The hydrogel patch was formed by dip‐coating MAP onto the sea anemone silk‐like protein (aneroin) hydrogel and casted into mesh shape to rapidly contact and absorb the blood plasma. In the case of electrospun nanofiber, MAP was only applied on single side to contact with bleeding site, and the other side was modulated to be hydrophobic for prevention the bleeding site from initial fouling or infection. Both hydrogel patch and electrospun nanofiber mat had demonstrated excellent hemostatic ability and appropriate biodegradation rate as topical hemostatic agent at in vivo rat liver damage model evaluation.
Application of cartilage extracellular matrix for enhancing the therapeutic efficacy of rheumatoid arthritis drug
1Pukyong National University
Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial inflammation. RA is still incurable, and treatment is aimed at relieving symptoms. Methotrexate (MTX) is the most used among disease‐modifying anti‐rheumatic drugs (DMARDs). However, side effects such as nausea and diarrhea exist and the treatment effect of the drug decreases when taken for a long time. So, it must take a high‐ dose drug or combination with other DMARDs for the treatment. Native cartilage consists of mainly collagen and glycosaminoglycan. We extracted the porcine cartilage extracellular matrix (CECM). Many papers were known to various tissue‐derived extracellular matrix can modulate inflammatory environment. The hypothesis is that low‐dose MTX with CECM can have a synergistic therapeutic effect on the RA model. To prove it, this study aim was to evaluate the CECM as a treatment CECM biomaterial. The experimental group was divided with Normal, Inflammation, 88μM MTX (high‐dose), 44μM MTX with CECM, 11μM MTX (low‐dose) with CECM. In the results, SW 982 in the high‐dose MTX group showed the cytotoxicity, but low‐dose MTX with CECM group did not showed the cytotoxicity based on cell proliferation assay. Low‐dose MTX with CECM group showed the depression of RAW‐264.7 activation and proliferation like high‐dose MTX. Expressions related inflammatory cytokines had been also suppressed by treatment, both high‐dose MTX and low‐dose MTX with CECM. In conclusion, the low‐dose MTX with CECM showed similar therapeutic effects to the high‐dose MTX. It means we could reduce the side effects of high concentration MTX currently used for RA treatment.
Functional skeletal muscle regeneration using muscle mimetic tissue fabricated by microvalve‐assisted coaxial 3D bioprinting
1Nano‐Bio Regenerative Medical Institute, College of Medicine, Hallym University, and Hallymdaehak‐ gil, Chuncheon, Gangwon‐do 24252, Republic of Korea
The three‐dimensional (3D) printed artificial skeletal muscle which mimics the structural and functional characteristics of native skeletal muscle is a promising treatment method for muscle reconstruction. Although various fabrication techniques for skeletal muscle using 3D bio‐printer have been studied, it is still difficult to build a functional muscle structure. We propose a strategy using microvalve‐assisted coaxial 3D bioprinting in consideration of a functional skeletal muscle fabrication. The unit (artificial muscle fascicle: AMF) of muscle mimetic tissue is composed of a core filled with medium‐based C2C12 myoblast aggregates as a role of muscle fibers and a photo‐crosslinkable hydrogel‐based shell as a role of connective tissue in muscles that enhances printability and cell adhesion and proliferation. Specially, microvalve system is applied for the core part with even cell distribution and strong cell‐cell interaction. This system enhances myotube formation consequently shows spontaneous contraction. A multi‐printed AMFs (artificial muscle tissue: AMT) as a piece of muscle is implanted into the anterior tibia (TA) muscle defect site of immunocompromised rats. In the result, the TA implanted AMT responds to electrical stimulation and represented histologically regenerated muscle tissue. This microvalve‐assisted coaxial 3D bioprinting system shows a great step forward to mimic native skeletal muscle tissue.
Double layered conductive nanoparticles for bioelectronics surface using mussel‐derived protein
1Department of Chemical Engineering, Pohang University of Science and Technology
There are increasing demands for conductive materials in biophysical environment, especially for bioelectronics device applications including neural and cardiac stimulation and recording, electroceuticals, and neural prostheses. In order to overcome the mismatch caused by the difference in physical properties and charge carriers between conductive materials and biological tissues, there have been many trials to bridge this interface. In this work, we developed novel double layered biocompatible nanoparticles with conductive components (for example, metal composites) and nature derived biomolecules. The first coating layer hyaluronic acid (HA) is widely distributed nature derived polysaccharide. HA is innately anionic in neutral pH condition, showing proper mechanical properties to use in biophysical condition, with biocompatibility and biodegradability. The second coating layer is bioengineered mussel adhesive protein (MAP), which contains rich DOPA residues playing critical role in adhesive and cohesive effects in mussel byssus and abundant lysine residues making protein positive net charge. We confirmed successful construction of double layered nanoparticles which contained conductive component and biomolecules. These double layered conductive nanoparticles will be applied as interface between electrode and target tissues.
Hybrid bio 3D printing technology using photocurable bio ink / poly‐ caprolactone for cartilage regeneration
1Nano‐Bio Regenerative Medical Institute, College of Medicine, Hallym University, 1 Hallymdaehak‐gil, Chuncheon, Gangwon‐do 24252, Republic of Korea, 2Department of Otorhinolaryngology‐Head and
Neck Surgery, Chuncheon Sacred Heart Hospital, School of Medicine, Hallym University, Chuncheon 24252, Republic of Korea
Cartilage is a structure without blood vessels and nerves. Therefore, when damaged, it lacks self‐healing ability and limit regenerate. Methods of manufacture, culture and transplantation are supported cartilage by bio 3D printing while satisfying the three conditions is being actively researched in recent years. Bio‐ ink is an essential material used in bio 3D printing technology. These bio‐inks require several essential conditions in terms of printability, biocompatibility and biomimetic properties, including structural and mechanical stability. Bio 3D printing technology fabricate it possible to complex organs quickly and easily. FDM type is printed by extruding the material. It is extruded by melting the solid material, and the hydrogel bio‐ink is extruded with air compression. We report to create 3D bio‐printed artificial larynx for whole‐laryngeal replacement. Our 3D bio‐printed larynx was generated using fused deposition method 3D bioprinter with rabbit's chondrocyte‐laden GelMA/ GMHA hybrid bio‐ink. We used polycaprolactone (PCL) outer framework incorporated with pores to achieve the structural strength of printed constructs. Notably, we established a novel fluidics supply (FS) system that simultaneously supplies basal medium together with bio 3D printing process, thereby improving cell survival during the printing process. Our results showed that the FS system enhanced post‐printing cell viability, which enables to generate large‐ scale cell‐laden artificial laryngeal framework. Additionally, incorporation of PCL outer framework with pores and inner hydrogel provides structural stability and sufficient nutrient/oxygen transport. The animal study confirmed that the transplanted 3D bio‐larynx successfully maintained airway.
Fabrication of graphene oxide composite ultra‐strong stretchable hydrogel with high conductivity and biocompatibility
1Hallym University
The goal of this study was to solve the disadvantages of the physical properties of naturally‐derived polymers by combining graphene oxide (GO) and polymers, and focused on improving the mechanical strength of composite conductive hydrogels and double network conductive hydrogels. In addition, graphene oxide composite methacrylation (Gelatin GelGOMA) an electrically conductive ultra‐strong hydrogel that can freely fabricate specific organs and tissues, was developed by modifying it with a photocurable bio‐ink capable of DLP 3D printing through the double bond of glycidyl methacrylate and methacrylate. To evaluate its physical properties, various analyzes such as compressive strength, tensile strength, electrical conductivity, and rheology were performed. In addition, real‐time cell proliferation using (INCELL machine) was observed for toxicity evaluation of hydrogel. The printability was evaluated by printing various microscopic structures using the solution of the optimal concentration of the finally made GelGOMA. Cell proliferation inside 3D hydrogel was confirmed by immunofluorescent staining of mouse neuroblastoma cell (Neuro2a) and immortalized mouse myoblast cell line by GO. By combining the material synthesis method described above and Bio 3D Printing technology, the insufficient physical properties of the existing natural polymer‐based hydrogel can be completely solved, and the desired shape of the supports can be freely produced using the 3D printing technology. In addition, it is possible to help the proliferation of muscle cells, cardiomyocytes, neuronal cells, etc. by securing electrical conductivity through the combination of graphene oxide as well as simply increasing physical properties.
3D printed fish‐derived extracellular matrix scaffolds for bone tissue engineering
1sungkyunkwan university
The limited self‐healing ability of the human being encouraged researchers to develop artificial substitutes (scaffolds) for restoring the major tissue defects. Various characteristics, such as biocompatibility, biodegradability, and structural features (porosity, pore interconnectivity, pore size, etc) of the scaffolds should be considered. Commonly, decellularized extracellular matrix (dECM) derived from mammalian has been accepted as an outstanding biomaterial for tissue engineering. However, problems such as auto‐immune responses, increased risk of viral and bacterial disease transmission, rejection by patients for religious reasons, and cost‐related issues can limit the use of mammalian‐based dECM. Recently, fish‐derived dECM showed the possibility as an alternative option to overcome these limitations. Therefore, in this study, we have investigated the potential of tilapia‐derived dECM (T‐ dECM) for tissue engineering. The components (collagen, glycosaminoglycan, elastin, and fatty acid) and characteristics (degradation rate, denaturation temperature, and biological activities) were compared with the mammalian‐derived collagen and dECM (M‐dECM). T‐dECM includes more polyunsaturated fatty acid than M‐dECM, which is known for angiogenesis accelerants. In addition, faster degradation and lower denaturation temperature were observed. The T‐dECM was prepared as a bio‐ink and applied to the 3D printing technique to control the structural features of scaffolds. To improve the osteogenesis, T‐ dECM/hydroxyapatite (HA) composite scaffolds were fabricated, and pre‐osteoblasts were seeded onto the scaffolds for the cellular activity evaluation (cell viability, proliferation, cell morphology, and osteogenesis). The results indicated that T‐dECM/HA scaffolds showed better osteogenesis than the conventional mammalian collagen/HA scaffolds.
Fabrication and characterization of a myrrh hydrocolloid dressing for dermal wound healing
1Hallym University
Common occurrence of dermal injuries has made wound dressing a critical component in wound healing. Hydrocolloid wound dressings have been developed as fanciful and effective wound dressing with ability for wound fluid control, adherence to wound site, and infection prevention. Effectiveness of topical application of myrrh resin to dermal wound has been well documented. We fabricated Mirderm hydrocolloid wound dressings with myrrh resin to enhance wound healing and improve on the mechanical characteristics of the hydrocolloid. The biocompatibility of Mirderm was assayed with CCK‐8 while the wound healing efficacy was tested in vivo. The physical and mechanical properties were checked to affirm the suitability of Mirderm as hydrocolloid wound dressing. We observed Mirderm showed superior wound healing and mechanical capability suitable for hydrocolloid polymer relative to investigated commercial hydrocolloid and medical Gauze. These are indications that Mirderm may be an improvement on standard dressings such as Gauze and some commercial hydrocolloids.
Multi‐functional microwell array platform for spontaneous glioblastoma spheroid formation and anticancer drug screening
1School of Integrative Engineering, Chung‐Ang University
In the evaluation stage of anticancer drugs, including discovery of new drugs and preclinical trials, the drug screening system for cancer cells cultured in three‐dimensional (3D) is in an important position. Herein, we report a composite platform capable of both 3D formation of cancer cells and anticancer drug screening. The highly conductive gold nanoparticle‐microwell array hybrid platform (H‐MIAH) combined with the highly conductive gold nanostructure (HCGN) developed through our previous study and a polymer microwell for inducing human glioblastoma spheroid formation generates 150 glioblastoma spheroids of uniform diameter (224.8 ± 11.36 μm) per unit area (1.13 cm2). Furthermore, human glioblastoma spheroids generated on H‐MIAH can be real‐time detected in a non‐destructive manner via electrochemical techniques. We used the H‐MIAH platform to evaluate the anticancer drug (hydroxyurea) against human glioblastoma spheroids with high sensitivity and cancer spheroid‐sphecific, which are difficult to detect by optical image analysis or traditional electrochemical detection platforms. Hence, it can be concluded that the H‐HIAH platform is highly promising and precise for anticancer drug screening.
Matrix stiffness dependent nuclear transport of STAT6 determines M2 activation of macrophages
1Korea University
Alternatively activated or M2 macrophages, as opposed to the well characterized pro‐inflammatory or M1 macrophages, vitally regulate anti‐inflammation, wound healing, and tissue repair to maintain tissue homeostasis. Although ubiquitous presence of macrophages in diverse tissues, exposed to different physical environments, infers distinct immune responses of M2 macrophages with high phenotypic heterogeneity, the underlying mechanism of how the varying extracellular mechanical conditions alter their immunological activation remains unclear. Here, we demonstrate that M2 activation requires a threshold mechanical cue from the extracellular microenvironment, and matrix rigidity‐dependent macrophage spreading is mediated by the F‐actin formation that is essential to regulate mechanosensitive M2 activation of macrophages. We identified a new mechanosensing function of STAT6 (signal transducer and activator of transcription 6), a key transcription factor for M2 activation, whose intranuclear transportation is promoted by the rigid matrix that facilitates the F‐actin formation. Our findings further highlight the critical role of mechanosensitive M2 activation of macrophages in long‐term adaptation to the extracellular microenvironment by bridging nuclear mechanosensation and immune responses.
Effect of silk fibroin/ nano‐hydroxyapatite composite on immune responses
1National University of Singapore
Silk fibroin (SF) has been incorporated with low crystallinity nano‐hydroxyapatite (SF/nHA) as a scaffold for bone tissue engineering. However, its long‐term non‐cytotoxicity and biodegradability have yet to be elucidated. Since the scaffold is made of degradable materials, there may be concerns about the local irritancy effects caused by its degradation products. Macrophages play a crucial role in dictating the long‐ term clinical outcome of biomaterials. They have high plasticity and can be polarized to pro‐inflammatory M1 or anti‐inflammatory M2 phenotypes in response to the local microenvironment. M1 macrophages secrete inflammatory cytokines (TNF‐α, IL‐6) to initiate the healing process and accelerate the degradation of biomaterial. Thereafter, macrophages switch to M2 phenotype and secrete anti‐ inflammatory cytokines (IL‐10, TGF‐β1) to facilitate tissue repair and regeneration. Therefore, the aim of the study is to investigate the pro‐inflammatory and anti‐inflammatory responses towards enzymatic degradation of SF/nHA material. The degradation products were cultured with mouse RAW 264.7 macrophages and expression of cytokines was measured using ELISA and PCR. Preliminary results have shown that macrophages expressed higher levels of TNF‐a in SF/nHA (34.21 pg/mL) than SF (0.07 pg/mL) in PBS solution. This means that nHA induced greater pro‐inflammation than SF. However, the macrophages expressed higher levels of TNF‐a in SF (96 pg/mL) than SF/nHA (37.07 pg/mL) in protease XIV solution, which means that amino acids degraded from SF induced greater inflammation than nHA. Nonetheless, further investigations in measuring the anti‐inflammatory responses of SF/nHA are required to predict pro‐/anti‐inflammatory responses in vivo and enhance its clinical translational potential.
Sprayable Ti3C2 MXene hydrogel for wound healing and skin cancer therapy
1Department of IT Convergence (Brain Korea Plus 21), Korea National University of Transportation, Chungju, 27469, Republic of Korea
Although skin wounds have excellent self‐healing ability, wound healing ability may be reduced due to repeated wounds or reduced immunity from aging. In addition, the skin most vulnerable to sun exposure is also at increased risk of developing skin cancers such as basal cell carcinoma and malignant melanoma. Recently, it has been reported that Ti3C2Tx MXene, a 2D nanomaterial, exhibits biological characteristics such as excellent photothermal conversion ability, antibacterial activity, and ROS (Reactive Oxygen Species) generation. This study evaluated the ability to heal wounds and remove skin cancer by forming a hydrogel using MXene and sodium alginate (SA), which has excellent biocompatibility and fast crosslinking. First, MXene was mixed with sodium alginate in a 1:1 ratio, transferred to a spray bottle, and sprayed with CaCl2 solution to form MXene/SA hydrogels. Then, to confirm the photothermal conversion capability, a near‐infrared (NIR) laser was irradiated to MXene/SA hydrogel to confirm that the MXene/SA hydrogel temperature increased rapidly. These photothermal conversion effects can be adjusted according to MXene concentration and NIR laser density and affect cancer cell fate. In addition, MXene/SA hydrogel has an antibacterial activity to help wound healing, promotes cell growth at specific concentrations, and proved to be biocompatible. As a result, MXene/SA hydrogel achieves effective wound healing and cancer cell removal based on the photothermal conversion ability, biocompatibility, and antibacterial effect. MXene/SA hydrogels represent promising potential materials for achieving practical tissue engineering in the future.
3D bioprinting LEGO system to construct large 3D‐tissues with complex property
1Division of Applied Chemistry, Graduate School of Engineering, Osaka University, Japan
Constructing three‐dimensional (3D) complex biostructure is promising in the medical and pharmaceutical fields. [1,2] 3D bioprinting as a powerful technology enables the fabrication of structures with a programmed feature, but the current state of this art still faces challenges to integrate multiple materials with different properties in one single printing. [3] Moreover, it is difficult to prepare features of large size due to the limited working space of the common commercial printer. [4] Inspired by LEGO, a game that to assemble small and simple pieces to build large and complex structures, we combine the 3D printing with the LEGO strategy to construct complex biostructures. The mixture of fibrinogen and thrombin was used as a bio‐glue to connect the printed pieces by formation of fibrin gel between LEGO pieces. Different materials with variable properties, or blocks bearing different types of cells can be individually fabricated as Lego units and then assembled to complex LEGO structure. This strategy may have a potential in both medical research and tissue engineering application.
[1] Z. Gu et al., Asian J. Pharm. Sci.,
[2] A. Lee et al., Science,
[3] Z. T. Xie et al., Soft Matter,
[4] E. Mirdamadi, et al., ACS Biomater Sci Eng,
Nanofilms constructed by cation‐dipole interaction to prevent cell migration for cell compartmentalization in 3D tissues
1Department of Applied Chemistry, Graduate School of Engineering, Osaka University
In the human body, basement membranes (BMs) provide cells and tissues support and act as platforms for complex signaling. The fabrication of artificial BMs (A‐BMs) is expected to administer cell functions and access, avoiding the deranging of precise cell locations in three‐dimensional (3D) due to cell migration during the cell culture period.[1] We recently reported an A‐BM through the layer‐by‐layer assembly of type IV collagen (Col‐IV) and laminin (LM).[2] The migration of endothelial cells was successfully prevented within 5 days by crosslinked Col‐IV/LM nanofilms.[3] Compared with fast‐ degraded protein nanofilms, biocompatible polyelectrolyte films exhibit higher stability co‐cultured with cells and biodegradability. We are surprised to find that poly‐L‐lysine (PLL)/dextran (D) nanofilms were helpful to prevent endothelial cell migration even extending to two weeks, which is longer than general polyelectrolyte films assembled by ions interactions. PLL/D nanofilms were alternately deposited on the surface of fibroblast layers by the cation‐dipole interaction, giving a positive surface charge. Fabricated PLL/D nanofilms provided a cell‐adhesive surface for the second layer of cell culture, maintaining cell morphology and functions well. The secretion and re‐assembly of Col‐IV between cell layers were detected, indicating the regeneration of natural BMs. 14‐day cell compartmentalization allows the regeneration of natural BMs, contributes to the highly organized 3D tissue construction, and advantages tissue engineering development.
[1] G. Perry, et al. Integr. Biol.
[3] J. Zeng, et al. Biomacromolecules
SUN1‐mediated nuclear tension determines nuclear wrinkling in progerin expressing cells
1Korea University
The cell nucleus is wrapped by nuclear lamina consisting of lamin proteins under nuclear envelope (NE), The nuclear lamina interacts with the intranuclear organization and cytoskeletal structure via the linker of the nucleoskeleton and cytoskeleton (LINC) complexes. Hutchinson‐Gilford progeria syndrome (HGPS) cuased by mutation in the LMNA gene, leading to increased production of truncated prelamin A, protein. As the HGPS patients display accumulated progerin at the nuclear membrane (NM) that results in abnormal nuclear morphology. Accumulation of progerin induces the increased level of SUN1, which connects the nucleoplasm with the cytoskeleton. The overexpression of SUN1 in HGPS cells alter nucleocytoskeletal connection and nuclear morphology, and both defects were rescued by reducing the increased level of SUN1. While an inner nuclear envelope protein SUN1 overexpression in HGPS cells correlated with nuclear defects, however, how the molecular mehanism of progerin expression induced SUN1 accumulation dependent nuclear deformation remains unclear. Here we present a time‐lapse nuclear deformation monitoring via doxycycline inducible progerin expression system. Using our system, we precisely monitor how nuclear morphology changes over time by progerin expression. Furthermore, by using nesprin tension sensor, we monitor that progerin expression leads to nuclear deformation in response to SUN1 overexpression induced alteration of myosin‐dependent nuclear tension. We expect that progerin‐inducible Tet‐On system will provide a new approach to investigate how the progerin expression alter the nucleus‐cytoskeletal connection, which is critical to find important role of nuclear lamina mediating mechanotransduction in biological aging process.
Development of elastin‐like protein derived from the domain of human elastin
1Department of Chemical Engineering, Pohang University of Science and Technology, 2Division Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology
Elastin is an attractive material owing to its unique properties including high resilience, long half‐life, and remarkable interaction with cells. Elastin provides structural integrity, biological cues, and persistent elasticity to a range of important tissues including vasculature and lungs. Elastin is also an important load‐bearing tissue in the bodies of vertebrates and used in places where mechanical energy is required to be stored. Human elastin consists of 36 domains and the structure consists of repeats of hydrophobic and crosslinking domains. The hydrophobic domain of elastin consists of motifs representing the physical properties of elastin and elastin‐like proteins (ELPs) have been developed by repeating these motifs. Although ELPs are attractive materials for tissue engineering applications where mechanical properties are of importance along with the ability to precisely control the physiochemical properties of the scaffolds, there are limitations compared to human elastin such as complex physical properties, in vivo maintenance, and cell affinity. In this research, we developed human elastin domain derived proteins (EDDPs) which can mimic the properties of human elastin and overcome the limits of ELPs.
Cellulose‐based tissue adhesive hydrogels for hemostatic application
1Yonsei University
In surgical treatments and wound healing process, the bleeding control is one of the most important considerations. Thus, the development of effective hemostatic agents is requested. The effective hemostatic agents should be activated instantly when exposed to bleeding area for reducing the blood loss in emergency situations. To meet these requirements, we modified carboxymethyl cellulose (CMC) with phenolic derivatives, and then formulated the modified CMC into adhesive hydrogel with remarkable hemostatic ability. The CMC‐based hemostatic formulations were verified to have sturdy modulus, wet tissue‐adhesiveness, and ability to be crosslinked within seconds for instant application. We additionally confirmed that the CMC‐based hydrogels exhibited strong hemostatic ability in vivo as a hemostatic agent. Moreover, we formulated CMC hydrogels into a patch form with further enhanced stiffness and adhesiveness for more effective hemostatic performance. The adhesive CMC patches exhibited superior hemostatic ability to a conventional hemostatic agent in a mouse liver hemorrhage model. Therefore, the developed CMC hydrogels in this study offer a prospective hemostatic agent for surgery and emergency.
Fabrication of perfusable and free‐form In vitro vascular model using a coaxial nozzle
1Department of Rural and Biosystems Engineering, College of Agriculture and Life Sciences, Chonnam National University, 2Chonnam National University
Conventional manufacturing techniques of an in vitro blood vessel (model BVM) using tissue engineering differ distinctively from native vessels in both physiological and morphological terms. As the conventional BVM has only been manufactured by two‐dimensional (2D) fabrication methods, limitations remain in synthesizing the multi‐layered structure of the blood vessel or managing the various diameters and curves.
To overcome such limitations, three‐dimensional (3D) bioprinting technology, a computer‐based additive manufacturing method, appears as the best candidate for synthesizing delicate vessels of various diameters of relatively free‐forms. Not only able to imitate an actual blood vessel's microenvironment, 3D bioprinting technology also enables anatomical and clinical applications, through the fabrication of an in vitro model tailored to each patient.
While different bioprinting methods to fabricate BVM have been developed, a free‐form blood vessel platform utilizing coaxial nozzle‐based bioprinting was proposed by Gao et al. [1]. The coaxial nozzle extrudes two materials simultaneously and concurrently prints out in concentric circle form to fabricate the desired shapes. Our project consists in applying this coaxial nozzle‐based bioprinting to establish the printing process of in vitro models of various shapes. The perfusable hollow tubular structure was fabricated using a sacrificial bioink in the core part and verified perfusion performance by diffusing fluorescence beads. This research focuses on developing a potential in vitro model in which tissues and blood vessels are interconnected through a perfusable blood vessel.
Reference
[1] Gao et al., Adv. Healthcare Mater., 7(23), 2018
Acknowledgment: This work was supported by No. NRF‐2019R1C1C1009606 and No.2021C300.
Neurotransmitter‐modified fibrous artificial 3D constructs for effective muscle regeneration
1Sungkyunkwan University
Therapeutic strategies to enhance muscle tissue repair have been actively studied due to its high clinical demand for overcoming incurable diseases, such as muscle defect, muscular dystrophy, and amyotrophic lateral sclerosis. Conventional research has focused on culture of the only muscle cells, yet these approaches cannot achieve the formation of neuromuscular junction through electrophysiological control of muscle tissue after implantation and highly depend on health condition of the patients. Thus, co‐culture technique of muscle and neural cell is important for efficient regeneration of muscle tissue. Nevertheless, biofabrication of 3D muscle tissues has been rarely reported. Here, we report an artificial 3D muscle tissue fabricated using neurotransmitter‐modified fibrous hydrogel inks for directional alignment of both muscle and neural cells and their effective synaptic connections. The microfiber‐based 3D muscle constructs showed more rapid formation of synaptic connection between muscle and neural cells than conventional bulk hydrogels simply encapsulating both muscle and neural cells. Our 3D muscle constructs will be further implanted in vivo disease model and their therapeutic effect will be demonstrated.
Self‐assembly small diameter vasculature via dragging 3D printing technique
1Wonkwang University, 2Pohang University of Science and Technology, 3Columbia University
The small diameter vasculature (SDV) diseases, such as carotid and peripheral artery disease are leading causes of mortality globally. Owing to the high clinical demand for synthetic SDV graft, several off‐the‐ shelf products (Dacron, Teflon, and GORE‐TEX) which are prepared using synthetic polymers (e.g., polyethylene terephthalate, polytetrafluoroethylene), have been developed to replacement. Despite development progress, clinical trial still challenging. The main reason of the clinical failure was that could be attributed to the fact that the reconstruction of SDVs causes thrombosis and intimal hyperplasia at the anastomotic lesion owing to the absence of the endothelium. Additionally, the surface thrombogenicity of the synthetic materials used for the synthesis of these graft and the creation of turbulence blood flow, which may cause platelet activation, limit the clinical effectiveness of small‐ diameter grafts. The main cellular compositions of native vascular tissue are endothelium and muscular layers. Therefore, build of the endothelium and muscular layers should be consider for successful reconstruction and replacement. In this study, we fabricated an engineered SDV construct with similar cellular layers by combining dragging 3D printing technique using synthetic polymer and bioprinting using cell‐laden bioink. The co‐culture test was carried out on the HUVECs‐ and human aortic smooth muscle cell (HAoSMC)‐encapsulated collagen bioinks to evaluate the effect of an oxygen concentration gradient between the inner and outer layer in a static culture environment on specific cell migration. The results revealed that the migration of the HUVECs inward facilitated the growth of endothelium into the vascular structure.
Development of a simple multi‐functional unidirectional freezing platform to engineer aligned scaffolds for tissue engineering
1Graduate School of Biomedical Engineering, University of New South Wales, 2Light Activated Biomaterials (LAB) Group, Department of Orthopaedic Surgery and Musculoskeletal Medicine, University of Otago
Cellular alignment contributes to the physical properties and unique biological functionality of aligned tissues such as skeletal and cardiac muscle. Therefore, biomaterial scaffolds for aligned tissue replacement or in vitro modelling should replicate the natural anisotropy of aligned tissue. Pore morphology also plays an important role in nutrient diffusion and scaffold vascularisation, further highlighting the importance of an easily optimisable pore structuring workflow. We present a custom‐ made unidirectional freezing setup, an ice templating technique that yields anisotropic ice crystals and by extension aligned pores in 3D biomaterials, made using simple additive manufacturing techniques. The setup can also regulate freezing rate and incorporate hydrogel fabrication into freezing workflow. Silk fibroin was frozen at different concentrations and freezing rates to study how different conditions impact final scaffold pore morphology.
Each condition yielded silk scaffolds with aligned pores. Concentration driven morphologies ranged from interwoven fibrous structures (2%w/v) to lamellae intertwined between fibres (4%w/v) to pores with fully defined lamellae (6 and 10%w/v). At slower freezing (0.5mm/min) rates, 4 and 6%w/v conditions presented no noticeable difference in pore morphology. Whereas the 2%w/v samples transitioned from a dense fibrillar mesh to hybrid structures of defined lamellae intertwined within fibrillar structures, demonstrating that slower freezing will sustain continuous displacement of polymer, resulting pores of improved resolution. Rapidly frozen (3mm/min) 2%w/v photoinitiated freeze‐dried hydrogels yielded well‐defined lamellae and no discernible strands. Final pore areas were tuneable between 100μm2 and 80,000μm2, and scaffolds supported cell adhesion with pore alignment directly correlating to aligned cellular morphology.
Selective modulation of single cell migration via double‐strand DNA rupture force
1Korea University
Cells sense surrounding micro‐environment through integrin‐mediated adhesion and alter their morphology dynamically to adapt to continuously changing external stimuli. Cell migration is one of the most essential features of diverse cellular functions such as wound healing, immune response, and cancer metastasis. Previous studies have shown that cell adhesion and spreading is determined by the integrin‐ mediated single molecular force. However, how the single molecular force between integrin and ligand determines cell behaviors such as cell differentiation and cell migration remain confusing. Here we present that the single molecular force across integrin determines the cell motility by regulating integrin expression and molecular force‐dependent protein activation. We precisely control the integrin‐mediated single molecular force precisely using double‐strand DNA rupture force. Our results show that cell spreading area decrease as the integrin‐mediated single molecular force becomes weak. We note that phosphorylation of focal adhesion kinase which regulated by integrin activation, is dependent on integrin‐ mediated single molecular force. Ultimately, adhesion‐dependent cells display distinct modes of migration in response to the single molecular force across integrin. We expect our results could provide new insight into the cell adhesion and migration in diverse in tissue environments.
Fabrication and characterisation of hybrid nanocollagen‐ gelatin thermoresponsive hydrogel for skin tissue engineering application
1Center for Tissue Engineering and Regenerative Medicine, The National University of Malaysia (Universiti Kebangsaan Malaysia), 2Department of Chemical and Process Engineering, The National University of Malaysia (Universiti Kebangsaan Malaysia)
Collagen is a polymer that has been used in tissue engineering as it is a natural biomaterial. However, collagen fabricated into nanocollagen could be a suitable carrier for any potential drugs or growth factors for deep skin tissue injury. Hence, this study aims to fabricate and characterise nanocollagen with gelatin hydrogel (NGH) for skin tissue engineering. Briefly, nanocollagen was fabricated via cryogenic milling, followed by mixing with graphene oxide (GO), producing three treatment groups: pure nanocollagen, nanocollagen with 0.005% GO, and nanocollagen with 0.01% GO. The fabricated nanocollagen was encapsulated with 7% gelatin hydrogel crosslinked with 0.1% genipin. It is characterised using Fourier transform infrared (FTIR) spectroscopy, X‐ray diffraction (XRD), energy dispersive X‐ray (EDX) spectroscopy, water vapor transmission rate (WVTR), mechanical stimulus test, tensile modulus test, particle size, polydispersity index (PDI) and zeta potential. The results demonstrated that the nanocollagen produced have particle sizes of less than 100 nm, and the addition of GO prevented agglomeration. FTIR spectroscopy results displayed Amide A, B, and I, II, III (1800 to 800 cm−1) in the nanocollagen treatment groups, together with C‐O‐C stretching characteristic to GO. MTT assay and Live and Dead assay were performed on the nanocollagen treatment groups alone and in combination with gelatin hydrogel using concentrations of 0.015, 0.0015 and 0.00015 mg/ml, demonstrated that as the nanocollagen concentration decreases, the cell viability increases. Based on current results, the fabrication process successfully produced nano‐sized collagen, with cell viability and metabolic assays suggesting that lower nanocollagen concentrations increases cellular viability.
Decellularised human umbilical arteries: exploring its potential as a readily available off‐the‐shelf coronary graft
1Universiti Kebangsaan Malaysia, 2Centre for Tissue Engineering and Regenerative Medicine (CTERM), Universiti Kebangsaan Malaysia (National University of Malaysia), 3Department of Surgery, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, 4Department of Obstetrics & Gynaecology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, 5Centre for Tissue Engineering and Regenerative Medicine (CTERM), Universiti Kebangsaan Malaysia
Coronary artery bypass grafting (CABG) remains the gold standard treatment option for patients with severe arterial occlusion, with autologous vessels such as the saphenous veins and internal thoracic arteries representing the most used bypass conduits to date. However, the complications of autograft surgery, which ranges from donor site infection to oedema and donor site morbidity warrants the need for an alternative bypass conduit. Advancements in tissue‐engineering techniques have allowed for the development of decellularised extracellular matrix (ECM)‐based vascular grafts that show great promise as functional alternatives to autologous vessels for use in CABG. Human umbilical arteries (hUA) are a widely available source of small‐diameter arteries that can easily be harvested with minimal complications. hUA segments were decellularised through the perfusion of varying concentrations of sodium dodecyl sulphate (SDS)‐based decellularisation solutions using a custom‐built perfusion bioreactor for different time periods, and the decellularisation efficiency was analysed and compared to the minimal decellularisation criteria. Histological analysis of decellularised hUA samples revealed an absence of cell nuclei, while DNA quantification and gel electrophoresis confirmed a significant reduction in DNA concentration and band sizes as opposed to native hUA samples. Collagen and elastin quantification assays were conducted to determine treatment effects on ECM constituents, and a negative correlation between treatment concentration and duration, and collagen and elastin concentrations were observed. These results proved that the decellularisation protocol was able to successfully decellularise hUA while preserving a majority of its ECM composition.
Dermal extracellular matrix‐derived nanoparticles improve the biological relevance of gelatine bioinks for future wound healing applications
1Centre For Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Cheras, Kuala Lumpur, 56000
Multifunctional biomaterials with high compositional values of instructive signals hold significant potential for improving cell growth, neighbouring cell recruitment, dimensional expansion, and boosting tissue morphological and functional maturation. Decellularized extracellular matrices (dECMs) are known for their promising pro‐regeneration capacity, offering a good source of cell‐free, proteins‐ and growth factors‐rich biomaterials. However, the 3D modelling of dECM‐derived biomaterials into tissue resembling architectures is still challenging. Thus, embedding dECM‐derived biomaterials as micro or nanoparticles into mouldable hydrogels may be the solution. In this study, human dermal dECM‐derived nanoparticles (dECM‐NPs) were fabricated via liquid nitrogen‐assisted cryomilling, and their compositional values and biocompatibility with human dermal fibroblasts (HDFs) were extensively evaluated. Furthermore, dECM‐NPs were employed to enhance the biological relevance of gelatine bioinks and bioprinted with HDFs into 3D multilayered scaffolds for in vitro wound healing. The results suggest that dECM‐NPs improved HDFs metabolic activity and mitochondrial amplification, increased HDFs mitochondria membrane permeability, and did not induce DNA damage, inflammation, or apoptosis. Post‐bioprinting, gelatine‐embedded dECM‐NP bioinks supported cell viability (>90%) and improved cell migration and proliferation (p < 0.0001) for 14 days in comparison to controls. Immunofluorescence staining showed enhanced cell organization and the formation of neo‐matrix secreted by HDFs. In conclusion, dECM‐NPs appear to substantially improve gelatine bioactivity, promote cell growth and cell‐material integration, and may represent a future turnover in biomaterials design into more relevant tissue‐mimicking models and transplants.
The development of multifunctional nerve guidance conduit using milk derived protein for peripheral nerve regeneration
1Department of Nanobiomedical Science & BK21 FOUR NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan, 31116, Republic of Korea and Center for Bio‐Medical Engineering Core Facility, Dankook University, Cheonan, 31116, Republic of Korea, 2Department of
Nanobiomedical Science & BK21 FOUR NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan, 31116, Republic of Korea, 3Department of Nanobiomedical Science &
BK21 FOUR NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan, 31116, Republic of Korea and Medical Laser Research Center, College of Medicine, Dankook University, Cheonan 31116, Republic of Korea
Peripheral nerve regeneration consists of a highly orchestrated phase: inflammation, proliferation and remodeling. The distal region of damaged nerve is cleared the tissue debris via M1 macrophage, which then polarizes to M2 macrophage that leads proliferation and migration of Schwann cell (SC). SC induces regenerative axonal sprouts and remyelination of axons for nerve remodeling. Recently, several studies reported that milk‐derived protein (MDP), casein was composed of various multifunctional bioactive peptides such as β‐casochemotide‐1, κ‐CAS, β‐casomorphins and FLPYPY that have abilities of immune modulation and stimulating neurite outgrowth. We hypothesized that the multifunctional effects of MDP would assist each phase of the peripheral nerve regeneration process. In this research, we designed a nerve guidance conduit (NGC) that was fabricated through a physical cross‐linking between MDP and polyvinyl alcohol (MDP‐NGC). Then, MDP‐NGC was surface‐modified with 3,4‐dihydroxy‐l‐ phenylalanine (DOPA) for improving cell attachment (MDP‐DOPA‐NGC). Both MDP‐NGC and MDP‐ DOPA‐NGC showed proper mechanical properties and degradation rate. Especially, MDP‐DOPA‐NGC showed immunomodulatory effect and neurogenic differentiation ability in in vitro. To prove the multifunctional effect of each NGC, we implanted NGC to rat sciatic nerve defect of 10 mm. In the early stage, MDP‐DOPA‐NGC showed lower expressions of M1 and higher expressions of M2‐related markers than autograft in 1 week. Furthermore, the MDP‐DOPA‐NGC showed a re‐bridged and highly remyelinated axon with increasing Schwann cell proliferation compared to MDP‐NGC after 8 weeks. This study successfully demonstrated the functionality of MDP‐DOPA‐NGC in nerve regeneration and the MDP‐DOPA platform would be suitable for utilization in treating another musculoskeletal disease.
Hydroxy‐conjugated bifacial scaffolds for localized drug delivery system
1Yonsei Univ.
Herein, we present a scaffold adhesive patch made of electrostatic conjugation between heparin and chitosan. Heparin is used to prevent blood clotting and prevent blood clots in nature. Therefore, It is potent for hydrophilic conditions like the human body against the side effects like blood coagulation of a scaffold. Chitosan is used in many pharmaceutical and medical applications including biocompatible, anti‐inflammatory, and anti‐oxidant. It has a positive charge by the primary amino group which can make electrostatic interaction negative charge of red blood cells in the blood. To address the disadvantages of chitosan, heparin assists in blocking blood clot formation in the human body. Also, we add to chitosan a bioadhesive group that makes the chitosan derivative soluble in neutral pH and helps to efficient electrostatic interaction with heparin in physiological pH. The bifacial layered scaffold showed distinctive physicochemical properties for each side, especially hydrophilicity and bio‐adhesiveness. In the current study, as a therapeutic aspect, adeno‐associated viral (AAV) vectors were chosen due to their safety, such as lower immunogenicity and non‐pathogenicity. And AAV has high infectious properties to most therapeutically relevant cells, including stem cells, cancer cells, or a variety of primary cells. In conclusion, the fabrication method discussed in the current study hold great potential as a gene delivering the vehicle for numerous human diseases (e.g., gene/cell therapy, cancer therapy, or regenerative medicine) via the synergistic combination of advanced scaffold technologies and promising gene carriers (i.e., AAV).
Engineering autologous vascularized thrombus implants for enhancing cutaneous wound healing
1UNIST (Ulsan National Institute of Science and Technology)
Autologous biomaterials have received great attention as a source of implantable scaffolds. Despite considerable efforts, current approaches for fabricating autologous scaffolds have the insufficient capability to construct microvascular networks uniformly in an implant to improve the treatment effectiveness. To address this unmet challenge, we developed an implantable vascularized engineered thrombus (IVET) using autologous whole blood, which enables a robust blood vessel reconstruction for effective wound healing. Micropillar arrays in a microfluidic channel induced shear stress along with continuous blood flows, resulting in the alignment of fibers (fibrin and von Willebrand factor (vWF)) along with the flow direction in the engineered thrombi. We confirmed that endothelial cells in IVETs are fully matured to perfusable microcapillary due to the vasculogenesis‐supportive environments constituted of aligned nanometer‐scale thrombus fibers and shear‐activated platelets. We employed these 3D fiber‐ aligned thrombi to construct a free‐standing IVET and observed superior recovery on full‐thickness skin wounds after implanting IVETs. The microcapillary in the implanted IVETs was anastomosed with host vascular networks, enabling rapid transportation of essential substances, including neutrophils, during the initial wound healing process. Furthermore, we confirmed that IVET‐treated wounds in a pro‐ inflammatory state rapidly transitioned to an anti‐inflammatory state by predominantly recruiting M2 phenotype macrophages. The wounds treated with IVETs recovered more rapidly and scarlessly, showing near‐complete re‐epithelialization. Most importantly, it also enhanced hair follicle regeneration and collagen deposition, much like normal dermis. Our work has an extensive utility to manufacture vascularized autologous implants, providing a useful tool to reveal unknown parameters involved in vascularized wound regeneration.
Fabrication of phycocyanin based fibrous membrane coated fish collagen for bone regeneration
1Pukyong National Univetsity, 2Pukyong national univetsity, 3National Marine Biodiversity Institute of Korea, 4Korea Institute of Ocean Science & Technology
Research on nano/micro fibrous membranes for help tissue regeneration has been extensively performed. Many studies have reported the application of tissue engineered fibrous membranes fabricated by various synthetic and natural polymers on bone tissue regeneration application. In this study, we fabricated a composite nano/micro fibrous membrane using phycocyanin extract from Spirulina maxima, polylactic acid (PLA), and sodium alginate (SA) and subsequent hydrophilize surface modification by fish atelocollagen extracted from Paralichthys ovlivaceus. In vitro studies showed that the phycocyanin was cytocompatible and increased the expression of alkaline phosphatase (ALP) and mineralization though MC3T3‐E1 cells. Micrographs of PLA, PLA/sodium alginate (PLA/SA), PLA/calcium alginate (PLA/CA), PLA/calcium alginate/phycocyanin (PLA/CA/PC), PLA/calcium alginate/atelocollagen/phycocyanin (PLA/CA/PC/Col) fibrous membranes obtained from scanning electron microscope showed uniform fibrous architecture with finely deposited collagen. The surface water contact angle of each fibrous membrane was gradually decreased from 118°, 64°, 50°, 24°, to 22° with respect to PLA, PLA/SA, PLA/CA, PLA/CA/PC and PLA/CA/PC/Col fibrous membranes. Cell adhesion was observed through fluorescence staining, and Alizarin Red S staining was used to confirm osteoblast differentiation and bone regeneration in the fibrous membranes. Micro‐CT and histomorphological evaluation of in vivo skull bone formation mice models further indicated an expedite bone tissue regeneration with respect to PLA/CA/PC/Col membrane compared to PLA membrane. Overall, the results confirmed that the PLA/CA/PC/Col fibrous membrane was a potential tissue engineered substitute for bone tissue regeneration applications.
A bio‐adhesive hyaluronic acid hydrogel for pH‐versatile biomedical applications
1Department of Biotechnology, Yonsei University, Seoul, Republic of Korea, 2Department of Biomedical Engineering, Sungkyunkwan University (SKKU), Suwon, Republic of Korea
Catechol, a key functional group for strong underwater adhesion of mussels, has led to the development of adhesive hydrogels for biomedical applications. However, its working mechanisms for interpolymer crosslinking and surface adhesion are highly dependent on oxidation‐based chemistries naturally favoring alkaline pH, which limits the use of such materials to only certain favorable environments. To overcome this limitation, this study introduces a mussel protein‐inspired polymer design, consisting of catechol and reactive pendant groups, to hyaluronic acid (HA) through dual modes of dopamine tethering to HA, allowing pH‐universal catechol‐amine chemistry. The catechol‐functionalized modified HA hydrogel exhibits not only fast gelation and strong adhesion but also active crosslinking under a wide range of pH conditions. Thus, the developed hydrogel enables rapid and robust wound sealing and hemostasis in neutral and alkaline conditions, respectively. In addition, it allows therapeutic cell and drug delivery through active crosslinking and firm adhesion even within dynamic and harsh environments such as motile heart and highly acidic stomach. Ultimately, the pH‐versatile bio‐adhesive hydrogel provides a promising biomaterial for various biomedical applications in a wide range of pH conditions in vivo. This work was supported by the Technology Innovation Program (Alchemist Project, 20012378) funded by the Ministry of Trade, Industry & Energy (MOTIE), Republic of Korea.
Acellular matrix film incorporating phlorotannins from Eckloinia cava suppressed post‐implantation inflammatory responses
1Pukyong National University, 2Inje University, 3Kangwon National University School of Medicine,
4National Marine Biodiversity Institute of Korea, 5Jeju Marine Research Center, Korea Institute of Ocean Science & Technology (KIOST)
Peritendinous adhesion mainly occurs between proliferating fibrous tissues and adjacent normal organs after surgery. Many physical barriers are applied to the implanted site to prevent peritendinous adhesion. However, these barriers often trigger inflammatory responses. Therefore, our study sought to develop phlorotannins‐loaded cartilage acellular matrix (CAM) films as a physical barrier and investigate their inhibitory effect on inflammatory responses, which are associated with the induction of postoperative peritendinous adhesion (PAA). Our findings indicated that incorporating phlorotannin into the CAM film did not affect its unique characteristics including its thermal and spectroscopic properties. Moreover, the phlorotannins‐loaded CAM films suppressed the expression of inflammatory mediators on RAW 264.7 macrophages stimulated using Escherichia coli lipopolysaccharides and exhibited an anti‐inflammatory effect when implanted subcutaneously in rats. Therefore, our results highlight the potential of phlorotannins‐loaded CAM films as a promising physical barrier for preventing PAA.
Fish collagen/PCL nanofibrous scaffolds with cross‐linked chitooligosaccharides for full‐thickness wound healing
1Pukyong National University, 2Korea Institute of Ocean Science & Technology, 3National Marine Biodiversity Institute of Korea
In wound‐healing applications, tissue‐engineered biodegradable artificial tissue substitutes with extracellular matrix‐mimicking features are of great interest. In present study, electrospinning was used to create novel bilayer nanofibrous scaffolds made of fish collagen (FC) and poly(‐caprolactone) (PCL), with chitooligosaccharides (COS) covalently attached by carbodiimide chemistry. Altering suitable electrospinning conditions, the architecture and fiber diameter of non‐cross‐linked nanofibrous scaffolds were maintained similar independent of polymer ratio, however the fiber diameter changed after cross‐ linking in response to FC concentration. According to Fourier‐transform infrared spectroscopy (FTIR) analysis, the biomaterial blend was homogeneous, with an increase in COS levels with increasing FC concentration in the nanofibrous scaffolds. According to cytocompatibility analysis, the nanofibrous scaffolds with high FC content were functionally active in response to normal human dermal fibroblast neonatal (NHDF‐neo) and HaCaT keratinocyte cells, resulting in the generation of a very effective tissue‐ engineered implant for full‐thickness wound‐healing applications. In addition, an evaluation of the suggested COS‐containing FC‐rich FC/PCL (FCP) nanofibrous scaffolds' swelling, hydrophilicity and mechanical characteristics verified that they have considerable promise for use as tissue‐engineered skin implants for skin tissue regeneration.
Ovine collagen type‐I (OTC‐I) biomatrix integrated with antibacterial coating for rapid treatment in diabetic wound care management
1Centre for Tissue Engineering and Regenerative Medicine, Universiti Kebangsaan Malaysia, 2Institute of Microengineering and Nanoelectronics, Universiti Kebangsaan Malaysia
Emerging research for chronic wound diseases such as diabetic ulcer (DU) involves fabricated ready‐to‐ use biomaterial that provides not only rapid treatment but also prevents infections and supports angiogenesis. This study focuses on evaluating an antibacterial extract (R)‐(‐)‐Carvone from mint in a monolayer ovine collagen type I (OTC‐I) biomatrix crosslinked with a natural crosslinker, genipin, for mechanical strength. Carvone has functional groups with antimicrobial effects that can be made into a suitable precursor to combine on freeze‐dried OTC‐I biomatrix via plasma polymerisation treatment. The composite biomatrix is fabricated and evaluated in vitro for physicochemical properties, antibacterial, angiogenic and cytocompatibility. Plasma polymerised carvone deposited on OTC‐I showed strong evidence of a suitable biomaterial in terms of physicochemical properties of porosity, contact angle, swelling ratio and biodegradation rates. The antibacterial assay showed that there was more than 60% cell death for both negative and positive gram bacteria post carvone coating. Angiogenesis assays displayed positive tubule formation of HUVEC cells posts carvone coating compared to control. SEM and cell attachment assay suggest more than 90% cell attachment with live/dead assay proved to be live cells 48 hours post‐seeded of human dermal fibroblasts. Proliferation assay indicates a 20% increment after 7 days. Further study in vivo is proceeding. In this work we present that crosslinked OTC‐I biomatrix coated with carvone conveys strong outcomes on wound healing as well as synergistic functions of acellular treatments, advancing future therapeutic use for DUs using tissue‐engineered substitutes.
Fabrication of antioxidant and anti‐inflammatory hydrogel based on fish skin gelatin/oxidized hyaluronate for accelerated wound healing
1Pukyong National University
Excessive inflammation is a major problem in chronic wounds caused by trauma, burns, infection, and medical conditions. In this study, we report a hydrogel that successfully accelerated wound healing by modulating inflammation. The hydrogels were cross‐linked with fish gelatin (FG), which has recently emerged as a substitute for gelatin derived from mammalian sources and oxidized hyaluronate (OHy), used as a natural macromolecular crosslinker. FG from Paralichthys olivaceus skin was extracted with hot water and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), hydroxyproline content, and Fourier transform infrared (FTIR) spectroscopy. Then, hydrogels with different ratios of FG and OHy were evaluated for morphological and rheological properties, swelling capacity, and degradability. In an in vitro studies, fabricated hydrogels were showed non‐cytotoxicity against human dermal fibroblasts (HDF) and RAW 264.7 macrophages. The hydrogels exhibit anti‐ inflammatory activity by inhibiting the nitric oxide (NO) production in lipopolysaccharide (LPS)‐ stimulated RAW 264.7 macrophages. Besides, the production of reactive oxygen species, the major indicators of free radicals producing oxidative stress, was reduced by fabricated hydrogels. These results demonstrated that FG/OHy hydrogels can be successfully used for chronic wound healing applications.
Fabrication of injectable iron(III) crosslinked hyaluronic acid/pectin hydrogel with antimicrobial activities
1Pukyong National University
Hyaluronic acid (HA) and pectin (PT) have been usually applied as base materials for hydrogel in tissue engineering and fabricate by crosslinking with multivalent ions. In this study, we successfully fabricated HA/PT hydrogels with various blending ratio (HA3PT7, HA5PT5, and HA7PT3) using Fe3+ ion cross‐ linking. Iron(III) crosslinked HA/PT hydrogel has a great potential for wound healing application due to its self‐healing ability through the interaction between Fe3+ and –COOH group of HA and PT. Self‐ healing hydrogel for wound dressing has a prolonged lifespan by defending on defects in mechanical properties with the possibility of autonomous self‐healing against damage. In addition, fabricated HA/PT hydrogels showed a antimicrobial activities against bacteria (gram‐negative Pseudomonas aeruginosa and gram‐positive Staphylococcus aureus) due to Fe3+ release after degradation. To investigate the characteristics of the HA/PT hydrogels were analyzed using Fouirer‐transform infrared spectroscopy, scanning electron microscope, and rheometer. Among them, the HA5PT5 hydrogel showed the highest mechanical properties and antibacterial activity without cytotoxicity. These results suggested that HA5PT5 hydrogel has a potential as injectable hydrogels for wound healing.
Characterisation of native tissue and development of multiphasic scaffolds for engineering of bone‐ligament interface
1University of Oxford
The enthesis, or interface, between bone and ligament, has a highly sophisticated biomaterial function. Injury to the enthesis often requires surgical intervention, however, due to poor regeneration during healing, this transitional tissue is prone to rupture recurrence. Tissue engineering offers the potential to recover the functional integrity of enthesis. However, its regeneration is limited by a lack of understanding of the structure‐function relationship. Therefore, focusing on the insertion site between the anterior cruciate ligament (ACL) and bone, the first half of this study evaluated the mechanical and compositional variations through the enthesis using histological staining, spectroscopy and both macro and nanoscale mechanical testing. These experiments confirmed the gradient increase in hardness and stiffness along the enthesis with increasing mineral content.
Utilising this critical insight into the structure‐function relationship, the second half of this study focused on developing collagen‐based multilayer scaffolds for ligament‐bone regeneration. Four collagen constructs were investigated to mimic the various regions: a ligament layer composed of type I monomeric collagen; an uncalcified fibrocartilage layer composed of type II monomeric collagen and chondroitin sulphate; a calcified fibrocartilage layer composed of type I monomeric collagen and less hydroxy apatite; a bone layer composed of type I monomeric collagen and hydroxy apatite. Multi‐layered structures comprised of these layers were shown to have a seamlessly integrated structure and high levels of porosity and pore interconnectivity.
Greater understanding of the native tissue allowed for the engineering of more complex and promising multi‐layered structures which could have great potential in future tissue engineering endeavours.
Decellularized plant and fungal‐based scaffolds for the in vitro production of bovine meat
1Yeungnam University
In vitro meat development from validated tissue engineering techniques has emerged as a more sustainable and ethical method of meat production. Plant‐ and fungal‐based scaffolds present many advantages over a variety of biomaterials. Although the plant parenchyma provides a comparatively porous and biocompatible substrate with stiffness values. The microchannels of the vascular tissue mimic the microenvironment and topographical cues for muscle cells. In this study, we fabricated plant‐ and fungal‐based scaffolds of jujube (DJF) and mushrooms (DMS), respectively that have the potential to contribute similar properties to the meat along with mechanical properties and nutritional benefits.
The decellularization strategy was performed for the formation of scaffolds. After decellularization of plant scaffolds, water was removed and 70% ethanol was added for sterilization, samples were carried out for further studies such as morphological analysis, stress‐strain, storage modulus, and loss modulus. The capacity of the decellularized plant‐ and fungal‐based scaffolds to enable myocyte adhesion, viability, and proliferation was tested in vitro. Morphological analysis of DMS was measured as 20‐73 μm diameter of porous structures and DJF were developed porous and patterned substrates, which are capable of recreating an aligned arrangement. The results of compressive strength were high in DJF compared to DMS. We found similar topography and mechanical properties in decellularized scaffolds. The proliferation of bovine myocyte cells on the DJF scaffold was significantly higher than on the DMS scaffold. This presents an inexpensive, sustainable scaffold material that is capable to get the desired property for the production of vegetative meat.
3D bioprinting of islet‐like aggregates using dual‐crosslinked hydrogel with promoted biofunctionality and enhanced shape stability
1Pohang University of Science and Technology (POSTECH)
Various external cues such as structural, mechanical and chemical cues are considered to have important role at guiding cellular behavior and maturation in tissue engineering. To achieve such affects, 3d bioprinting technologies are being developed over conventional tissue engineering approaches. Microextrusion‐based bioprinting, which is one of bioprinting strategies, can recapitulate the physiologically relevant architectures of organs by applying cells with biomaterials into tissue‐speciifc structure. Here, the biomaterial should possess biocompatibility, shape stability, and printability. In this study, we developed dual‐crosslinked hydrogel with promoted biofunctionality and shape stability using decellularized extracellular matrix (dECM) and alginate. The ECM‐based biomaterials are well‐known to provide mircroenvironment for cells but have low shape‐stability, which limits the 3D tissue printing. To overcome the limitation, we adopted the alginate and pre‐crosslinked with calcium gluconate. The relatively slow crosslinking time of calcium gluconate enabled dECM to blend with alginate. The rheological analysis result showed that the shape‐stability of alginate/dECM bioink was significantly increased while the printability was maintained compared to dECM only. The bioprinted tissue construct could maintain its shape without any support bath or structure. The encapsulated cells were viable and proliferated well in alginate/dECM bioink and the functional aspect was promoted.
Dual controlled photocrosslinkable and photodegradable gelatin‐based hydrogel
1Department of Materials Science and Engineering, National Tsing Hua University
Implantable fillers have been widely used in clinical and biomedical applications, such as skeletal muscle repair, tissue regeneration, drug release, etc. However, it is not always satisfied to patients after implantation and needs another surgery to remove implants. The ideal fillers would not only be selected by its biocompatibility, but also by its tunable degradability and stiffness according to clinical needs. Recently, many types of hydrogels with controllable stiffness have been developed, but most of them only can irreversibly increase or decrease of its stiffness. It is not easy to post‐tune hydrogel stiffness after gelation process, which limits its applications to mimic physiochemistry microenvironments in animals.
Here, we conjugated dibenzocyclooctyne (DBCO), nitrobenzyl‐azide, (NBazide), photodegradable molecules, and methacrylamide (MA), photocrosslinkablle molecules, on gelatin molecules to synthesis gelatin‐based hydrogels. First, the hydrogel was crosslinked by the click reaction between DBCO and NBazide. To achieve one or locally patterned hydrogels with bidirectionally tunable mechanical properties, the hydrogels were exposed to different light source under the designed mask. Then, human mesenchymal stem cells (MSCs) were encapsulated into this hydrogel to evaluate the effect of dynamic regulation of hydrogel mechanical properties on cell behavior. Results demonstrated that this gelatin‐ based hydrogel is biocompatible, and byproduct during crosslinking and degradation after exposing to UV light was shown harmless to cells. Cell proliferation rate and spreading areas could be reversibly controlled by adjusting the mechanical property of the hydrogel, representing our gels have great potential in regenerative medicine and tissue engineering.
In situ forming and copper‐containing hydrogel as a controlled nitric oxide‐releasing scaffold for tissue engineering
1Department of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of Korea (*kdp@ajou.ac.kr)
Nitric oxide (NO), an endogenous gas molecule, has been indicated as a critical modulator for various therapeutic applications, such as the treatment of vascular disorders, wound healing, and cancer treatment. However, the short half‐life of NO in living systems is a great challenge to its clinical applications. Herein, copper (Cu) ions incorporated in phenol‐rich gelatin‐based hydrogel (GH/Cu) were prepared for in situ generations of NO in the presence of endogenous NO donors. During the enzymatic reactions of horseradish peroxidase (HRP) and tyrosinase, cross‐linked networks of GH and Cu nanoparticles were formed simultaneously. The physicochemical properties of GH/Cu hydrogels such as gelation time, mechanical strength, and degradation rate were well controlled with varying the concentrations of HRP and H2O2. Most importantly, in vitro release profile of NO from hydrogel matrices was precisely controlled with varying the concentration of Cu ions over 2 weeks. Moreover, migration and tube formation activities of human umbilical vein endothelial cells were stimulated in the presence of GH/Cu hydrogels. The ex ovo chick chorioallantoic membrane assay and in vivo subcutaneous injection assay showed that the NO‐releasing GH/Cu hydrogels promoted neovascularization and host tissue infiltration. As a result, we successfully developed NO‐releasing gelatin‐based hydrogels as injectable and dynamic matrices for tissue engineering applications.
3D printing of biohybrid electrical stimulation platform to promote insulin secretion of pancreatic β cell
1POSTECH, Pohang, Gyeongbuk, South Korea
The electrophysiological activities of the pancreatic β cells are crucial for appropriate insulin secretion and blood glucose level control. In recent studies, it is confirmed that external electrical stimulation can be applied to regulate the intracellular calcium dynamics promoting insulin secretion. From the physiological point of view, long‐term analysis is needed for a better understanding of the circadian rhythm of insulin secretion. The in vitro platform benefits in continuous culturing with electrical stimulation and observation of the effects on the cells. The advancements in 3D printing technology facilitated the fabrication of electrode integrated platforms with conductive materials. In this study, we 3D printed a biohybrid tissue stimulation platform where the electrical stimulation can be applied through electrodes printed with carbon nanomaterial‐filled polymer ink. The developed conductive ink was 3D printed for electrical stimulation with high conductivity, biocompatibility, and printability. To demonstrate the ability to apply electrical stimulation to the tissue, the MIN6 cells are cultured with biphasic electrical pulse stimulation. The cell showed high cell viability, and the calcium activity inside the cell was observed. We confirmed the ability to construct a biohybrid 3D‐printed platform for electrical stimulation and the possibility to apply electrical stimulation to elevate insulin secretion. Replicating the electrophysiological property contributes to the higher performance of in vitro and in vivo pancreatic tissue. In addition, the biohybrid platform may provide a large opportunity to apply to other tissues such as cardiac, muscular, and neural tissues.
Enzyme‐based on‐demand photo‐cross‐linkable hydrogel for image‐ guided vascular embolization
1Donga Univ
Vascular embolization provides an effective approach to treating bleeding, aneurysm, and other vascular abnormalities. However, there is a problem with the lack of visibility of blood vessels to approach the target site during surgery. Current embolization materials, such as metal coils and liquid embolizes, are limited in that they cannot provide safe consistently controlled embolization. In this study, we developed polyphenol‐based carbon dots that can induce images and then report injectable hydrogels that can be crosslinked using a photo‐initiator in the field. Polyphenol‐based carbon dots are nanoparticles with visible optical properties and anti‐inflammation. The HA‐EGCG conjugates produced by the combination of HA and EGCG are expected to exhibit not only viscoelastic, biocompatible, and biodegradable properties of HA but also various biological activities, including anti‐inflammatory properties of EGCG. Photo‐activated tyrosinase was designed to allow quick photo‐crosslinking by exposure to UV. Polyphenol‐based injectable hydrogels are easy to distribute through needles and catheters, demonstrating a fast transition from liquid to solid via cross‐linked bonds at target sites in a short time. Additionally, the optical properties of polyphenol carbon dots make it quite easy to track the location of the material during surgery because it is visible in the image. From this study, we expect that polyphenol‐based carbon dots with hyaluronic acid provide a viable, biocompatible, and efficient alternative to existing embolic material limitations.
Development of light‐blocking nanofiber membrane for a three‐ dimensional in vitro angiogenesis model capable of real‐time selective imaging
1Kyungpook national university, 2Kyungpook national university hospital, 3Pohang University of Science and Technology (POSTECH)
Electrospun nanofiber membranes have great potential for modeling angiogenesis in vitro as they involve degradation and invasion through the basement membrane. In angiogenesis, vascular endothelial cell invasion is known as one of the important mechanisms. Nanofiber membranes have translucent properties, and these features overlap images of invaded and non‐invaded cells, preventing clear image acquisition and accurate analysis. To overcome the limitation, we developed a carbon black‐ polycaprolactone (CB‐PCL) nanofiber membrane that completely blocks light and in vitro angiogenesis platform using it. The CB‐PCL nanofiber membrane was manufactured as a 24‐well insert type with a thickness of 42.88 ± 5.95 μm and a pore size of 4.70 ± 1.32 μm. Vascular endothelial cells were cultured on the upper surface of the 24‐well insert with the fabricated CB‐PCL and then treated with vascular endothelial growth factor (VEGF) on the basolateral side. At 100 ng/ml VEGF, cell invasion was time‐ dependent, and cells were completely invaded at 7 days. Also, it was demonstrated that cell invasion was inhibited by treatment with 10 ug/ml anti‐VEGF in the angiogenesis‐induced model. In cell invasion assay using fluorescence microscopy, the CB‐PCL nanofibrous membranes can clearly discriminate between apical to basolateral invaded cells through angiogenesis compared to synthetic porous and PCL nanofibrous membranes. The 24‐well insert‐based platform containing the CB‐PCL nanofiber membrane selectively visualizes only invaded cells so that even non‐experienced researchers can easily use and analyze it. Besides, fluorescence intensity‐based invasion cell analysis can provide rapid and meaningful results in the large‐volume screening of drug candidates.
Development of silk‐based cultured meat scaffold with aligned fibrous texture
1National University of Singapore
Cultured meat is an innovation that is touted to be able to overcome inefficiencies and environmental impact of current meat production practises and is expected to have significant impact on society to tackle the rising demand for meat. However, technical challenges have made the translation of cultured meat into commercialisation difficult, mainly due to difficulties in recapitulating several key characteristics that gastronomically define meat such as texture, flavour and colour. While there has been success in replicating the flavour and colour of meat using additives, the same cannot be said of texture, which is hindered mainly by challenges in scalability. To tackle this, we present a method to produce aligned texture fibrous scaffolds using Silk Fibroin (SF), herein termed ‘vortex spinning’. Containing proteins as its main constituent, SF is a bio‐material that exhibits the ability to be processed into various forms and has been widely used in muscle tissue engineering. The production method processes SF to form aligned fibrous scaffolds without the use of toxic solvents or reagents. The size and thickness of scaffolds produced using this method can be adjusted to the desired size by modifying the size of the collector base. Texture profile analysis and mechanical testing of the SF‐based scaffolds show similar mechanical profiles compared to cooked meat. C2C12 cells were seeded on these scaffolds, and it was demonstrated that the seeded cells could proliferate on these scaffolds. This work shows that cultured meat can be produced using SF and can replicate fibrous texture found in meat.
Harnessing the in vivo inflammatory response for tissue engineering
1University of Manchester
The inflammatory response to implanted biological material can create a proregenerative immune environment which can be harnessed for tissue engineering [1]. Using the surgical concept of pre‐ fabrication [2], we created a pro‐angiogenic in vivo rodent flow through surgical model to evaluate the vasculogenic properties of biological materials, with respect to macrophage recruitment and polarisation, neovasculature as well as neo‐tissue formation.
We evaluated a range of biomaterials, consisting of synthetic self‐assembling peptide hydrogels (Manchester BIOGEL alpha 4), commercially available decellularized matrix (Jellagen), synthesised porcine adipose tissue decellularized matrix [3] as well as a widely used photocrosslinkable hydrogel (Gelatin Methacrylate 7.5% w.t.) [4]. The flow through models was created on Sprague‐Dawley rats hind limbs where the femoral vessels were dissected and placed within 3D printed chambers containing the biomaterials. Short term evaluation of the tissue response (2 weeks) will be reported.
1. Dziki, J.L., et al., Extracellular Matrix Bioscaffolds as Immunomodulatory Biomaterials. Tissue Eng Part A, 2017.
2. Erol, O.O. and M. Spira, New Capillary Bed Formation with a Surgically Constructed Arteriovenous‐ Fistula. Plastic and Reconstructive Surgery, 1980.
3. Poon, C.J., et al., Preparation of an adipogenic hydrogel from subcutaneous adipose tissue. Acta biomaterialia, 2013.
4. Shirahama, H., et al., Precise Tuning of Facile One‐Pot Gelatin Methacryloyl (GelMA) Synthesis. Sci Rep, 2016.
3D chondrogenic differentiation of human stem cells in reprogramming factor‐based injectable hydrogel for cartilage tissue engineering
1Dong‐A University
Reprogramming factors from non‐human vertebrate have been investigated to induce human stem cell differentiation into chondrocytes as they have potential to regenerate tissues such as bone, cartilage, and nerve. In this study, we have specified chondrogenic factors from lizard which can regenerate amputated tails. We verified the target molecule, endoplasmin protein (ENPL) as chondrogenic factor in 2D human tonsil‐derived stem cells (hTSCs). Additionally, we designed cartilage‐mimetic hydrogel composed of hyaluronic acid (HA) and chondroitin sulfate (CS) for delivering ENPL and hTSCs. The hTSCs in 3D hydrogel structure were successfully differentiated into chondrogenic cells. Additionally, the synergistic effect of cartilage regeneration of the hydrogel with the protein ENPL was confirmed in mouse osteoarthritic model. This study demonstrates that ECM‐based ENPL‐loaded hydrogels can provide a therapeutic option for cartilage tissue engineering.
In this study, we present options for cartilage therapy of bioactive hydrogel that induce in situ implantation, systems forming cross‐links, and chondrogenesis. Here, we introduce an enzyme‐mediated crosslinking method to fabricate injectable hydrogels. HA and CS are commonly used in tissue engineering and provide the biological and chemical environment of cartilage tissue. HA‐TA and CS‐TA produced by conjugating tyramine here are advantageous for tyrosinase‐mediated crosslinking. Enzymatic reactions using tyrosinase can provide cells, proteins, and tissues with biocompatibility, cell affinity, and a biomimetic environment. In addition, we targeted HSP90b1, which is related to the survival and proliferation of chondrocytes, by paying attention to the mechanism of lizard tail regeneration in which cartilage is regenerated instead of bone to induce cartilage differentiation.
Natural‐origin injectable hydrogel for acellular skin wound treatment
1Universiti Kebangsaan Malaysia
The enhancement of skin wound cases is a great attention in healthcare sector. Unfortunately, the available treatment options nowadays are still not effective. In this research, an in situ forming genipin‐ crosslinked gelatin hydrogel, namely gelipin, has been produced. Hydroxytyrosol (HT), a natural antibacterial agent, is added into it. The formulations with three minutes gelation time have been selected to be characterised and observed their efficacy in mouse model of skin wound. Physical characteristics have been evaluated through biodegradation and swelling test in the collagenase and buffer solution, respectively. The findings revealed that gelipin hydrogel was durable for 2 weeks with good swelling ability (>100%). It also has been proven to be compatible with human dermal fibroblasts through migration and viability checking by using cell tracker and MTT assay, respectively. Animal study in BALB/c mouse with skin wound has shown that HT‐incorporated gelipin hydrogel was able to heal the wound. All the accumulated data represented good capability of HT‐loaded gelipin hydrogel to be used as a new promising alternative in wound management.
Transplantation of the cultured human corneal endothelial cells with decellularized extracellular matrix in the corneal endothelial dysfunction rabbit model
1Department of Ophthalmology, Asan Medical Center, University of Ulsan College of Medicine, 2Biomedical Engineering Research Center, Asan Institute for Life Sciences, Asan Medical Center, 3Department of Mechanical and Biomedical Engineering, Kangwon National University
The corneal endothelium is a single layer of hexagonal morphology found between the aqueous humor and the Descemet's membrane. Corneal endothelial cells (CECs) show no regeneration in vivo because of contact inhibition at the G1 phase of the cell cycle. The subsequent loss of CECs due to injury or disease is compensated by the migration of the remaining CECs. However, if endothelial cell density decreases below 500 cells/mm2, corneal physiological deterioration occurs, leading to bullous keratopathy and corneal endothelial dysfunction (CED). The bullous keratopathy can only be treated by corneal transplantation. However, a lack of suitable donor tissue and graft rejection have been the main problems preventing corneal endothelial failure‐related blindness. CECs cultured and expanded in vitro were spin‐ down onto the steel wire‐attached decellularized extracellular matrix (dECM) obtained from decellularized porcine corneal stroma as carriers of CECs. They were transplanted into the anterior chamber via a magnetic control system in the CED rabbit model. Transplantation of the CECs seeded on the dECM containing a steel wire under a magnetic control system in the CED rabbit model resulted in improved corneal transparency at 16 weeks postoperative when compared with the control group. Clarity did not improve when the steel wire‐attached dECM transplanted CED rabbit model was observed for 16 weeks. Our finding suggests that transplantation of dECM with CECs under a magnetic control system can be a new treatment option for treating the CED.
Bio‐adhesive complex coacervate‐mediated localized AAV delivery
1Yonsei University, 2Yonsei University, South Korea
Adeno‐associated viral (AAV) vector has emerged as an efficient therapeutic tool for treating rare diseases. AAV‐based therapeutics are one‐time treatments and show long‐term therapeutic effects and high transduction efficiency. However, several issues such as off‐targeting and immune response still need further investigation. Herein, a liquid phase bioadhesive AAV delivery system by complex coacervation between a catechol derivative polymer and a biocompatible polysaccharide is discussed. Complex coacervation is a liquid‐liquid phase separation phenomenon by non‐covalent interactions such as poly‐ion electrostatic interactions. A complex coacervate‐mediated gene delivery system is advantageous to address the aforementioned issues because the low immunogenicity of the materials can avoid immune responses, which are the main issues of AAV delivery. Catechol group has shown its potential in localized drug delivery due to its bio adhesiveness. In addition, the AAV can be loaded with high efficiency by the interaction with the catechol groups with viral capsid protein. To evaluate the delivering performances, the physicochemical properties of the complex coacervate system are discussed. Utilization of bio‐adhesive coacervates for delivering AAV vectors showed localized transgene expression, decreased off‐target effects, and low immunogenicity.
Thiol‐ene clickable silk fibroin bio‐ink for digital light processing bio‐ printing
1National University of Singapore
Bio‐printing is an emerging tissue engineering technique that can produce patient‐specific tissues to repair or replace damaged tissues. This is particularly the case for digital light processing (DLP) methods, where precision of bio‐printed constructs is crucial. However, the main challenge hindering it from clinical translation is in proper bio‐ink design, which is encapsulated in the ‘Biofabrication Paradigm’. Silk Fibroin (SF) is a potential bio‐ink candidate due to its desirable mechanical properties and processibility. However, SF solution itself cannot be easily bio‐printed as it has no readily available functional groups that can be used in conjunction with DLP. While SF has been functionalised as bio‐inks for DLP, such functionalisation only allows for chain‐growth polymerisation which results in crosslinked constructs with uncontrollable polymer length and may have cytotoxic effects on embedded cells. Herein, this work presents a method to functionalise SF for DLP which allows for step‐growth polymerisation, which may allow SF bio‐inks to overcome these challenges. This method involves functionalising SF with an ether group (called Sil‐E) and by using a photoinitiator in conjunction with dithiothreitol, Sil‐E could form constructs with as designed through the bio‐printer. Characterisation of Sil‐E demonstrated that this method resulted in about 50% degree of substitution. Sil‐E was shown to have mechanical properties comparable to SF functionalised with methacrylate groups (Sil‐M). Further, when embedded with primary chondrocytes, Sil‐E demonstrated cytocompatibility and allowed embedded cells to proliferate.
Biomaterials text mining: A comparative study of methods on the biocompatible polymer polydioxanone
1Institute of Biomedical Engineering, University of Oxford, 2Department of Material Science and Engineering, Universitat Politu00e8cnica de Catalunya, 3Barcelona Supercomputing Center (BSC),
4Department of Material Science and Engineering, Universitat Politu00e8cnica de Catalunya; Barcelona Supercomputing Center (BSC)
The rapid increase in the volume of biomaterials research has exceeded the capacity of researchers to manually survey all the relevant literature. Text mining tools enable the quick extraction of information from text, in an automated and accurate manner; nevertheless, there is little precedence of using these tools in the biomaterials field.
We compared the ability of various text mining methods to extract useful information from biomaterials abstracts. Focusing on polydioxanone, a biodegradable polymer with a relatively small volume of scientific publications, we used supervised and unsupervised machine learning, semantic annotations and bag‐of‐words analysis tools. Results were analysed alongside manual review of the literature, including systematic reviews and meta‐analyses.
Our findings show that existing text mining tools can be highly efficient and powerful for mapping biomaterials text, albeit different tools revealed differing aspects of polydioxanone. Specifically, they enabled detecting evolution and frequency of specific terms, such as ‘graft’ and ‘surgery’; as well as identifying main topics within the collection, for instance ‘biocompatibility’ and ‘biodegradable’. Some of the tools used in this study required substantial computational skills, limiting their use by the wider research community, pointing to a clear need for biomaterials‐specific, user‐friendly and automated tools.
The text mining tools provided a wider, broader and open‐ended view of polydioxanone. Importantly, our analysis could be performed and updated rapidly. We also identified a significant challenge in mining biomaterials articles: heterogeneous term usage.
Our work shows the potential value of using automated Natural Language (Processing NLP) and, in particular, domain‐specific tools to extract and organise biomaterials data.
Anti‐inflammatory, dry adhesive patches based on catechol‐modified sulfated hyaluronic acid for multipurpose application
1Seoul National University, 2Johns Hopkins School of Medicine
Sulfated hyaluronic acid(sHA) has attracted much interest in the field of biomedical engineering because of its stable in vivo retention, immunomodulation, and efficient drug loading ability. However, repulsion between sHA chains due to its highly negative charge makes the sHA‐based materials hard to form networks and decreases the mechanical properties. To solve this problem, we conjugated catechol moieties to sHA (sHA‐CA) and fabricated the sHA‐CA dry patch via crosslinking mediated by auto‐ oxidation of catechols and freeze ‐drying. The formation of ice crystals during freezing increased local concentrations of sHA‐CA and made efficient crosslinking between catechols. This network formation mechanism intensified the robustness of the sHA‐CA patch. Furthermore, the fast water absorption of the sHA‐CA dry patch removed water molecules on target surfaces and showed a synergic effect with adhesive catechol groups on the adhesive strength of the patch. Also, this adhesive dry patch can be used as a drug delivery system for cationic drugs due to its highly negative charge, which showed synergistic effects with the anti‐inflammatory properties of sHA in various diseases, including diabetic wounds and liver hemorrhage. In conclusion, we demonstrated that catechol conjugation of sHA and sHA‐based materials can improve the mechanical properties of sHA‐based materials and be used as effective therapeutic materials for various diseases.
Strong adhesive hemostatic agent based on catechol‐chitosan and hyaluronic acid
1Seoul National University, 2Republic of Korea Army
Uncontrolled massive hemorrhage caused by major vascular injury killed 2 million people worldwide each year. Hemorrhage accounts for 50% of preventable mortality in battlefield situations. It is also the second leading cause of death in the civilians after multiple injuries, with nearly 31% of civilians dying from bleeding and its complications. In this situations, rapid hemostasis is paramount. The ideal hemostatic agent should be able to be applied to the bleeding site quickly and easily, can stop bleeding in a rapid time, and have no side effects.
In this project, we aim to develop hemostatic agents based on chitosan and hyaluornic acid. Catechol is a functional group of dopamine that interacts with the surface in various binding methods to exhibit adhesive strength. Oxidation reaction turns catechol into a quinone, further announcing the adhesive strength. We mixed catechol with chitosan so that when applied to a bleeding environment, oxidative catechol made cross‐linking and adhesive properties. Not only that, but the catechol group made chitosan well soluble in the aqueous solution. In addition, the mechanical properties were strengthened by oxidizing hyaluronic acid to form a network with the amine group of chitosan. Above the two bio‐based polymers were mixed to form a network, lyophilized and made into a dry patch for rapid hemostasis. This biocompatible dry patch is easy to apply to bleeding sites, has a fast adhesive and hemostatic effect.
Bioadhesive cryogel for non‐compressible haemostasis during orthopaedic surgical procedure
1School of Chemical & Biological Engineering, Institute of Chemical Process, Seoul National University, Seoul 08826, South Korea, 2Institute of Engineering Research, Seoul National University, Seoul 08826, South Korea
Haemorrhages during bone tumour resection and trauma pose a significant problem for orthopaedic surgeons in controlling blood loss and they pose threat to the patient's life. Traditional haemostatic agents such as Gelfoam, Surgicel and Actifoam achieve rapid haemostasis by compression in the bleeding site, which is not suitable in most orthopaedic producers where compression can't be applied. Bone wax, a commonly used bone haemostatic agent is non‐absorbable, causes an inflammatory reaction and interferes with the bone healing process. Thus, a non‐compressible haemostatic material is required for stopping the bone haemorrhage. Herein, we propose a composite cryogel made of silanized polymer and bone biomineral. The developed cryogel shows tissue adhesion, cytocompatibility and hemocompatibility. Thus, the composite cryogel provides non‐compressible haemostasis during orthopaedic surgeries.
Integrating endothelialized microchannels with mesenchymal stem cell spheroids in a 3D‐printed construct for ischemic disease therapy
1UNIST
Ischemia is a restriction of blood supply to organs or tissues that results in irreversible damage and tissue necrosis by oxygen and nutrient insufficiency. In severe cases, local hypoxia caused by ischemia leads to heart failure, stroke, or limb ischemia. To induce blood rescue, researchers utilized delivery of various biomaterials such as growth factors, nucleic acids, stem cells, etc. However, those injection‐based treatment methods resulted in an inconsistent therapeutic effect and unexpected side effects because of the off‐target distribution. Here, we propose the fabrication of a pre‐vascularized graft consisting of endothelialized microchannels and mesenchymal stem cell spheroids encapsulated in hydrogel for ischemic disease treatment. MSC spheroids with enhanced paracrine effect were formed using dot‐ printing technology. While endothelialized microchannels that matured with in vitro culture time were patterned between spheroids with a distance for optimal intercellular interaction. As a result, the MSC spheroids pattern guided the formation of micro‐vessels sprouting from adjacent endothelialized microchannels. Moreover, bioprinted constructs with MSC spheroids showed higher angiogenic factors secretion ability. To in vivo examine the therapeutic effect of our pre‐vascularized grafts of different designs, we performed a mouse hind limb ischemia model assay. To conclude, we believe that our technique can produce a functional pre‐vascularized construct that has great potential in tissue regeneration and ischemia therapy.
Ceramic loaded tissue adhesive composite gel for rapid hemostasis in osteo‐surgeries
1School of Chemical and Biological Engineering, The Institute of Chemical Processes, Seoul National
University, Seoul, 08826, 2The Institute of Engineering Research, Seoul National University, Seoul, 08826
Blood loss is one of the major concerns during various osteo‐surgeries such as femoral fracture repair, spinal fusions, pelvic osteotomy, and maxillo‐facial surgeries. Some of these procedures can lead up to 1500‐2500 mL of blood when uncontrolled which requires extensive blood transfusions. Further, secondary bleeding also can occur even up to 7‐10days post‐surgery, when the surgical sites are not sealed properly. Therefore, it is necessary to utilize a hemostatic agent that can act rapidly during the surgery, adhere well to the surgical site and prevent the delayed secondary bleeding, thereby reducing morbidity and mortality. It would also be advantageous if the adhesive hemostat is compatible with the bone tissue on which surgery has been performed such that it could promote tissue healing. Herein we propose a composite gel for rapid hemostasis during bone surgeries. The gel is composited with bone mimicking calcium magnesium phosphate ceramic particles, the ions of which will aid in initiating the clotting cascade. The ceramic particles are coated with gelatin which would aid in tissue adhesion and healing post‐surgery. These gelatin‐coated ceramic particles will be further decorated with catechol groups through chemical linkages, which aid in the enhanced tissue adhesion and retention of the gel in the surgical sites for a prolonged duration. Thus the composite hemostatic gel will provide rapid and prolonged blood clotting both during and post‐surgery of bone, preventing profuse blood loss, thus promoting quicker recovery for the patients.
Efficient activation of dendritic cells with CpG‐coated functional nanoparticle
1Sungkyunkwan University, 2Seoul National University
Modulation of the immunosuppressive tumor microenvironment (TME) is crucial for effective tumor treatment. Herein, we demonstrate immune‐modulating functional magnetic nanoparticles (f‐MNPs). These f‐MNPs were coated with CpG oligonucleotide (CpG*f‐MNPs), capable of maturating dendritic cells (DCs) by upregulating activation markers such as CD80, CD86, CD40, and MHC‐2. For sustained and local delivery of the prepared CpG*f‐MNPs to the tumor resection site, CpG*f‐MNPs were embedded in a methacrylate‐modified hyaluronic acid hydrogel. The hydrogel containing CpG*f‐MNPs was implanted in the 4T1 tumor resected model in vivo. The implantation of the CpG*f‐MNPs could be able to effectively activate the DCs in the vicinity of the tumor resection site, resulting in the activation of cytotoxic T cells. We believe this strategy has great potential for the effective prevention of tumor recurrence and metastasis.
Modulating sepsis‐associated NETosis dysregulation using bioinspired DNase‐I‐coated polymeric nanospheres
1Seoul National University College of Medicine, 2Sungkyunkwan University
Neutrophils are the first line of defense of the immune system. Upon infection, activated neutrophils generate neutrophil extracellular traps (NETs), which trap microorganisms and pathogens, and then release cytotoxic enzymes into the NET structures to kill them. During sepsis, substances released from apoptotic cells, such as cfDNA, histones, and HMGB1, cause severe vascular inflammation and multiple organ dysfunction. Circulating DNase‐1 is known to suppress these inflammatory mediators and regulate the activation of blood coagulation, which suggests DNAse‐1 as a potential treatment option. In this study, we hypothesize that long‐circulating nanoparticles coated with DNase‐I can further suppress the inflammatory mediators, thereby preventing and/or treating sepsis in the mouse model. In this study, we prepared polymeric NPs that were coated with PEG and DNase‐I to pursue their prolonged circulation in the bloodstream and destroy NETs in the body, respectively. The prepared DNase‐I coated NPs exhibited a spherical shape with approximately 180 nm in size. When tested under in vitro environments, the DNase‐I coated polymeric NPs degraded DNA even at low concentrations (1.5 ∼ 2mg/ml), suggesting that the DNase‐I coated NP could be applicable for NET digestion in vivo. Therefore, we propose that preventing NET formation by the DNase‐I coated NPs in inflammatory or thrombotic diseases can be a potential strategy for sepsis treatment. Given this, we envision that the NET degradation with the DNase‐I coated NPs may also reduce the lung injury, thereby improving the survival rate.
Engineered endothelium model ensures direct EC‐pericytes interactions via polyvinyl alcohol/ECM‐based artificial basement membrane
1Center for Biomaterials, Korea Institute of Science and Technology
Endothelial cells (ECs) play a crucial role in the regulation of vascular functions. Pathophysiology of the vascular system would lead to endothelial dysfunctions and eventually develop life‐threatening vascular diseases (i.e., atherosclerosis). To this end, a biomimetic endothelium model in vitro is a very powerful tool in studying vascular biology and in recapitulating vascular diseases toward development of novel therapeutics. In this study, we propose an artificial basement membrane (aBM) that is fabricated by combining polyvinyl alcohol (PVA) with cell‐derived, decellularized ECM on each side, followed by further process to create a porous structure of aBM (p‐PVA). ECs and mesenchymal stem cells (MSCs) were seeded on each side of p‐PVA and they were then cultivated in a growth medium upon a freestanding co‐culture setting. Our results demonstrated that our endothelium model facilitates robust barrier function assessed via vascular endothelial cadherin (VE‐cadherin) and permeability assay using FITC‐Dextran. We also found out that the robust barrier function is driven by direct ECs‐pericyte interaction through p‐PVA via hemoglobin‐α1 and N‐cadherin, leading to higher secretion of nitric oxide. Another result showed a resilient property of endothelium when treated with a proinflammatory cytokine, TNF‐α as observed via the recovery of VE‐cad structure with time. More interestingly, we investigate the early stage of atherosclerosis using our endothelium model, monitoring the transmigration of THP‐1 monocytes inside the p‐PVA and confirming foam cell development. The proposed aBM has the potential for in vitro atherosclerosis model as well as possible application for in vivo graft model.
Nano‐graphene oxide crosslinking improves in vivo durability of decellularized scaffold through MMP suppression and immunomodulation
1Seoul National University
Decellularized extracellular matrix (dECM) scaffold, widely utilized for organ engineering, often undergoes ECM decomposition after transplantation and produces ECM byproducts that cause inflammation, which can lead to clinical failure. Hence, a new strategy using nano‐graphene oxide (NGO) is proposed to modify the biophysical properties of dECM. Notably, the NGO‐crosslinked scaffolds show high resistance to enzymatic degradation via direct inhibition of the activity of matrix metalloproteinases (MMPs) as well as increased mechanical rigidity. NGOs also promote macrophage polarization toward anti‐inflammatory M2 within transplanted scaffolds, which reduces graft‐elicited inflammation. Moreover, low MMP activities that are attributed to both NGOs and the tissue inhibitors of metalloproteinases expressed by M2c can protect NGO‐crosslinked scaffolds against in vivo degradation. Lastly, bioengineered livers fabricated with NGO‐crosslinked scaffolds are highly preserved and remain functional, thereby effectively regenerating the damaged livers after transplantation into mouse models of acute and chronic liver failure. Overall, NGO crosslinking of dECM scaffold prolongs allograft survival and eventually improves therapeutic effects of bioengineered livers, which may offer an alternative for donor organs.
Development of poly(lactide‐co‐caprolactone) film combined with mesenchymal stem cell‐derived matrix for corneal endothelial cells transplantation
1University of Science and Technology, Korea Institute of Science and Technology, 2Department of ophthalmology, Dongguk University
Human corneal endothelial cells (hCECs) play a crucial role in maintaining hydration in the cornea and work as the pathway that transfers nutrients to the stroma through ions and water pumps. Once the hCECs are damaged, tight cell junctions would collapse and lead to corneal edema and eventually compromise the corneal transparency. In this study, we propose a new hCECs delivery carrier that is transparent, very thin, and biocompatible. To that end, mesenchymal stem cell (MSC) derived matrix was tightly embedded on the poly(lactide‐co‐caprolactone) (PLCL) where we anticipated stable hCECs adhesion and phenotype maintenance, due to given microenvironments for natural cell‐matrix interactions. To fabricate, PLCL solution was poured onto the decellularized matrix obtained from in vitro cultured umbilical cord blood MSCs. The ECM‐PLCL film was 20 μm thick, transparent, rich in fibronectin and collagen type IV, and easy to handle. Surface characterizations exhibited that ECM‐PLCL was very rough (54.0 ± 4.50 nm), uniformly covered by ECM, and retained a positive surface charge (65.2 ± 57.8 mV), as assessed via atomic force microscope. The hCECs on the ECM‐PLCL showed good cell attachment, with the cell density similar to the normal cornea. The essential markers of hCECs (ZO‐1, N‐cadherin, Na+/K+‐ ATPase) were also identified via immunofluorescence, western blot, and RT‐qPCR, respectively. Moreover, ECM‐PLCL transplantation into the anterior chamber of the rabbit eye for 8 weeks proved maintenance of normal cornea properties. Taken together, this study demonstrates that our ECM‐PLCL can be a promising cornea endothelium graft with an excellent ECM microenvironment for CECs.
Enzyme‐mediated redox system for tissue engineering
1Dong‐A University
Enzyme‐mediated redox system has been a promising approach for mild and biocompatible hydrogel crosslinking strategy in the field of tissue engineering. Inspired by skin melanin synthesis and marine mussel adhesion, tyrosinase‐mediated hydrogel crosslinking has been exploited as cell‐friendly reactions and explicit reaction mechanisms. Hydrogel prepared by tyrosinase exhibits appealing properties as a dynamic scaffold for cell delivery and as a bioink for 3D bioprinting. Recapitulating the structure of the native extracellular matrix (ECM), innovative tyrosinase‐mediated hydrogel crosslinking has now shifted to the field of translational medicine. Biomimetic hydrogel with in situ tyrosinase crosslinking can be efficiently and easily applicable to the disease model for therapeutic purposes. We demonstrate that the novel enzyme‐based crosslinking hydrogel has a robust potential in tissue engineering and regenerative medicine.
Development of PDRN loaded alginate/silica hybrid hydrogel scaffold using 3D printing for enhanced diabetic wound healing
1The Catholic University of Korea
Patients with diabetes suffer considerably delayed wound healing resulting from uncontrolled blood sugar levels. As a way to effective healing, hydrogel‐based wound dressing materials have been extensively utilized since they could provide moist environment to the wound sites. In addition to material itself, versatile biomolecules have been incorporated to accelerate restoration. Here, alginate/silica hybrid hydrogel system was developed by sol‐gel method. Prepared alginate/silica composite ink was used for extrusion based 3D printing. Printing parameters regarding silica contents were set and properties of fabricated alginate/SiO2 hybrid hydrogel scaffolds were tested. Based on the results of the alginate/silica support, polydeoxyribonucleotide (PDRN) was incorporated into the optimized condition of the alginate/silica bio‐ink for improving tissue regeneration. The physical properties, degradation, swelling, and release behavior of the fabricated PDRN loaded alginate/silica hybrid hydrogel scaffold were examined. Additionally, in vitro cell adhesion and migration using fibroblasts and in vivo animal experiments using a diabetic mouse model were conducted to verify the effectiveness of PDRN loaded alginate/silica scaffolds.
Fabrication of biomimetic microneedle patches with anti‐microbial and enhanced wound healing ability using DLP‐based 4D printing
1The Catholic University of Korea
Thermo‐responsive shape memory polymers (SMPs) have been intensively researched because of shape recovery ability following environmental temperature. However, fabrication of SMPs has mostly relied on molding or extrusion methods. Recent studies focused on 4D printing which combines 3D printing technique and shape memory effect (SME) to create highly complicated structures. Especially, digital light processing (DLP) technique, which is characterized by high precision and accuracy, has been utilized for 4D printing by photopolymerization of diverse acrylates. Present study adopted mixture of two acrylates for 4D printing. In addition, polydeoxyribonucleotide (PDRN) was loaded to improve tissue regeneration ability. For introducing anti‐microbial properties, Zn ion was embedded into the surface of microneedles by sputtering‐based plasma immersion ion implantation (S‐PIII). Shape recovery ability was analyzed using specifically designed zig and needle. Surface hardness and compression tests were carried out to evaluate the effect of Zn S‐PIII process. In vitro cell adhesion and proliferation tests was also carried out. Effectiveness of microneedle patches was also verified through in vivo tests using diabetic mouse model.
Investigation of shear flow effect on vascular endothelium under a dynamic flow system
1KIST
Endothelial cells (ECs) are constantly exposed to the blood flow and resultant shear stress. Understanding of how ECs can sense blood flow has been a critical subject for many years to elucidate the underlying mechanisms of vascular pathophysiology. To investigate such mechanisms, multiple in vitro endothelium models (EM) have been developed over the past few decades. Here, we propose a novel circulation model that can mimic physiological conditions while combining engineered endothelium and dynamic flow system. In order to imitate mature blood vessels, our EM consists of porous polyvinyl alcohol (p‐PVA) covered on both sides with decellularized extracellular matrix for the stable anchorage of vascular cells, such as human umbilical vein endothelial cells (HUVEC) and smooth muscle cell (SMC) on each side. Our early works confirmed secure cell attachment of our EM under dynamic flow condition. The flow system was built with polydimethylsiloxane in two separate parts, specifically engineered to provide sufficient and different levels of shear stress (SS) while maintaining a continuous laminar flow of culture media. Current setup is a closed circuit that allows flow without disturbing the EM samples. While running our system at 12 dynes/cm2 SS, we expect to confirm previously reported changes in ECs as compared to the static condition, such as increased level of KLF2, Piezo1, hemoglobin alpha subunit, underexpression of VCAM‐1 and changes in the cell morphology from polygonal to spindle‐like shape. Our system is very effective in investigating EC‐SMC interactions in physiologically relevant environment and elucidating EC mechanosensing mechanisms as well.
Composite scaffolds of gelatin and Fe3O4 nanoparticles for magnetic hyperthermia‐based breast cancer treatment and adipose tissue regeneration
1a.Research Center for Functional Materials, National Institute for Materials Science; b.School of Pure and Applied Sciences, University of Tsukuba, 2Research Center for Functional Materials, National Institute for Materials Science
Breast cancer is the most common cause of cancer‐related death in women worldwide. There remains a challenge in completely eradicating breast cancer cells and reconstructing the tumor‐initiated breast defect after surgical intervention. In recent years, bifunctional composite scaffolds with hyperthermal and tissue regeneration functions play an essential role in the efficient cancer treatment. In this work, gelatin was conjugated with folic acid (FA) to obtain FA‐modified gelatin, and then hybridized with Fe3O4 nanoparticles (NPs) to prepare bifunctional composite scaffolds. Pre‐prepared ice particulates were used as a porogen material to control the pore structures. The composite scaffolds had well‐interconnected spherical large pores that allowed cell migration and infiltration. Due to the presence of folic acid, the composite scaffolds could capture FA receptor‐expressing breast cancer cells. The composite scaffolds possessed a high magnetic‐thermal conversion capacity and could completely kill breast cancer cells under AMF irradiation in vitro cell culture and in vivo animal experiments. Furthermore, culture of human bone marrow‐derived mesenchymal stem cells (hMSCs) in the composite scaffolds showed that the composite scaffolds facilitated proliferation and adipogenic differentiation of hMSCs. The results indicated that the composite scaffolds could not only offer magnetic hyperthermia therapy for elimination of breast cancer cells but also guide the adipogenic differentiation of hMSCs for adipose tissue regeneration. The composite scaffolds could serve as a good platform to exert anticancer effects and to promote the reconstruction of adipose tissue.
Development of PLA/sirolimus coated biodegradable PCL/SiO2 stents fabricated by 3D printing
1Catholic University of Korea, 2CG Bio Co., Ltd
Metallic coronary stents, which are made of CoCr, NiTi, and stainless steel, have been utilized to manage coronary stenosis. However, those permanent metallic stents adversely affect the blood vessels causing thrombosis and restenosis. As an alternative candidate for stent materials, biodegradable poly(ɛ‐ caprolactone) (PCL) has been extensively utilized. However, enhancement in mechanical properties and biocompatibility is still necessary. Here, we propose an advanced composite system by combining sol– gel‐derived SiO2 and PCL and 3D printing of composite to generate customized biodegradable stents. Furthermore, poly(lactic acid) (PLA) coating including immunosuppressive drug was supplemented to as‐ fabricated PCL/SiO2 composite stents for prohibiting the proliferation of smooth muscle cells (SMCs). Properties of PCL/SiO2 composites were firstly assessed and printability of PCL/SiO2 composites was evaluated through hot‐melt extrusion printing technique. Effectiveness of PLA/drug coated PCL/SiO2 composites was also verified through in vitro tests. Further examinations were also followed to PLA/drug coated PCL/SiO2 composite stents.
Development of drug‐eluting bullets with controlled drug release and radiopacity for anti‐cancer treatment
1Catholic University of Korea, 2Sungkyunkwan University
In chemotherapy, the injection method has been regarded as a golden standard for anti‐cancer drug treatment. However, continuous drug injection has caused pain and inconvenience to patients. Various methods have been developed to efficiently deliver anticancer drugs without repetitive penetration. Inserting drug loaded scaffolds into the cancer site is acknowledged as one of the promising ways to achieve abovementioned goal. Nonetheless, the scaffolds should be observed from the outside to monitor whether they were well‐placed. In this study, drug‐eluting bullets (DEBs) that can be used as a fiducial marker with controlled drug release through external stimuli were developed. Fabricated DEBs are radiopaque by including titanium in poly(lactic acid) (PLA) matrix, and it is possible to control the drug release from DEBs by locating the drug incorporated phase change materials (PCMs) inside the bullet which could be melted through raising the temperature by irradiating the near‐infrared (NIR) laser. Physicochemical analyses, radiopacity tests, and monitoring drug delivery behavior were conducted to confirm the effectiveness of this novel drug delivery system. In in vitro and in vivo studies, developed DEBs were proven to be effective to prohibit the growth of cancer and even to reduce the size of cancer.
Magnetic nanoparticles‐based specific enrichment system for biomarker concentration of transplant rejection in the blood
1Department of Biomedical Engineering, Ulsan National Institute Science and Technology (UNIST)
Magnetic nanoparticles have been commonly used in various biomedical applications., including magnetic hyperthermia, drug delivery, and molecular diagnostics. Among those applications, magnetic enrichment has great potential as a pretreatment method for isolating the specific components such as protein biomarkers and pathogens in the biofluids. However, biomarkers in the blood are most difficult to be detected due to the complex components including abundant blood cells, cell‐free DNA, and proteins. For these reasons, the biomarker isolation from whole blood samples has limited separation efficiency and accuracy are still challenging. Herein, we report a large‐scale synthesis process of superparamagnetic nanoparticles with excellent crystallinity and uniform size distribution. The magnetic nanoparticles are composed of ∼20 nm‐sized primary nanoparticles which are self‐assembled to form clusters and eligible to isolate biomarkers from the complex blood samples due to superior magnetic property. With the magnetic capture under biological fluid (e.g., blood or urine), one can efficiently separate very low abundant target molecules and enrich them over 10‐fold rapidly with great accuracy. Granzyme B, a biomarker for transplant rejection in the blood, is detected using the magnetic nanoparticle‐based enrichment process. Due to the very low concentration (∼ pg/ml) found even in the early onset of the transplant rejection, it is hardly detected in previous studies, however, the magnetic separation is useful to concentrate the granzyme B. This research presents the potential for early diagnosis of transplant rejection in the whole blood samples by concentrating on the large volume of the complex biofluid under the continuous capture system.
Influence of viscosity on osteogenesis and adipogenesis of mesenchymal stem cells with controlled morphology
1Tissue Regeneration Materials Group, Research Center for Functional Materials, National Institute for Materials Science, Department of Materials Science and Engineering, Graduate School of Pure and Applied Sciences, University of Tsukuba, 2Tissue Regeneration Materials Group, Research Center for Functional Materials, National Institute for Materials Science
Recent researches have shown that the along with other mechanical cues of the cellular microenvironment, viscosity has an influence on stem cell fate. However, the conventional methods for investigating the influences of viscosity on cell functions normally cannot exclude the influence of cell morphology. In this study, micropatterned surfaces were used to control the cell morphology and the effect of viscosity on stem cell functions with controlled cell morphology was investigated. micropatterned surfaces with circular micropatterns of different sizes and elliptic micropatterns of different aspect ratios were used to control the cell size and elongation. Induction media with different viscosities for cell culture were preaperd by adding polyethylene glycol of different molecular weight but the same concentration. The osteogenic and adipogenic differentiation of human mesenchymal stem cells (hMSCs) in different viscosities were compared. High viscosity enhanced the osteogenic differentiation of large and elongated hMSCs while inhibited the adipogenic differentiation. However, the osteogenic and adipogenic differentiation of small hMSCs were not affected by viscosity. The effect of viscosity on differentiation of hMSCs was correlated with the actin filament organization in different viscosity medium.
Fabrication and evaluation of a powder‐type hemostatic agent with effective adhesion property
1Kumoh National Institute of Technology
Blood is a body fluid that is 6‐8% of our body and maintains homeostasis and carries oxygen and waste products. The phenomenon that blood leaks from a blood vessel due to trauma or internal bleeding is called bleeding. In case of extreme bleeding, hemostasis is essential for survival. In order to fabricate a powder‐type hemostatic agent with effective hemostatic properties, the blood absorption rate was improved by crosslinking chitosan and starch, and thrombin and Ca2+ ions were introduced to induce rapid blood clotting upon contact with blood. Polyvinylpyrrolidone (PVP) was introduced to improve tissue adhesion property and prevent rebleeding. The PVP coating improved tissue adhesion, reduced the coagulation time by 3.5 times compare to due to the introduction of blood coagulation factors, and showed the formation of 7‐8 times more thrombus. In the plasma recalcification experiment, fibrin networks were formed 4‐5 times faster in the case of containing a blood coagulation factors than in the control group. On the other hand, it was confirmed that PVP coating does not inhibit the blood coagulation reaction by the blood coagulation factors. In cell culture experiments, the powder coated with PVP less than 5 wt% showed negligible cytotoxicity. In vivo animal studies using a liver bleeding model in SD rats showed faster hemostasis and less bleeding. The chitosan/starch/PVP powder has promising hemostatic and tissue adhesion properties. It is expected to be applied as a surgical local hemostatic agent.
Fabrication and characterization of a powder‐type anti‐adhesion agent with improved adhesiveness using hyaluronic acid
1Kumoh National Institute of Technology
Adhesion between organs and tissues after surgery is the most common complication after surgery. Adhesion formation causes symptomatic diseases, such as chronic pelvic pain, infertility, bowel pain, and intestinal obstruction. In this study, the powder‐type anti‐adhesion agent made of hyaluronic acid (HA) and poly(vinyl pyrrolidone) (PVP) was fabricated and evaluated the effect of tissue adhesion prevention using the rat peritoneum/cecum defect model. HA is a promising material because of its biocompatibility and biodegradability but has weak mechanical properties. To overcome these disadvantages, crosslinked HA powder using glyoxal as a crosslinker is designed for a novel powder‐type adhesion barrier. PVP was incorporated into three‐dimensional network chain of HA as a semi‐IPN structure to increase the adhesion of powder to the application site. Through FTIR analysis, the presence or absence of a crosslinking reaction of the powder was confirmed, and the tissue adhesion of the powder was measured through a rheometer. The cross‐linked HA‐PVP powder was completely degraded after 120 hours by hyaluronidase treatment, and the prepared x‐HA/x‐HA/PVP powder showed negligible cytotoxicity through the in vitro cell viability test. From the results of animal study, it was confirmed that the powder‐type anti‐adhesive agents prepared in this study has an excellent tissue adhesion prevention effect. Crosslinked HA‐PVP powder could be proposed as a useful candidate for reducing unwanted post‐operative tissue adhesion.
Development of a powder‐type adhesive hemostatic agent containing blood coagulation agent
1Kumoh National Institute of Technology
Hemostasis must be done because bleeding caused by external or internal factors can lead to death in severe cases. Recently, development of powder‐type hemostatic agent with improved tissue adhesion property is essential because laparoscopic and endoscopic surgery are frequently performed. In this study, we fabricated a dialdehyde starch (DAS)/hyaluronic acid (HA) powder‐type adhesive hemostatic agent containing blood coagulation agents. The DAS/HA powder was crosslinked by glyoxal. The blood absorption ability was controlled through the DAS/HA composition, reaction time and volume of crosslinking agent and particle size. Crosslinking was analyzed by using Fourier‐transform infrared spectroscopy (FT‐IR) and X‐ray photoelectron spectroscopy (XPS). Calcium ions and thrombin as blood coagulation agents were introduced and their introduction was confirmed through inductively coupled plasma optical emission spectroscopy (ICP/OES), Bradford assay and chromogenic assay with S‐2238 substrate. In vitro blood coagulating experiments showed that the DAS/HA/CaCl2/thrombin powder reduced the clotting time compared with the control group. Interactive dynamic coagulation process of the DAS/HA/CaCl2/thrombin powder was measured by thromboelastography (TEG). In vivo animal test using rat hepatic hemorrhage model, the DAS/HA/CaCl2/thrombin powder considerably reduced bleeding time and hemorrhage quantity than control group and commercial products. The tissue adhesive DAS/HA powder containing blood coagulation agents will provide a valuable hemostatic agent for laparoscopic and endoscopic surgery.
Detection of cancer using carbon dot‐based conductive hydrogels with controlled pH‐sensitivity through boronate ester bonds
1Department of IT and Energy Convergence (BK21 FOUR), Korea National University of Transportation,
2Department of Chemical and Biological Engineering, Korea National University of Transportation,
3Department of Green Bio Engineering, Korea National University of Transportation
A tumor microenvironment‐responsive wireless strain‐pressure hydrogel sensor based on pH‐induced controllable nanoparticles was designed for cancer detection in vitro–in vivo model, which show excellent ability to distinguish between cancer and normal cells. The pH‐responsive nanoparticles (CD‐ PNB), comprising diol–diol crosslinked semiconducting carbon dots (CDs) and non‐conductive polymer (PNB), are sensitive to acidic tumor microenvironments and play crucial role in demonstrating tumor‐ selective strain‐pressure responses. Upon application of strain and pressure, CD‐PNB@PVA hydrogel produced distinct electronic signals in the presence of cancer cells (HeLa, PC‐3), compared to the normal cells (MDCK, CHO‐K1). The strain and pressure gage factors for cancer‐cell‐treated CD‐PNB@PVA hydrogel were found to be 0.7439 and 5.3052 kPa−1 respectively, which were higher than normal‐cell‐ treated CD‐PNB@PVA hydrogel (0.5009 and 4.2720 kPa−1). CD‐PNB@PVA hydrogel demonstrated excellent sensing response in tumor‐bearing mice based on in situ and ex situ measurements, with no inflammation during hydrogel implantation. Moreover, wireless sensing system was used along with CD‐ PNB@PVA hydrogel to simplify monitoring process and obtain real‐time conductivity and strain– pressure profiles on smartphone. Thus, this approach constructs a tumor microenvironment‐responsive strain–pressure hydrogel sensor and presents potential for sensitive and selective tumor detection in point‐ of‐care diagnostic applications.
GSH responsive carbon dots incorporated flexible and stretchable skin sensor with wireless monitoring of pressure strain response in cancer condition
1Department of IT and Energy Convergence (BK21 PLUS), Korea National University of Transportation,
2Department of Chemical and Biological Engineering, Korea National University of Transportation,
3Department of Green Bio Engineering, Korea National University of Transportation
Fabrication of a flexible and stretchable skin sensor comprising of GSH responsive carbon dots loaded with polydopamine (PDA‐CD) was carried out which could distinguish between normal and cancer cells based on distinct pressure and strain responses. The selective behaviour of the carbon dots towards the CD44 receptor was vital for the pressure‐strain response. The PDA released in the high GSH cancer environment was responsible for the tunable conductivity and the H‐bonding assisted self‐healing behaviour. The higher conductive behaviour in presence of cancer cells assisted in generating a higher pressure and strain sensing response compared to that of normal cells, thereby helping in the detection process. Additionally, the sensor when attached to a wireless system could relay the response generated via a smartphone. This provides a pathway for a more straightforward and simple cancer detection process, leading to rapid diagnosis.
Real‐time wireless detection of tumor cells using a ROS‐sensitive sensor comprising a diselenide polymer dot‐coated surface
1Department of Chemical and Biological Engineering, Korea National University of Transportation, 2Department of IT and Energy Convergence (BK21 FOUR), Korea National University of Transportation
A real‐time electrochemical sensor was designed based on diselenide functionalized dopamine‐ conjugated hyaluronic acid polymer dot (PD(HA/DP)‐ DiSe) coated surface for the selective tumor diagnosis by modifying the coated surface morphology. Owing to the high levels of ROS in tumor cells, the diselenide bond is cleaved thereby changing the morphology of PD(HA/DP)‐ DiSe coated surface from a sphere‐like to a sheet‐like structure which in turn changes the electrochemical property of the system. The change in fluorescence to a brighter intensity following treatment with H2O2 is attributed to the selectivity and sensitivity of a diselenide polymer dot‐coated surface towards ROS. Moreover, when connected to a wireless system, the system demonstrated the ability to detect ROS in real time, indicating that it has significant promise for tumor diagnosis in the future. This work was supported by Korea National University of Transportation in 2021.
Tumor microenvironment‐dependent maturation of hepatocarcinoma cells spheroids formed within microfluidics‐generated 3D microgels for chemotherapeutics testing
1Ulsan National Institute of Science and Technology
Tumor tissue models have been highlighted as a platform to investigate tumor physiologies such as progression, proliferation, vascularization, and maturation as well as chemotherapeutics screening and sensitivity testing. Among multiple factors to determine tumor spheroid fate, the importance of microenvironmental factors such as extracellular matrix is well recognized but has not been widely investigated. Here, the different subtype of hepatocarcinoma cell (HCC) spheroids was formed and matured within an even size of microgel fabricated by flow‐focusing microfluidics device. Microgel was designed to control its physical and biochemical properties for changes in the tumor microenvironment. Stiffness around HCC was controlled in a wide range by polymer concentration. In addition, hyaluronic acid, composed of liver extracellular matrix as bioactive glycosaminoglycans, was added to the microgel to investigate HCC spheroid fate depending on stiffness and biomaterial composition. Their influence on tumor physiology and maturation was exhibited to be highly dependent on cell subtype, HEPG2, and HEP3B cell line. Using these spheroids, chemotherapeutic screening was performed to confirm various chemoresistance effects for each cell subtype, resulting from the different microenvironments guidance.
Preparation of multiscale biomedical scaffold by assembling self‐healable hydrogel modules
1Yonsei University
Over the past decade, self‐healing hydrogels have been a favorite subject due to their long‐term stability, self‐healable aspects and wide range of application vary from artificial tissue fabrication, wound healing/tissue regeneration, drug/cell delivery, electrical devices, to adhesive glue. Despite these several advantages, self‐healing hydrogels have a limitation with cell encapsulation because gels are brittle, have weak mechanical strength and there is no moiety for cells to attach. Herein, to overcome this problem, a double‐network hydrogel was synthesized by crosslinking a collagen (COL) type l within a self‐healing hydrogel based on N‐Carboxyethyl Chitosan (CEC) and Oxidized Dextran (OD). These CEC‐OD‐COL hydrogels have shown sufficient self‐healing ability both in lab condition and physiological condition. Also, it showed no cytotoxicity and enhanced cell adhesion as well as elongation in both normal cells and cancer cells. For the possible biomedical applications, assemblies of resultant self‐healing hydrogel modules were prepared for spatiotemporal drug delivery and co‐culture models that could mimic angiogenesis in tumor environments. These results demonstrated that CEC‐OD‐COL self‐healing hydrogel can be used as 3D cell culturing scaffolds to study cell‐cell interaction and chemotaxis.
Fabrication of biomimetic scaffold for glycosaminoglycan (GAG)‐rich tissue
1Tissue Engineering Laboratory, Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong, China
Proteoglycan (PG) is an important extracellular matrix (ECM), which is composed of long unbranched, and highly negatively chard glycosaminoglycan (GAG) chains. G The PGs showed a ‘bottlebrush’ like structure that the GAGs were covalently attached to the hyaluronic acid (HA) backbone, which fill the space of collagen meshwork. In common musculoskeletal diseases such as disc degeneration and osteoarthritis, typical pathophysiological changes include significant loss of GAG, followed by loss of the normal function of tissues. Here, we aimed to fabricate a biomimetic scaffold for GAG‐rich tissue, such as nucleus pulposus with the GAG/ hydroxyproline (HYP, marker of collagen, 13.5% w/w of the collagen) ratio of 27:1. And we hypothesized that mimicking the GAG content by chemically modifying certain ECM components, including collagen (Col) and hyaluronic acid (HA) to positively charged, is possible to fabricate a scaffold that mimics the complex ECM of GAG‐rich tissues. In this work, we demonstrated the fabrication of the biomimetic scaffold, aminated collagen (aCol)‐aminated HA(aHA)‐ GAG. The fabricated aCol‐aHA‐GAG showed a high and controllable GAG/HYP ratio up to 39.1:1, achieving the quality of GAG‐rich tissue as that of young adult NP (27:1). The aCol‐aHA‐GAG scaffold showed biomimetic ‘nanobeads' like structure in SEM and ‘bottlebrush’ structure under TEM, recapitulating the ultrastructure of the native disc. In addition, the aCol‐aHA‐GAG maintained the bNPCs morphology and GAG content after bNPCs encapsulation. These results suggested that aCol‐aHA‐GAG was a biomimetic scaffold for NP. This work contributes to developing a biomimetic scaffold for NP tissue engineering.
Gelatin scaffold with lipid‐PLGA microparticles for sustained curcumin release and corneal tissue engineering
1National Tsing Hua University
Corneal transplantation is currently the only approach to cure corneal blindness. Cell‐based strategies that employ corneal endothelial cells (CECs) grown on supporting biomaterials hold great promise as possible alternative therapies for treating corneal endothelial dysfunction. Nevertheless, most biomaterials are used merely because of their robust mechanical properties, providing passive physical support for the transplantation of CEC monolayers. Based on the versatility of curcumin in ophthalmic applications, this study aims to develop a multifunctional scaffold system that can not only support the function and transplantation of CECs but also prevents post‐engraftment complications by sustained curcumin release, thus enhancing the long‐term success of CEC engraftment. Curcumin‐loaded lipid‐poly(lactic‐co‐glycolic acid) (PLGA; Cur@MPs) hybrid microparticles (MPs) fabricated using an oil‐in‐water single emulsion method are embedded into gelatin‐based scaffolds. The anti‐inflammatory, antioxidative, and anti‐ angiogenic potentials of the developed scaffolds and their capacity in supporting CEC monolayer formation are evaluated. The Cur@MPs are capable of promoting CEC proliferation, protecting CECs from oxidative stress‐induced cell death via modulating Nrf2/HO‐1 signaling axis, suppressing the secretion of pro‐inflammatory cytokines by macrophages, and inhibiting the migration and angiogenesis of vascular endothelial cells. By incorporating the Cur@MPs into a thin gelatin membrane, the fabricated scaffold is able to support the growth and organization of CECs into a polygonal morphology with tight junctions. These experimental results demonstrate the potential of the Cur@MPs‐loaded gelatin scaffold for actively supporting the survival and function of CEC monolayers after transplantation.
Development of cell‐laden α‐TCP/GeLMA 3D construct for hard tissue regeneration
1University of Science and Technology (UST), 2Korea Institute of Materials Science (KIMS)
Due to its potential to deposit numerous types of cells into a defined region, hydrogel based cell‐laden scaffolds have been intensively researched in various tissue engineering field. They are, however, largely used in soft tissue engineering because of their low mechanical strength. For that reason, in this study, we attempted to manufacture a 3D construct suitable for hard tissue regeneration by adding alpha‐tricalcium phosphate (α‐TCP) to a photo‐crosslinkable gelatin methacrylate (GelMA) for enhancing mechanical strength. According to the results, there was significantly improved the mechanical property of the hydrogel via α‐TCP cement reaction. Furthermore, compare to the hydrogel without α‐TCP, the encapsulated cells in the α‐TCP/GelMA hydrogel showed identical cell proliferation and enhanced osteogenesis differentiation. Also, it was investigated that the possibility of printing of cell‐laden α‐ TCP/GelMA hydrogel with high cell viability and high fidelity of printing. Therefore, we anticipated the hydrogel composite developed in this work, can potentially be used for bone tissue regeneration.
Separable double‐layer microneedle codelivery of Dox and LPS for treating subcutaneous glioma tumor via immunochemotherapy
1National Taiwan University of Science and Technology, 2Buddhist Tzu Chi Medical Foundation
Microneedle patch (MNP) is a novel biomedical device for transdermal drug delivery system which are considered to be an alternative approach instead of injection. MNP can penetrate human's stratum corneum and deliver the drugs into dermis, further, achieve the therapeutic effect. Thus, we designed a separable MNP that is constructed from an interpenetrating polymer network (IPN) hydrogel, and manufacture by sodium alginate and zwitterionic polymer crosslinking by calcium ions and photo‐ initiator with crosslinkers. The lipopolysaccharide (LPS) and doxorubicin (DOX) have been coloaded in the needle part for a synergistic immunochemotherapeutic outcome for cancer treatment. In the compression and tensile test, MNP has been designed as interpenetrating hydrogel (IPN) and then optimized its mechanical strength significantly to penetrate the skin. For separable backbone part of MNP, the IPN hydrogel was crosslinked with disulfide bond and calcium crosslinking agents that can be degraded by adding 1,4‐dithiothreitol (DTT) and ethylenediaminetetraacetic acid (ETDA) mixture solution. In the dissolution test, the MNP rapid degradation happened and sustained release the loaded drug. The IC50 value of DOX@MNs revealed that the drug loaded onto the MNs was effectively released to kill the CT‐2A‐Luc cell line at concentration of 1.088 μg/ml. Moreover, we confirmed that the dual drug‐loaded MNs (LPS/DOX@MNs) present the excellent immune response and tumor inhibition in the mice bearing with C57BL6 cancers experiment. Therefore, the effectively immunochemotherapy for cancer inhibition by MNP, which provide a new treatment method for the future therapy.
Synergistic composite for wound healing by delivery of fibroblast growth factor
1UNIST
In a process of wound healing, basic fibroblast growth factor (bFGF) is one of the key biomolecules and promotes proliferation of fibroblast, which forms collagen, fibrin, and fibronectin located in extracellular matrix, thus plays a major role in tissue regeneration. However, such biomolecules are administered by oral and intravenous route. Moreover, it requires sufficiently large amount to scarless wound healing, due to its rapid inactivation in vivo. In order to prolong therapeutic effect and achieve the higher recovery rate and quality, one can suggest the loading of bFGF payloads into micro/nanoparticles. Here, we report human serum albumin (HSA)‐derived nanoparticles and porous silicon microparticles for up‐graded stability of the bFGF. The porous silicon microparticles can help wound healing by stimulating prolonged angiogenesis. The bFGF can be loaded into mesoporous pore of the silicon microparticles. There are two application methods, one is direct drop casting and the other one is dressing with silicon microparticle embedded hydrogel (GelMA). Through these methods to wound skin (∼2 cm in a diameter) with a Tegaderm cover, the result showed significantly rapid and enhanced tissue regeneration. The degree of wound recovery and re‐epithelization area was much higher and larger compared to those of free bFGF administration. These combinatorial application of bFGF using micro/nanoparticles can reduce the risk of wound infection and thus it can be further effective when used with other drugs such as antibiotics, antimicrobial peptides and various dressing methods.
Zinc ion‐releasing tissue adhesives for wound management
1Incheon National University
Tissue adhesives have attracted much attention in clinical applications with multifunctional properties, including tissue adhesion and therapeutic effects. While various bioadhesives have been developed, there are still some problems, such as the inadequate mechanical properties, poor adhesion strength, and low bioactivities. Recently, many researchers have endeavored to develop bioactive adhesives delivering therapeutic agents like drugs, growth factors, and metal ions for improving wound healing. Growing evidence demonstrates that zinc ion (Zn2+) plays an important role in wound healing, including proliferation, angiogenesis, and collagen deposition. Herein, we represent Zn2+‐releasing adhesive hydrogels via a ZnO2‐mediated crosslinking reaction. Bioactive hydrogels had controllable physicochemical properties with gelation time (8 ‐ 26 sec), mechanical strength (520 ‐ 810 Pa), and proteolytic degradation (completely degraded in 24 hours). Notably, we also demonstrated that hydrogels had strong adhesive strength (35 ‐ 81 kPa) and were effectively bound to various tissue surfaces. Our hydrogels exhibited the prolonged Zn2+ release behavior in a sustained manner for up to 28 days. In addition, we confirmed that the bioactive matrices showed excellent cyto‐ and tissue compatibility. Moreover, we evaluated the hemostatic ability of the hydrogels in vivo, showing a high absorption capacity that enhanced the physical barrier effect and induced the efficient hemostatic effect with good tissue adhesive performance. We investigated the capability of hydrogels to promote fibroblast proliferation and the expression of angiogenic factors. In sum, our Zn2+‐releasing adhesive hydrogels possess great potential as advanced tissue adhesives for wound management.
Zinc ion‐releasing in situ crosslinkable hydrogels for endogenous tissue regeneration
1Incheon National University
Inorganic ion‐releasing hydrogels have been extensively studied in biomedical applications owing to their tissue regeneration ability. Recently, zinc ion (Zn2+) has drawn increasing attention among the reported inorganic ions. It is well known that Zn2+ regulates the wound healing process, such as antibacterial effect, immune modulation, cell proliferation, angiogenesis, and collagen deposition. Although various Zn2+‐releasing biomaterials have been developed, they have some limitations, including complex procedures and burst ion release. Herein, we design a new type of Zn2+‐releasing hydrogels via zinc peroxide (ZnO2)‐mediated disulfide forming cross‐linking reaction. The Zn2+‐releasing hydrogels have controllable physicochemical properties, such as phase transition time (1 min 30 sec‐23 min), elastic modulus (10‐2640 Pa), and proteolytic degradation (6 hour‐3 day) depending on ZnO2 concentrations (0‐0.5 wt%). Also, the Zn2+‐release behaviors are investigated, showing that the hydrogels can release Zn2+ for up to 14 days (0.2‐0.57 mM). We next perform the cytotoxicity test using human dermal fibroblasts, exhibiting excellent cell viability (>100 %) compared to the control group. Moreover, we analyze the pathological changes of the major organs after hydrogel degradation, demonstrating that our hydrogels have excellent tissue compatibility. Finally, we treat our hydrogels in the mouse skin defect model, showing that our bioactive hydrogels enhance the wound healing process with cell proliferation, angiogenesis, hair follicle development, and collagen deposition. In conclusion, we suggest that our Zn2+‐ releasing hydrogels hold great potentials as promising materials for wound management and tissue regeneration.
Assessing jellyfish collagen hydrogel for supporting human osteoblasts
1University College Londo (UCL), 2Jellagen Ltd
Large bone defects can be caused by trauma, disease, surgery or tumour resection where the bone is unable to heal as normal due to the size of the defect or fracture. To understand and resolve this clinical problem, regenerative medicine approaches, particularly tissue engineering and the use of biomaterial, have been widely studied and used. A more recent type of collagen that has emerged is jellyfish collagen (Jellagen), which is collagen type 0, non‐cytotoxic and biocompatible. In this project, we are investigating whether a jellyfish collagen hydrogel can provide a natural 3D microenvironment for bone formation.
Established methods such as Cell Viability Reagent, Live/Dead ® Viability/Cytotoxicity Kit, DNA quantification, histology and immunostaining were used to human osteoblast viability, proliferation and migration.
The viability results showed that the hydrogel supported cell viability, with the confirmation of Live/Dead images showing viable and proliferating osteoblasts within and on top of the hydrogel. It was also evident that osteoblasts within the hydrogel were distributed throughout the hydrogel across 7 days. This is a promising result that suggests the cells will be able to proliferate long‐term in a 3D microenvironment.
The early experiments of this project have shown that a jellyfish collagen hydrogel can support human osteoblasts and encourage their growth over 7 days. Some results have shown that cells are viable within the hydrogel at 21 days, giving a good indication that cells can be cultured for longer. The next step would be to assess bone formation up to 28 days.
Gelatin‐based dual delivery matrices releasing calcium and oxygen to facilitate vascularized bone tissue regeneration
1Incheon National University
Recent trends in developing biomaterials for bone tissue regeneration are to create scaffolds with cells or bioactive molecules for vascularized bone tissue regeneration. It is still challenging to develop the biomaterials for regenerating the complex vascularized structure of the native bone tissue. Toward this, cryogel has attracted much attention as a scaffold for delivering therapeutic agents, such as cells, nanoparticles, growth factors, and metal ions. Moreover, it has a highly interconnected porous structure, high swelling and adsorption capacity, and improved mechanical properties. Therefore, many types of cryogels releasing various bioactive molecules have been developed for facilitating tissue regeneration. Among these molecules, calcium ion (Ca2+) and oxygen (O2) are critical elements to improve osteogenesis and angiogenesis, such as proliferation, differentiation of osteogenic cells, matrix mineralization, and vascularization. Herein, we report gelatin‐based cyrogels as dual delivery matrices releasing Ca2+ and O2 for vascularized bone tissue regeneration. These bioactive cryogels are fabricated by calcium peroxide (CaO2)‐mediated oxidative crosslinking reaction and lyophilization. We characterized their physicochemical properties demonstrating that our cyrogels sustainably release Ca2+ for 28 days (1.6‐8.8 mM) and rapidly release O2 (DOmax: 24‐58 pO2%) for 2 days. Notably, the cryogels expedite the enhanced proliferation activity of pre‐osteoblast (MC3T3‐E1). Taken together, we suggest that our Ca2+ and O2‐releasing cryogels are promising matrices for facilitating vascularized bone tissue regeneration.
Silk‐collagen hydrogel improves therapeutic effects of mesenchymal stem cells on neovascularization in hindlimb ischemia via FAK/Src axis
1Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 31116, Republic of Korea. Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Republic of Korea, 2Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 31116, Republic of Korea.
Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Republic of Korea. Department of Biomaterials Science, School of Dentistry, Dankook University, Cheonan 31116, Republic of Korea. UCL Eastman‐Korea
Dental Medicine Innovation Centre, Dankook University, Cheonan 31116, Republic of Korea
Chondrocyte‐mimicking microspheres for osteochondral defect repair
1Institute of Tissue Regeneration Engineering (ITREN), Dankook University, South Korea; Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine,
Dankook University, South Korea, 2Institute of Tissue Regeneration Engineering (ITREN), Dankook University, South Korea, 3Institute of Tissue Regeneration Engineering (ITREN), Dankook University,
South Korea; Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, South Korea; Department of Biomaterials Science, School of Dentistry, Dankook University, South Korea; UCL Eastman‐Korea Dental Medicine Innovation Centre, Dankook University, South Korea; Cell & Matter Institute, Dankook University, Cheonan, 31116, South
Korea, 4Institute of Tissue Regeneration Engineering (ITREN), Dankook University, South Korea; Cell & Matter Institute, Dankook University, Cheonan, 31116, South Korea
Oxygen‐supplying syringe to create hyperoxia‐inducible hydrogels for in situ tissue regeneration
1Incheon National University
Recent trends in designing regenerative materials are to develop bioactive matrices to leverage the innate healing capacity of the body using various biophysicochemical stimuli of materials (defined as in situ tissue regeneration). Hyperoxia, high oxygen (O2) tension over 21% pO2, is a well‐known therapeutic factor for tissue regeneration. Although various peroxide materials with catalase have been utilized as O2‐ generating agents, their use still has some cytotoxic issues, such as (i) excessive oxidative stress of hydrogen peroxide (H2O2), an intermediate molecule in the peroxide decomposition, and (ii) the cellular signaling suppression of the catalase at high concentrations. Herein, we report a new type of O2‐ supplying platform, catalase‐immobilized syringes (defined as an Oxyringe), to overcome these limitations. This Oxyringe is fabricated through calcium peroxide‐mediated dopamine oxidation and catalase immobilization, minimizing the catalase incorporation into hydrogel solution to prevent cytotoxic issues of catalase. Our Oxyringe reveals controllable O2 generation up to 88.8% pO2 via catalase‐ mediated H2O2 decomposition in vitro. Utilizing Oxyringe, we fabricate cytocompatible O2‐releasing hydrogels, inducing transient hyperoxia (up to 47.9% pO2) in vivo. Interestingly, our hyperoxia‐inducible hydrogel expedites tissue regeneration, including hemostatic effect, boosted macrophage infiltration, promoted cell proliferation, improved neovascularization, and enhanced tissue maturation through transient oxidative stress. In sum, we suggest that our Oxyringe has excellent potential as an advanced O2‐supplying system to create hyperoxia‐inducing matrices for in situ tissue regeneration.
Long‐term maintenance of viable adipocytes and enhanced blood vessel infiltration in vivo using spheroid‐based bioprinted construct
1Ulsan National Institute of Science and Technology (UNIST)
Engineering volumetric vascularized adipose tissue is of major clinical importance for soft tissue reconstruction, post trauma or tumor abscission. However, this is hindered by limited vascularization especially in larger constructs. Inadequate vascularization in both autologous and engineered adipose grafts results in high levels of resorption and ultimate cell death. Here, we propose a novel method for the fabrication of a vascularized adipose tissue construct by integrating adipose‐derived stem cell (ADSC) spheroids with isolated fat tissue. Spheroids were fabricated by encapsulating ADSCs in an alginate matrix. The lack of adhesion sites in the matrix enabled cells to adhere to one another and generate spheroids within the construct in a simple one‐step manner. Separately, adipose tissue was mechanically dissociated into micro‐fat to facilitate the integration with the printed construct and implanted into subcutaneous region of nude mouse. In vivo, first, the effect of spheroid number on blood vessel infiltration into the construct was investigated. Histological results showed that 9 spheroids were the optimal number for both blood vessel infiltration and adipose tissue survival after 2 weeks of implantation. Then, long term maintenance of viable adipocytes were confirmed after 4 and 8 weeks. The results consistently showed that the incorporation of spheroids significantly enhanced the blood vessel infiltration and thus aided in long‐term survival of the implanted adipocytes. In conclusion, our integrated construct represents a novel approach to achieve vascularization within an adipose tissue construct. This could potentially open the door for the generation of scalable volumetric adipose tissue constructs.
Controlled drug release by a tough and adhesive bilayer hydrogel with external stimulation
1Jeonbuk National University
Electroactive nanomaterials have gained significant interest in the field of tissue engineering and regenerative medicine due to their intrinsic conductivity, tunability, and low‐cost processability. In this work, piezoelectric bilayer hydrogel is fabricated as an alternative treatment method for skin tissue regeneration. The top layer is composed of a tough and self‐healing poly(vinyl alcohol) (PVA) hydrogel that mimics the mechanical strength of the skin. Piezoelectric barium titanate (BTO) nanoparticles are incorporated into this layer, and subsequent changes in physicochemical, as well as current and voltage output, are observed in a concentration‐dependent manner. The bottom layer, which is in direct contact with the wounded area, consists of an adhesive chitosan hydrogel loaded with L‐ascorbic acid. We apply low‐intensity pulsed ultrasound (LIPUS) to the hydrogel system to trigger cell growth and simultaneously control the transdermal delivery of L‐ascorbic acid. Furthermore, we confirm the adhesion of the chitosan layer to a rat tissue sample and the top layer. Together, our findings elucidate the potential use of this piezoelectric composite scaffold, in combination with LIPUS stimulation, for future clinical trials.
Bio‐reprinting technique as an advanced method for micro‐scaled tissue structure fabrication
1Tech University of Korea
In human body, organs are organized with hierarchical structures with microstructures. To mimic these microstructures of native tissue in vitro, bioprinting technologies have been widely used. Digital light process (DLP) and stereolithography (SLA)‐based bioprinting technology have high resolution which is enough to fabricate microstructure of native tissue however, the photoinitiators used in these technologies have cytotoxicity which can lead to failure of artificial tissue or organ fabrication. The extrusion‐based bioprinting technology, a representative bioprinting technology, is applied in artificial tissue fabrication using viscous bioinks. In this strategy, there is a limitation on mimic the native tissue`s microstructure due to the shear stress during extrusion process and low mechanical properties of bioinks. Therefore, in this research, we developed the bio‐reprinting technique which can fabricate complex, and micro‐scaled structures of native tissue. The main point of bio‐reprinting technique is repetition of pre‐set extrusion bioprinting technology, we developed before. The precursor cartridge is fabricated by commercial 3D printing system and filled with bioinks. Then the bioinks in precursor cartridge extruded to another precursor cartridge. In this process, the pattern of cross section of origin precursor cartridge is miniaturized to tens of micrometer scale. With this technique, we can successfully fabricate multicellular, heterogeneous micro‐tissue structure such as hepatic lobules and muscle fiber. The bio‐reprinting technique developed in this research have high potential in the fabrication of a variety of tissue or organs.
A multiple surface modification used in Ti, and 3D‐printed Ti alloy scaffold to regulate osteoimmunology, angiogenesis and osteogenesis for orthopaedic and dental implant application
1Department of Dentistry, National Yang Ming Chiao Tung University, Taipei, Taiwan
Titanium (Ti) and Ti alloy are one of the most used metallic materials for orthopaedic and dental implant application. Over the past few decades, different kinds of surface modifications, including physical, chemical, electrochemical and biological surface treatment, have been developed to improve osteointegration of Ti and Ti alloys. As we know, the interaction between osteoimmunology (especially M1/M2 macrophage polarization) angiogenesis and osteogenesis plays important role in osteointegration. Therefore, developing an appropriate surface modification to regulate the aforementioned biological responses is a great concern. In this study, a multiple surface modification combining acid‐ etching/alkaline‐etching/procyanidin cross‐linking type I collagen immobilization was developed to mimic the 2D surface morphology, the 3D structure and biomolecule property of extracellular matrix (ECM). The current results showed that a hydrophilic ECM‐like 3D fibrous network structure was fabricated on the both Ti and electron beam melted (EBM) 3D‐printed Ti alloy scaffold. This unique surface structure not only enhanced the angiogenic responses of human umbilical vein endothelial cells (HUVEC) such as cell migration and tube formation but also the osteogenic differentiation of human mesenchymal stem cells from bone marrow (hBMSCs) through regulating M1/M2 polarization. Moreover, this multiple surface modification directly improved ECM mineralization and osteogenic marker expression of hBMSCs. We concluded that this multiple surface modification used in Ti and EBM 3D‐printed Ti alloy scaffold showed great potential for orthopaedic and dental implant application.
pH‐sensitive photonic crystal patch for wound healing monitoring
1Unist
The wound healing process is organized step by step and has a property that the local pH changes according to the causal relationship and metabolism of the biomolecule step by step. Since the recovery process of the wound is linked, if additional infection occurs, the wound becomes a local alkaline state due to biomolecular metabolism, which slows recovery and makes it vulnerable to chronic inflammation and external infection, making the patient's life more threatening. Therefore, it is necessary to continuously monitor wound recovery and quickly inform patients of the infection. During the wound healing process, the pH changes are the indicative signal of inflammation by infection. Herein, to monitor the wound healing, we proposed a pH‐sensitive rugate filter sensor which is electrochemically etched and has optical properties showing visible color alteration upon microscopic changes in the porous interior. Initially, the green‐colored sensor gradually changes to purple within 36 hr while the inflammatory wound shows a rapid color shift within 4 hr. Due to the alkalinity of the chronic wound, the photonic rugate filter can inform the alternative treatment required to achieve scarless healing. In addition, the hydrogel‐ embedded rugate film patch is loaded with bFGF, and consistently release to accelerate skin recovery, and also released Si ion accelerates tissue reconstruction. The photonic rugate filter patch will be a promising tool for non‐invasive monitoring of skin recovery by providing key information on acute inflammation with naked eyes under ambient conditions while alleviating the pain of a skin‐damaged patient.
Bio‐ink and 3D printing‐based to mimic of three‐dimensional skin complex with internal blood vessels
1Korea Institute of Machinery and Materials, 2Seoul National University
3D bio‐printing techniques have been developed to generate cell positioning and using various bio‐ink such as Gelatin, alginate and PEG. There are many challenges to find stable printability and maintained high cell viability. The purpose of this study was to develop a 3D artificial composite tissue material and manufacturing technology that simulates blood vessel‐derived fat/skin tissue. By combining 1. printing‐ based skin tissue production technology for fat/skin complex tissue development and 2. spheroid‐based adipose tissue production technology, Finally, 3. To establish a 3D artificial composite tissue production and culture technology that simulates adipose tissue linked blood vessels. Printing was carried out using PCL, and printing was carried out based on the void control technology devised in the first year so that the spheroids could be placed stably. Printing was carried out using micro‐particles of the same size before confirming the actual placement of spheroids, and through this, it was possible to confirm the possibility of stable placement of spheroids. Securing a manufacturing process technology that allows skin tissue to be printed on adipose tissue structures by printing with ink. The specific cell morphology of dermal fibroblasts was observed in GelMA and GelMA‐dECM groups. A denser cell morphology was observed in the skin bio‐ink mixed with dECM, suggesting that the proliferation rate of cells was improved by cytokines and growth factors of the dECM component. The skin layer with blood vessels and the fat layer were fabricated as a double layer, making it similar to the actual skin tissue.
3D conduit model bio printing for mimicking the human intestine
1Korea institute of machinery and materials
The human intestine, an important part of the digestive system, has a complex structure. In adults, if there is a problem with the intestine, a part of it can be excised and sutured, but in the case of congenital intestine atresia in fetuses and infants, organ transplantation in a intestine model is unavoidable.
To solve this problem, in this study, after producing a collagen‐based bio‐ink containing myo‐fibroblast CCD‐18Co cells, a intestine with a length of 20 mm, an inner diameter of 2 mm, and an outer diameter of
2.4 mm was created using the bio‐ink in three dimensions. We would like to introduce bio‐printing technology that can be manufactured in the form of a conduit.
In addition, we mimicked the complex structure of the intestine by mixing Caco‐2 cells, which are epithelial human colons, with Matrigel and seeding them.
Live&dead assay, CCK‐8 Assay, and ALP/Protein Assay were performed to evaluate the biological properties of the manufactured conduit intestine model, and the formation of 3D micro‐villi.
In addition, in order to evaluate the biological properties of Caco‐2 cells, a bio‐ink with CCD‐18Co cells was used as an experimental group and a bio‐ink without CCD‐18Co cells as a control when producing bio‐ink, and whether CCD‐18Co cells were present According to this, the growth of Caco‐2 cells was different.
In addition, 3D microvilli were well expressed when Caco‐2 cells were co‐cultured with CCD‐18Co cells. These results will show that the three‐dimensional conduit intestine model is most suitable for mimicking the human intestine.
Stent‐based electrode for radiofrequency ablation in the rat esophagus: A proof‐of concept study
1Biomedical Engineering Research Center, Asan Institute for Life Sciences, Asan Medical Center
Endoluminal radiofrequency (RF) electrodes have been developed for the management of inoperable biliopancreatic ductal cancers and Barrett's esophagus, the formation of a uniform ablation zone is still challenging. The monopolar nitinol RF stent‐based electrode (SE) was newly developed to deliver uniform RF energy into the inner wall of endoluminal organs. The purpose of this study was to investigate technical feasibility and efficacy of RF ablation with use of a novel SE in the rat esophagus. The RF temperature in the exposed rat esophagus reached at 70 °C in 89 sec at 30 W, 59 sec at 40 W, and 34 sec at 50 W, respectively with thin‐walled mucosal injuries. Eighteen of 21 rats underwent stent‐based RF ablation at 40 W and the remaining three rats underwent a sham procedure. Histological changes were analyzed and compared at immediately (n = 6), 1 week (n = 6), and 2 weeks (n = 6) in H&E and MT staining, and HSP70 and TUNEL. The RF ablation was successfully performed in 16 (88.8%) of the 18 rats. The degrees of RF‐induced fibrotic changes and heat shock protein changes in the RF‐ablated rat esophagus were significantly and gradually increased compared with the sham control at 1 and 2 weeks (all p < 0.05). TUNEL‐positive deposition was significantly increased immediately after RF ablation (p < 0.001) and gradually decreased. The stent‐based RF ablation is technically feasible and effective in achieving uniform thermal damages in the rat esophagus. It might represent a promising new approach for the treatment of endoluminal malignancies in non‐vascular organs.
Intragastric satiety‐inducing device combined with photodynamic therapy to treat obesity
1Asan Medical Center
An intragastric satiety‐inducing device (ISD) is a minimally invasive approach to induce satiety by continuously pressing the cardia portion of the stomach and stimulating ghrelin‐producing cells. To enhance the therapeutic effects of ISD, photodynamic therapy (PDT) can be combined by generating singlet oxygen under laser irradiation. Chlorin e6 (Ce6), as a photosensitizer (PS), was coated on the ISD surface for singlet oxygen production to stimulate or destroy cells. This study aimed to investigate the efficacy and safety of PDT with Ce6‐embedded ISD to suppress weight gain in mini pig stomach. A total of 12 minipigs were divided into four groups. PDT group (n = 3) received single PDT using Ce6‐ embedded ISD. ISD group (n = 3) received ISD placement. ISD plus PDT group (n = 3) received single PDT with Ce6‐embedded ISD placement. The remaining three weight and age‐matched healthy pigs were used as a control group. Anti‐ghrelin and TUNEL‐positive deposition were significantly increased in PDT, ISD, and ISD plus PDT groups compared to the control group (all p < 0.001). The weight gain was observed to decrease in the order of PDT, ISD, and ISD plus PDT groups compared to the control group, and the ISD plus PDT proved to be the most effective for wight loss. In addition, damaged cells by single PDT were observed to rapidly and gradually regenerated over time. The simple and unique operation extends the point of view in PDT and is expected to be a novel bariatric therapy.
Development of 3D printed thermo‐responsive skin‐derived decellularized extracellular matrix hydrogel adhesive patch with controllable shrinkage behavior
1POSTECH, Pohang, Gyeongnuk, 37666
Extracellular matrix (ECM) supports surrounding cells and provides a microenvironment that helps cell adhesion, growth, and development. It has been used as a biomaterial for tissue regeneration, treatment, and recovery in tissue engineering field. However, there is a problem that the physical properties are weak and the stimulus is not responsive. Alternatively, studies on the use of poly(N‐isopropylacrylamide) (PNIPAM), a thermo‐responsive polymer, have been reported for the purpose of delivering therapeutic agents in vivo. However, PNIPAM has relatively low bioactivity and provide only isotropic shrinkage direction, making it difficult to optimize their shrinkage behavior.
we developed a functional dECM material with shrinkage, adhesion, and high bioactivity. Furthermore, we fabricated a controllable, thermo‐responsive skin‐derived decellularized extracellular matrix (skin dECM) hydrogel adhesive patch through 3D printing with this material. For the development of the material, polymer network formation was controlled through the preparation and mixing of PNIPAM, chitosan‐catechol, and skin dECM. Through this, we developed an adhesive, thermo‐responsive skin dECM hydrogel with different shrinkage. As skin dECM is used, the developed material provides a tissue‐specific microenvironment and has relatively high bioactivity. A multi‐material hydrogel patch was produced with this functional dECM by 3D printing technology. This patch can implement a variety of controllable shrinkage behavior through the patterning and concentration of PNIPAM. To verify, the measurement of adhesiveness, thermo‐responsiveness, and mechanical properties of the patch were performed. These results can be applied to hydrogel patches that help recovery according to wound types in the field of skin wound healing in the future.
Development of 3D printing‐based tendon‐derived stem cell‐laden 3D microtissues for tendon tissue engineering
1Soonchunhyang Institute of Medi‐bio Science (SIMS), Soonchunhyang University, 2Department of Anesthesiology and Pain Medicine, Soonchunhyang University Bucheon Hospital
Although a large number of patients have been suffering from traumatic tendon injuries, including tearing of elbow, shoulder rotator cuff, and Achilles tendons, due to devastating injury and age‐ associated degeneration, an efficient method for regenerating damaged tendon is still a daunting task. Thus, we aim to develop a novel strategy that could promote tendon regeneration using tendon‐derived stem cells‐laden three‐dimensional biomimetic scaffolds with 3D printable tendon‐ specific microstructures. Our initial findings demonstrated that cells isolated from tendon showed stem cell‐specific surface markers and these cells could undergo in vitro tenogenic differentiation. In addition, we employed 3D printing techniques to create biomimetic scaffolds with topological cues and tendon‐derived stem cells cultured on these 3D printed biomimetic matrix could undergo cellular alignment along with the direction of the micropatterns including uniaxially and randomly oriented microfibers. Such a cell culture platform can offer novel strategies to achieve functional tenocytes from a patient‐specific stem cells for tendon tissue regeneration.
Development of anti‐fouling and anti‐thrombogenic surface using visible light cross‐linked zwitterionic hydrogel coatings for implantable medical devices
1Korea Institute of Science and Technology, 2Research Institute for Convergence Science, Seoul National University
Bacterial infections and thrombogenicity are common concerns for blood‐contacting devices since they can lead to significant inflammatory responses in the human body. Thus, anti‐fouling and anti‐ thrombogenic properties are crucial for developing bio‐interfacing materials. While several strategies including super‐hydrophilic surfaces or hydrogel coatings have been proposed, there remain some challenges in immobilizing hydrogels on surfaces and improving the stability of hydrogels.
Herein, we reported a facile zwitterionic hydrogel coating method using visible light. Briefly, the hydrogel layer composed of the zwitterionic polymer, poly (sulfobetaine methacrylate) (PSBMA), is introduced on surfaces via photo‐crosslinking, which allows any surface to be uniformly coated regardless of the surface structures. PSBMA with equal cationic and anionic groups strengthens the electrostatically induced hydration layer by binding water molecules. This characteristic of PSBMA invested the fabricated hydrogel surface with essential physical properties such as long‐term stability and super‐hydrophilicity. On the basis of these advantages, current studies focus on the characterization of the hydrogel coated‐surfaces by analyzing mechanical and biological properties depending on the types of substrates. Furthermore, we validated the effectiveness of the fabricated surfaces by protein and platelet adsorption assessment.
Visible light activated collagen based hydrogel for rotator cuff regeneration
1Korea Institute of Science and Technology, 2Chung‐ang University
Currently, drugs (steroids, NSAIDs) and surgical treatments are used to treat rotator cuff tears. However, it takes a considerable amount of time to regenerate once damaged. Even if it is regenerated, the same function as before is not fully restored. Collagen plays a dominant role in restoring wound healing, especially collagen type 1 is the major component in tendons, bones, muscles, and other connective tissues. Herein, we use modified collagen hydrogels for rotator cuff regeneration. We mixed ruthenium complex (Ru(II)bpy32+) and sodium persulfate to collagen‐based hydrogels and exposed blue light for gelation. With these hydrogels, cell cytotoxicity and tendon regeneration effects in rotator cuff tear models were evaluated. As a result, it was confirmed that modified collagen‐based hydrogels were biocompatible and enhanced cell growth. Also, from the animal defects models, rotator cuff tear was regenerated and restored compared to the non‐treating group. Therefore, it is considered that modified collagen‐based hydrogel could be a suitable application for treating rotator cuff tears.
Polycaprolactone scaffolds with improved mechanical properties and structural stability fabricated by a screw extrusion‐type 3D printer
1Medifab Co, Ltd.
3D bioprinter including screw extruder was developed and the PCL (Polycaprolactone) grafts fabricated by screw‐type and pneumatic pressure‐type bioprinters were comparatively evaluated. The density and tensile strength of the single layers printed by the screw‐type were 14.07% and 34.76% higher, respectively, than those of the single layers produced by the pneumatic pressure‐type. The adhesive force and tensile strength of the PCL grafts printed by screw‐type were 2.72 times and 29.89% higher, respectively, than those of the PCL grafts prepared by pneumatic pressure‐type. As a result of evaluating the consistency with the original image of the PCL grafts, it had a value of about 98.35%. The layer width of the printing structure was 485.2 ± 0.004919um, which was 99.5 to 101.8% compared to the set value
(500cm2), indicating high accuracy and uniformity. The printed graft had no cytotoxicity, and there were no impurities in the extract test. In the in‐vivo studies, the tensile strength of the sample 12 months after
implantation was reduced by 50.37% and 85.43% compared to the initial point of the sample printed by the screw‐type and the pneumatic pressure‐type, respectively. As a result of observing the fractures of the samples at 9‐ and 12‐month samples, the PCL grafts prepared by the screw‐type had better in‐vivo stability. Therefore, it is considered that our printing system developed in this study can be used as a treatment for regenerative medicine.
Development of a superhydrophilic surface for antifouling and antithrombotic properties using layer‐by‐layer assembly of laponite and heparin
1Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), 2Department of Dental Materials, School of Dentistry, Kyung Hee University
Implantable medical devices can cause acute thrombosis and inflammation due to adhesion and denaturation of plasma proteins when exposed to blood, thereby impairing the performance of the medical devices. In order to overcome this problem, we plan to manufacture a superhydrophilic surface that is attracting attention due to its excellent biocompatibility as well as reduced foreign‐body reactions attributing to prevent of adhesion and activation of non‐specific proteins. The superhydrophilic surface was developed using laponite and heparin, i.e., a disk‐shaped clay particle and a typical antithrombotic material, respectively. Laponite and heparin were alternately deposited based on the electrostatic interaction using layer‐by‐layer assembly. Several layers of coating were formed with high stability, and the thickness was precisely controlled according to the number of deposition layers. Heparin was coated as the outermost layer to inhibit thrombus formation. The fabricated coatings were characterized by FT‐ IR, Alpha‐step, and Water Contact Angle. Furthermore, in order to evaluate the antifouling and antithrombotic properties of the surface, inhibition of cells, plasma protein, and platelet adsorption was confirmed through FITC‐BSA, SEM, and QCM‐D. These coating can be applied to implantable medical devices including biosensors, or drug‐releasing bio‐devices.
Fabrication of 3D bioprinted tumor cell‐laden scaffold using photo‐ crosslinkable bioink
1Laboratory of Tissue Engineering, Korea Institute of Radiological and Medical Sciences, 2Program in Biomicro System Technology, Korea University, 3Research Team of Radiological Physics & Engineering, Korea Institute of Radiological and Medical Sciences
Because two‐dimensional (2D) models cannot completely mimic the biochemical and biophysical signals of the tumor environment, a three‐dimensional (3D) tumor model using 3D bioprinting is a promising approach to fabricate the sophisticated in vitro tumor model. This study demonstrates the fabrication of scaffolds containing tumor cells or spheroids using 3D bioprinting. First, we prepared methacrylate to gelatin (GelMA) and hyaluronic acid (HyA‐MA) to make a photo‐crosslinkable bioink. Then tumor cells or spheroids were combined with the photo‐crosslinkable bioink. The rheological properties of GelMA/HyA‐MA showed an increase of the complex shear modulus compared to GelMA alone, and Live/Dead assay with HDF cells indicated a cytocompatibility of the photo‐crosslinkable bioink. We also optimized 3D bioprinting conditions such as pneumatic pressure and printing speed for the fabrication of cell‐ or spheroid‐laden scaffolds. A proliferation of cells was evaluated through DNA assay and confocal microscopy. These results suggest that the bioink is a promising material for making various 3D structures through various manufacturing strategies, and 3D bioprinting using a photo‐crosslinkable bioink is suitable for mimicking tumor microenvironment. Furthermore, as this technology is applicable to various tumors, they can be considered as in vitro tumor models with the potential to be used as patient‐specific tissue scaffolds.
In‐vivo biological safety and longevity study of thermal‐sensitive chitosan dermal filler
1Medifab
Chitosan dermal filler with thermo‐sensitive was developed using ‘Liquid to Gel’ technology. That had high viscoelasticity and recovery rate into the body. Also, chitosan filler was crosslinked with non‐toxic crosslinking agents. The biocompatibility of the chitosan filler was evaluated through an in‐vitro and in‐ vivo study. The removal of crustacean tropomyosin, the cause of crustacean allergies, was confirmed through ELISA analysis. In‐vivo persistence of chitosan filler was performed by MRI observations for 12 months. Compared the volume after 1 month of subcutaneous injection in mice, the HA filler swelled about twice, and the chitosan filler decreased to about 60%. This volume difference gradually decreased and showed a similar after 9 months. Chitosan filler was maintained at 35% after 12 months, which is seen similar levels to highly crosslinked HA filler. That is, the chitosan filler has a smaller volume change than the HA filler, and it can be said that it is slowly biodegradable and continues in the body. In addition, the distribution of chitosan in vivo was observed by labeling fluorescent materials. The strongest fluorescent signal in the kidney was continuously observed until the 14th day, and after then on, fluorescent signals similar to normal animals were observed in all organs. As a result, it was confirmed that chitosan was discharged to urine through the kidney and did not accumulate in the body. Therefore, we expect that chitosan filler is bio‐safety material and will be applicable as a novel medical device for the dermal tissue.
Development of a 3D culture hydrogel and artificial skin model based on alginate‐decellularized extracellular matrix
1MediFab Co.,Ltd
Alternative animal testing is increasing as animal testing for new cosmetics and therapeutic drugs is prohibited. Two‐dimensional (2D) culture has limitation to mimic in‐vivo condition. In this study, an artificial skin model was developed using a material containing alginate hydrogel based on double crosslinking technology and decellularized extracellular matrix prepared using a supercritical fluid process. The 3D skin model had a compressive modulus of 46 kPa and showed physical properties similar to those of human skin. When dermal fibroblasts were encapsulated in a hydrogel and cultured, the rate of shape change was less than 85% for 4 weeks and had morphological stability without shrinkage. As a result of culturing keratinocytes, the proliferation of cells cultured in our material was improved compared to the negative control. As a result of applying the wrinkle improvement efficacy evaluation method of the manufactured artificial dermis, it was confirmed that it was suitable for the standards of the Korea Ministry of Food and Drug Safety (KFDA). Therefore, it is expected that our platform technology can be utilized as an in‐vitro screening system for cosmetics and drugs.
Cationic N,N,N‐trimethyl chitosan biomaterial‐mediated modulation of inflammatory cytokines for wound healing and tissue regeneration
1Beckman Laser Institute Korea, Dankook University College of Medicine, 2Interdisciplinary Program for Medical Laser, Dankook University, 3Department of Otorhinolaryngology‐Head and Neck Surgery, Dankook University College of Medicine, 4Department of Surgery, Dankook University College of Medicine
N,N,N‐trimethyl chitosan chloride (TMC), a quaternary chitosan derivative, has recently emerged as an attractive, versatile, and functional polymer. This quaternary chitosan derivative possesses a positive charge and is soluble over a wide range of pH with better mucoadhesive, permeation, drug delivery, and DNA delivery properties. We developed a new biomaterial using TMC. TMC possesses highly effective bactericidal properties with hemostatic and anti‐adhesion properties. TMC showed marked regulation of pro‐ and anti‐inflammatory cytokines at different observation periods corresponding to specific time points in wound healing. Reduced pro‐inflammatory cytokine protein and gene expressions relevant to the wound healing cascade were observed. TMC reduced the expression of pro‐inflammatory cytokines such as TNFα and IL‐6 in the early phase of treatment, which increased in later days to induce cell migration. A significant reduction in anti‐inflammatory cytokine expression of IL‐10 and IL‐13 was also observed after TMC treatment in a wound‐healing animal model. TMC was also found to induce changes on macrophage polarization. The reduction of pro‐inflammatory cytokines correlated with the increase in anti‐inflammatory cytokine expressions, and the macrophage polarization effects support the hypothesis that TMC can modulate inflammatory signals. This study supports the potential for further developing TMC‐based biomaterials in the biomedical field.
Design of artificial human keloid skin equivalents with collagen‐based hydrogels
1Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and (Technology KIST), 2NBIT, KU‐KIST Graduate School of Converging Science and Technology, Korea University
Keloid scars are abnormal tissues in which the connective tissues of skin proliferate uncontrollably and pathologically, making hard bumps. Although many studies are being conducted on the mechanism and treatment of keloid tissue, there are fundamental limitations because it is different from the actual keloid human tissue that can be implemented in animal models or cell models. In this study, we designed artificial human keloid skin platforms with collagen‐based hydrogels and human keratinocytes that could compensate for animal models or two‐dimensional cell culture models. First, we fabricated the dermis layer with keloid fibroblasts in hydrogels mixed with collagen and fibrin and then seeded keratinocytes to form the epidermis layer in the air‐liquid interface for full‐thickness artificial human keloid skin equivalents. We optimized the hydrogel compositions, cell ratios, cell densities, and medium compositions for controlling hydrogel contractility, fixing internal structure supporting keloid fibroblasts, and stabling multi layers. By histological studies and mechanical analysis, it was confirmed that keloid tissues produced in the platform showed keloid‐specific microstructures, abundant secretion of vimentin, and stiff mechanical properties compared to normal skin tissues. Simultaneously, by comparing the dermis/epidermis thickness and surface area with the normal skin equivalents, the correlation between cell proliferation and contractility was also revealed. In conclusion, this presented the 3D scar model with keloid characteristics in an easier and simpler way and by developing this, we suggest that this keloid model has the potential to make up the animal models.
The effect of PLCL nerve guidance conduit and electrical stimulation on facial nerve regeneration
1Department of Otorhinolaryngology Head and Neck Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea, 2Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul, Republic of Korea, 3Korea Institute of Science and Technology
This study aimed to investigate the effect of poly(lactide‐co‐ɛ‐caprolactone) (PLCL) nerve guidance conduit (NGC) and electrical stimulus in promoting recovery of the facial function and nerve regeneration after facial nerve (FN) section in a rat model. The experimental group used PLCL conduit and applied transcutaneous electrical stimulation (TES) at the same time, and the control group only connected the conduit with a silicon tube or PLCL. For electrical stimulation, two commonly used direct current (DC) and charge‐balanced pulse stimulations were used. The electrical stimulation group (DC and Pulse) showed much superior whisker movement than the non‐electrical stimulation group at both the fourth and eighth weeks. In IF staining, it was investigated that myelination in the Pulse group and DC group was thicker and more homogeneous than in other groups. Also, regeneration of neuronal constituent proteins such as DAPI, beta‐tubulin, and S‐100 was also achieved in the Pulse and DC group better than in the control group. This study confirmed behavioral and functional FN recovery according to PLCL and electrical stimulation types. And also, from a histopathological point of view, pulse and DC stimulation promoted and accelerated the recovery of FN compared to the PLCL group without electrical stimulation. Therefore, we proved that nerve regeneration occurs much more efficiently when TES is used together than simply using NGC in the facial nerve that has been physically damaged.
Layer‐by‐layer coatings for the enhanced biological performance of orthopaedic implants
1Newcastle University
Implementing orthopaedic implants for bone fracture has resulted in different outcomes, including implant failure, which leads to biofilm formation and pathological bone loss due to infection and aseptic loosening. The National Health Service (NHS) spends £3 billion annually on treating implant failure, which fatally causes death. Currently, no proven therapeutic approaches efficiently guarantee the eradication of those effects. Thus, developing strategies to combat the infection and poor bone integration is crucial. This research aims to establish functional coatings by incorporating antibacterial agents and natural‐derived materials in a controlled technique through various in vitro tests of mesenchymal stem cells (MSCs) for affordable applications in clinical settings. Superficial modification of titanium alloy implant substrates via Layer‐by‐Layer assembly technology illustrates an innovative solution on the surface coating to inhibit bacteria adherence and improve cell attachment. The titanium implant alloy undergoes the incorporation of collagen and hyaluronic acid as polyelectrolytes with the methylglyoxal (MGO) incorporation as antibacterial. The sample's wettability through contact angle measurement showed hydrophilic surfaces for all groups of layers, with or without MGO. The metabolic activity analysis of the coated samples showed increasing values in the first five days while decreasing in the next few days. In addition, the immunostaining evidenced cellular spindle shape, typical of MSCs, spread throughout the different coated samples' surfaces. The results reflect the cells' ability to withstand the antibacterial effects of MGO. In addition, the cells can attach and grow on the different coated samples compared to controls, suggesting a suitable cytocompatibility without compromising MSCs' biofunction.
A senolytic‐eluting coronary stent for the prevention of in‐stent restenosis
1Seoul National University, 2Yonsei University College of Medicine
The vast majority of drug‐eluting stents (DES) elute either sirolimus or one of its analogues. While limus drugs stymie vascular smooth muscle cell (VSMC) proliferation to prevent in‐stent restenosis, their antiproliferative nature is indiscriminate and limit healing of the endothelium in stented vessels, increasing the risk of late‐stent thrombosis. Oxidative stress, which is associated with vascular injury from stent implantation, can induce VSMCs to undergo senescence and senescent VSMCs can produce pro‐inflammatory cytokines capable of inducing proliferation of neighboring non‐senescent VSMCs. We explored the potential of senolytic therapy, which involves the selective elimination of senescent cells, in the form of a senolytic‐eluting stent (SES) for interventional cardiology. We found that in vitro, a H2O2‐ treated VSMCs underwent senescence and that the conditioned medium (CM) of H2O2‐treated senescent VSMCs triggered the proliferation of quiescent VSMCs. ABT263 reverted H2O2‐mediated senescence and the proliferative capacity of senescent VSMC CM. Unlike everolimus, ABT263 did not affect endothelial cell migration and/or proliferation. Senolytic‐eluting stents, but not everolimus‐eluting stents, significantly lowered stenosis area in vivo compared with bare‐metal stents. Our study shows the potential of senolytic‐eluting stents as an alternative to current forms of DES.
Genetic fusion of a human serum albumin‐specific protein binder significantly increases the biological functionality and blood circulation time of human interleukin‐15
1Gyeongsang National Univ.
Human interleukin‐15 (hIL‐15) has attracted considerable attention as an anti‐cancer agent due to its functionality to directly stimulate the proliferation and cytotoxic activity of NK and T cells. Nevertheless, a relatively short half‐life of IL‐15 requires repeated administration and higher doses, causing serious side effects. Here, we demonstrate that the genetic fusion of a human serum albumin‐specific protein binder significantly increased the biological functionality and blood circulation time of hIL‐15. The fusion construct was shown to maintain respective binding activities for hIL‐15 receptor α and human serum albumin. The protein binder‐fused hIL‐15 was shown to result in a significant increase in the secretion of Granzyme B and INF‐γ by immune cells compare to free hIL‐15. Furthermore, the protein binder‐fused hIL‐15 enhanced the population of activated T cell subsets such as CD4+ and CD8+ T cells. The terminal half‐life of the protein binder‐fused hIL‐15 was prolonged by around 40‐fold in transgenic mice expressing human serum albumin, compared to free hIL‐15 and an off‐target protein binder‐fused hIL‐15. The protein binder‐fused hIL‐15 exhibited distinct anti‐tumor activities in xenograft and allograft melanoma mouse models through activation of NK and CD8+ T cells. The protein binder‐fused hIL‐15 is expected to be used in cancer immunotherapy, assisting in the development of other cytokines as immunotherapeutic agents with greater efficacy.
Tumor intracellular microenvironment‐responsive nanoparticles for magnetically targeted chemotherapy
1Department of Biomedical Sciences, Chonnam National University Medical School, 264, Seoyang‐ro, Jeollanam‐do, 58128, Republic of Korea., 22tDepartment of Polymer Science and Engineering,
Chungnam National University, 99 Daehakro, Yuseong‐gu, Daejeon, 34134, Republic of Korea.
Nanoparticles (NPs) with responsive modalities in biological microenvironments and external stimuli have received great attention as highly efficient and precise cancer therapy agents. In this study, tumor intracellular microenvironment‐responsive NPs co‐assembled from poly(ethylene glycol) poly(aspartic acid) [PEG‐P(Asp)] copolymer, doxorubicin (DOX), and superparamagnetic iron oxide NPs (SPIONs), termed as PEG‐P(Asp)/DOX/SPIONs, were prepared for tumor intracellular microenvironment (enzyme and pH)‐responsive and magnetically targeted chemotherapy. The NPs exhibited not only enzyme‐ responsive degradation in the presence of protease, but also triggered the release of DOX at pH 5, which is an acidity similar to endolysosomal microenvironments in tumor cells. Furthermore, the PEG, P (Asp)
/DOX/SPIONs showed a contrast effect in magnetic resonance imaging. In vitro viability assays showed that PEG‐P(Asp)/DOX/SPIONs could effectively augment the cytocompatibility of DOX compared to free DOX without a change in magnetic forces. Fluorescence microscopy images indicated that the fabricated NPs efficiently increased the targeted uptake and release of DOX within cells. Overall, this hybrid NP system could be a favorable biomedical agent for effective tumor‐targeted anti‐cancer therapy.
Capsulation technique‐based intercellular organelle transfer for osteoarthritis therapy
1CHA University
Chondrocytes are the represented cells existed in mature articular cartilage. When the articular cartilage breaks down by mechanical and inflammatory factors, osteoarthritis (OA) is progressed. In this study, isolated fresh organelle has been delivered to the OA chondrocytes by fusogenic liposome. Therapy targeting organelle dysfunction can be a potential treatment for OA.
Isolated organelle from human mesenchymal stem cells (hMSCs) and rat L6 myoblasts is encapsulated by fusogenic liposomes. Fusogenic organelle capsules (FOCs) are transferred to OA chondrocytes. Lipopolysaccharide (LPS) was used to stimulate human chondrocytes (C28/I2) to construct an OA model in vitro. Fusogenic liposomes were synthesized by the film hydration method using different ratios of neutral lipid, cationic lipid, and aromatic lipid.
The fusogenic liposomes had a positive charge due to cationic lipid and showed that organelle encapsulation efficiency was proportional to the amount of cationic lipid in the liposome. Organelle encapsulation efficiency was measured by FRET (Fluorescence Resonance Energy Transfer). FOCs showed higher organelle transfer efficiency than naked organelle. Transferred organelle suppressed LPS‐ induced inflammatory responses and reduction of collagen synthesis in OA chondrocytes. Inflammatory cytokine markers and ECM degradation markers were measured via qRT‐PCR and western blotting. Furthermore, FOCs can avoid the formation of organelle intracellular‐endosomes and thus can escape from pH damage.
In summary, FOCs has the advantage of faster and more stable delivery of large amounts of organelle. The transplanted organelle helps to recovery of LPS‐induced chondrocytes.
Fabrication of a polymeric inhibitor of membrane‐type co‐localized enzymes for synergistic inhibition of cancer cell metabolism
1Osaka University
Simultaneous inhibition of metabolically and spatiotemporally related proteins/enzymes is a promising strategy for improving therapeutic interventions in cancer therapy because the characteristic cancer cell metabolism including survival/proliferation is dependent on a limited number of molecular mechanisms [1]. Carbonic anhydrase IX (CAIX) is a transmembrane enzyme that neutralizes intracellular pH acidified by glycolysis to promote tumor cell survival [2]. Recent interactome studies revealed that zinc‐dependent metalloproteinases such as MMP14 and ADAM17 are metabolically and spatiotemporally related to CAIX [3]. Herein, we report a polymer ligand functionalized with inhibitors for CAIX and MMPs/ADAMs which synergistically inhibits the proliferation/survival and migration of the human breast cancer cell line MDA‐MB‐231. We synthesized a CAIX‐ and MMPs/ADAMs‐ targeted poly (L‐ glutamic acid ;PGA) derivative (PGA‐UT) by functionalizing PGA with a CAIX inhibitor (U‐104) and a broad‐spectrum inhibitor of MMPs and ADAMs (TAPI‐2). PGA‐UT synergistically inhibited the cell proliferation than a mixture of small‐molecule inhibitors and that of PGA derivatives functionalized with either U‐104 and TAPI‐2. PGA‐UT also inhibited the cell migration compared to small‐molecule inhibitors and the other PGA derivatives. These results indicate that PGA‐UT synergistically inhibits cell metabolisms of MDA‐MB‐231 via targeting the co‐localized enzymes. This study provides a potential therapeutic strategy using polymer‐based molecular‐targeted drugs for synergistic inhibition of cancer cells.
[1] T.W. Chen et al., Biomater. Sci.
[3] M. Pastorekova et al., Br. J. Cancer
Cell‐favorable protein‐based adhesive microcapsules for NK cells‐ mediated cancer immunotherapy
1POSTECH
Cancer immunotherapy, which uses body's own immune system to attack cancer cells, has been developed as a novel alternative to conventional cytotoxic therapies that can result in cytotoxicity against normal cells and resistance. Especially, natural killer (NK) cell adoptive therapy exhibits rapid and potent anti‐tumor immunity by secreting cytotoxic cytokines and directly killing tumor cells without the requirement for the prior antigen presentation. However, insufficient localization of delivered NK cells to target tumor site and a lack of in vivo persistence by systemic injection necessitates the development of functional biomaterial‐based NK cell carrier. Here, we developed cell‐favorable adhesive microcapsules capable of adhering to cancer tissues using bioengineered mussel adhesive protein (MAP) to achieve efficient NK cell‐mediated cancer immunotherapy. Highly uniform microcapsules with a hydrated inner microenvironment which is beneficial for the suspension cell encapsulation were fabricated by microfluidic process. The MAP microcapsules exhibited mechanical stability with a great injectability as well as NK cell‐favorable biocompatibility. In particular, the microcapsules showed effective anticancer therapeutic activity by releasing secreted antitumoric cytokines from NK cells and subsequently tumor‐ killing NK cells as well. Thus, the MAP microcapsules with adhesive properties can be promisingly employed as efficient lymphocyte carrier system for localized adoptive anticancer immunotherapy.
Click chemistry complex drug delivery system using tissue extracellular matrix for the anti‐tumor therapy
1Industry 4.0 Convergence Bionics Engineering, Pukyong National University, 2Department of Display Engineering, Pukyong National University
This study developed novel stimuli responsive injectable hydrogels composed of a biocompatible and anti‐adhesive cartilage acellularized matrix (CAM) and a diselenide bridge‐containing cross‐linker by using norbornene (Nb)‐tetrazine (Tz) click chemistry. The crosslinking reaction between CAM‐Nb and the Tz‐cross‐linker evolved nitrogen gas and resulted in injectable hydrogels with highly porous structures. The synthesized hydrogels demonstrated high drug loading efficiencies, good swelling ratios, and better rheological properties. The doxorubicin (DOX)‐loaded hydrogels released minimal amounts of DOX in simulated physiological medium, however, sustained release of DOX was detected in reducing conditions, revealing more than 90% DOX release after 96 h. Interestingly, the indocyanine green (ICG) incorporated hydrogels produced reactive oxygen species upon exposure to NIR light and exhibited burst release (> 50% DOX release) of DOX during the first 4 h, followed by a sustained release phase. The in vitro biocompatibility studies showed that the synthesized CAM‐Nb and hydrogels are essentially non‐ toxic to human fibroblast cells and human colorectal adenocarcinoma cells. Furthermore, DOX‐loaded and DOX+ICG‐loaded hydrogels inhibited the metabolic activities of cancer cells after glutathione (GSH) or NIR exposure, and induced anti‐tumor effects, which were similar to that of the DOX only. And we expand to in vivo studies. Results showed that NIR exposed ICG and DOX loaded hydrogels have thermal effect and suppressive tumor growing. Therefore, these cartilage extracellular matrix and click chemistry complex injectable hydrogels could be promising candidates for minimally invasive local delivery of cancer therapeutics.
Transdermal delivery of hyaluronate based upconverting nanoparticle
1Pusan National University
Among the visible light, short wavelength like blue with sufficient energy for manipulating biosystems can be widely exploited in photomedicine. However, there are some limitations for practical applications, because of low penetration into tissue and cell damages. To resolve this issue, upconversion (UC), an anti‐stock process that converts two or more low‐frequency photons into a single photon of a higher frequency, is attracting attention as an attractive alternative. Here, we encapsulate organic based red‐to‐ blue UC dyes, palladium( II ) tetraphenyl‐tetra benzoporphyrin (PdTPBP) as a photosensitizer, and perylene as an acceptor, into polycaprolactone grafted hyaluronic acid nanoparticles (HA‐PCL/UC NPs). The successful preparation of HA‐PCL/UC NPs is confirmed by DLS and TEM, and stability is confirmed by UV‐visible spectroscopy. In the porcine skin penetration experiment, the 635 nm laser penetrated into the dermis converts to blue light in the UC NPs complex. Based on these results, our HA‐ based NPs with upconversion dye as a light source that can convert light with low energy (red) into high energy (blue) are appropriated for transdermal delivery of UC dye and it is expected to be an effective photomedicine platform.
Doxycycline‐eluting core‐shell type nanofiber‐covered trachea stent for inhibition of cellular metalloproteinase and its related fibrotic stenosis
1Department of Biomedical Science, BK21 FOUR Program in Biomedical Science and Engineering, Inha University College of Medicine, Incheon 22212, Korea, 2Department of Biomedical Science,
Translational Research center, Inha University Hospital, Incheon, Korea, 3Inha Institute of Aerospace Medicine, Inha University College of Medicine, Incheon 22332, Korea
An endotracheal stent covered by a doxycycline (doxy)‐eluting nanofiber is fabricated to prevent the intubation related tissue fibrosis and re‐stenosis. Poly (D,L‐lactide) was chosen for the shell and polyurethane was used as core forming materials. Changing the electro‐spray of every polymeric solution via compositional ratio of the core to shell was adjusted to 1:0, 1:2, and 1:4 and microscopic observation of nanofibers using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and the fluorescence microscopy proved core‐shell structure of nanofibers. The in vitro release study suggested that the release of doxy could be controlled by increasing the compositional ratio of the shell. The growth of HT1080 fibrosarcoma cells was inhibited by the 10% doxy‐containing nanofiber. The doxy‐eluting nanofiber inhibited the expression of MMP‐2 and MMP‐9. Our results demonstrated that a doxy‐releasing nanofiber stent can prevent fibrotic deformation of tracheal tissues after stent intubation
Improved properties of polymeric micelles via hydrophobic core‐ clustering of superparamagnetic iron oxide nanoparticles
1Department of Biomedical Science, BK21 FOUR Program in Biomedical Science and Engineering, Inha University College of Medicine, Incheon 22212
Superparamagnetic iron oxide nanoparticles (SPIO) have been studied to design theranostic polymeric micelles for targeted cancer therapy and diagnostic MR imaging over decades. Nevertheless, the effects of SPIO on the physicochemical and biological properties of polymeric micelles have not been fully characterized to this day. Herein, we investigated potential effect of SPIO on the physical and biological properties of theranostic polymeric micelles using representative cancer drug (doxorubicin; Doxo) and polymer carrier (i.e., poly (ethylene glycol)‐co‐poly(D,L‐lactide), PEG‐PLA). SPIO were synthesized from Fe(acetyl acetonate)3 in an aryl ether, subsequently, SPIO and Doxo were loaded into the polymeric micelles. The effect of SPIO‐clustering on drug loading, micelle size, thermodynamic stability, and theranostic property of PEG‐PLA polymeric micelles were observed. Moreover, cellular uptake behaviors, pharmacokinetic and biodistribution study have been carried out. SPIO formed hydrophobic geometric cavity in the micelle core and affected the integrity of micelles in terms of micelle size, Doxo loading, critical micelle concentration (CMC) and in vitro dissociation. In vivo pharmacokinetic studies also showed the enhanced Area Under Curve (AUC) and elongated the half‐life of Doxo. Thus, clustered SPIO in micelles largely affects MR imaging properties in addition, influences biological and physical properties of polymeric micelles.
An organ‐on‐a‐chip approach for efficient phage display biopanning under physiological conditions
1Ulsan National Institute of Science and Technology, 2Sungkyunkwan University
Cell‐based phage display screening is a powerful technique for identifying ligands binding with a specific receptor, which can be exploited for selective drug delivery. Particularly, it has drawn a substantial interest in discovering novel peptide ligands targeting organ‐specific vascular‐bed. However, this approach has met with little success due to the incompetency of traditional cell‐based model for peptide screening, which fails to replicate the unique features of a vascular‐bed of a specific organ. The vascular endothelium lining the inner surface of blood vessel is subjected to blood flow‐induced fluid shear stress, which enables maintenance of structural and functional features of endothelial cells through mechano‐ transduction. Thus, an organ‐on‐a‐chip recapitulating blood flow in vascular endothelium has emerged as a promising platform that provides a better recapitulation of physiological features such as cytoskeletal organization, gene expressions, and intracellular trafficking dynamics than a traditional static model. Herein, we describe how an organ‐on‐a‐chip technology allows reliable and efficient selection of an organ‐targeting shuttle peptides. To demonstrate this, phage display selections were compared using a microfluidic brain endothelium model as opposed to a static transwell‐based model. The influence of fluid flow on the cellular activities of brain endothelial cells was shown by a comparison of the transcriptome profiles of two models, which revealed distinct patterns of gene expression. We found that shuttle peptides identified using microfluidic brain endothelium model exhibited superior brain targeting capabilities in vivo due to their increased physiological relevance, indicating that an organ‐on‐a‐chip approach offers a promising benefit for successful phage display panning.
Fabrication of dual‐drugs loading liposomes stimulated by physical activation
1CHA University
Liposomes, composed of a phospholipid bilayer and loading both hydrophilic and hydrophobic drugs have been famous for their applications in drug delivery system. By changing the components constituting the liposome, the size and permeability of the liposome membrane can be controlled, resulted in the applications as a vector effective for drug delivery with less toxicity to cells.
In many cases, drugs with hydrophobic properties are loaded into the liposome in relatively small amounts. To overcome this, the dose of the drug administered must be increased. And because liposomes, which are biomaterials, easily bind to cells, they need ligands that can recognize target cells.
Recently, many studies have been focused on dual‐drugs which loaded into liposomes. However, liposomes have also been studied for their characteristics. During the preparation of them, the liposomes can be changed their physical capacity by physical stimulation, such as ultrasonic wave, electrical shock, and photo‐irradiation. Their physical stimulation will be changed the conformation of their outer or inner hydrophobic or hydrophilic moieties. This conformational change of the liposome will have the ability to load more drugs into the liposome.
In this study, a novel method has been applied for more reliable loading the drugs and targeting to cells. By preparing liposomes using pH‐sensitive phosphatidylethanolamine, more drugs are loaded by physical stimulating the liposome. Thus, more loading drugs and increasing pH sensitivity, effective drug delivery‐ treatment is possible. This study could solve the problem of hydrophobic drug loading efficiency, cause to the disadvantage of drugs of dual‐drug loaded liposomes.
Regulation of cell membrane permeability and photo‐processing DNPs behavior for LED‐mediated gene delivery
1CHA University
Recently, light‐emitting diodes (LEDs) have recently been attracting attention as physical regulators for controlling various cellular behaviors. In this study, we show the effect of LED as physical regulators for gene delivery to stem cells by regulating cell membrane, nanomaterials behavior and enhancing transfection efficiency. For the effective delivery of gene into tissue and cells by LED, we fabricated Dexamethasone (DEX) loaded poly (poly (DL‐lactic‐co‐glycolic acid) (PLGA) nanoparticles complexed with cationic polymer of polyethyleneimine (PEI). By complexed with PEI, the gene can ionic interacted with nanoparticles. Also, we confirmed various voltage and time of LED irradiation to establish suitable conditions for gene delivery, and directly stimulated on stem cell and nanoparticles, confirming the transfection process. By changing the wavelengths of LEDs (Blue, Green, Red and Near‐InfraRed (NIR)), we assessed cell membrane permeability via confocal laser microscopy, FACS and nanoparticle's behavior using DLS, SEM, and Nano sight. Then transfection efficiency using confocal laser microscopy and western blotting. Results demonstrate that NIR wavelength irradiation increased the cell membrane permeability by enhancing internalization of extracellular substance and behavior of nanoparticles by increasing the diffusion and decreasing the size. On the other hand, Blue wavelength has a significant impact on gene delivery and protein expression through rapid escape from endosomes located in the cytoplasm. Here, we show important role of LED in controlling nanoparticle's behavior and new opportunities for gene therapy.
Hydroxytyrosol: In vitro study on its feasibility and efficacy in attenuating intimal hyperplasia progression
1Department of Surgery, Hospital Canselor Tuanku Mukhriz, Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia, 2Center of Tissue Engineering and Regenerative Medicine, Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia
Intimal hyperplasia (IH) is a vascular thickening that occurs due to excessive proliferation and migration of smooth muscle cells (SMCs), often responding to a traumatic stimulus. IH decreases the patency of bypass graft post Coronary artery bypass graft surgery (CABG) and threatens angioplasty procedure. Therapies employing antiproliferative drugs have inhibited SMCs proliferation but concurrently impede re‐endothelisation. Hydroxytyrosol is an olive‐derived polyphenol. Its effect on IH attenuation has not been extensively studied. Therefore, we aim to study the effect of HT on endothelial cell proliferation and SMC inhibition in‐vitro before its application in an ex‐vivo IH model. We isolated ECs and SMCs from saphenous veins of patients undergoing CABG surgery. The half‐maximal inhibitory concentration (IC50) of HT‐treated SMC is 300mM. EDU assay detected 40mM up to 320mM significantly (p < 0.05) decreases PDGF‐BB induced SMCs proliferation. A dose from 20 mM‐80mM suppressed the wound closure and migration of PDGF induced SMCs by 54% (p < 0.001). HT downregulated vimentin, a synthetic phenotype marker in PDGF‐BB induced SMCs. Besides, up to 50mM, HT could maintain EC proliferation. VEGFR2 protein, an angiogenesis marker was highly expressed in HT‐treated EC. Hydroxytyrosol also salvages TNF‐α induced cytoxicity and apoptosis in ECs. Following this, we also established the IH ex‐vivo model by mechanically scraping the ECs in in‐vitro artificial conditions where the intimal media thickness was significantly higher than the native vessel. Therefore, these preliminary findings enable the study of HT combinatory effect on both EC and SMC in an ex‐vivo IH model to be further elucidated soon.
Adeno‐associated viral vector delivery system for the regeneration of hypoxia ischemic encephalopathy injured brain
1Yonsei University
AAV has been widely utilized in many research studies both in vitro and in vivo, as well as in clinical trials for the development of gene therapy products. Several AAV‐based gene therapy products include Glybera, Zolgensma, and Luxturna. However, one of the main hurdles in commercializing them is their high dosage requirement. The development of highly efficient AAV delivery system is crucial in reducing the dosage and off‐targeting issues, thereby minimizing cost and toxicity issues. Herein, we aimed to enhance the efficacy of AAV transduction by treating it with biomimetic phenolic molecules. Previously reported to have improved cardiac transduction, we hereby demonstrate that the phenolic‐AAV enhanced the local transduction in HIE injured brain. The effect of the phenolic molecules in the transduction efficiency of AAV towards damaged brain regions may aid in the development of a highly potent therapeutic product for the treatment of various central nervous system (CNS) diseases.
Enzyme‐mediated oxygen releasing polyphenol particles for ischemia treatment
1Dong‐A University
Peripheral arterial disease (PAD) around the world is a major health problem with about 230 million patients. PAD is an angiopathology in which narrowed arteries reduce blood flow to peripheral ends and organs such as legs, arms, and heads. In this regard, Ischemia disease does not provide oxygen and nutrients to tissues, resulting in necrosis. To solve this problem, by supplying oxygen to tissues through polyphenol particles, it is possible to prevent tissue necrosis and induce vascular production at the same time, thereby increasing cell survival rate. EGCG particles loaded with Tyrosinase, an enzyme that induces hydroxylation of the phenol group, produce hydrogen peroxide. Additionally, catalase, an enzyme that decomposes hydrogen peroxide into water and oxygen, was incorporated into the surface residue to form particles that release oxygen. By applying these particles to hindlimb ischemia, a sustainable oxygen release system establish to generate blood vessels through oxygen supply and improve tissue regeneration ability so that damaged tissue can be stably regenerated through the anti‐inflammatory reaction of polyphenol. This study provides new insights into the potential for the reliable treatment of ischemic diseases.
Disturbed flow‐targeting nanovesicles for early theragnosis of atherosclerosis
1Chung‐Ang University, 2Yonsei University, 3Sookmyung Women's University
Despite the high mortality rate and irreversibility of atherosclerosis, conventional diagnoses and therapeutics have limited capability of detecting and preventing early pathogenic events. As atherosclerosis is reported as a type of inflammatory disease, mesenchymal stem cells (MSC) or their derivatives have emerged as new therapeutic candidates due to their anti‐inflammatory and pro‐ regenerative features. Here, physical breakdown of cells with micropore filtration followed by self‐ assembly is conducted to achieve high‐throughput of therapeutic exosome‐mimetic nanovesicles (MSC‐ NVs). To target early atherogenic lesion represented by disturbed blood flow site, the surface of MSC‐ NVs were functionalized by GSPREYTSYMPH (PREY) peptide which was previously screened from 108 candidates by phage display method. Our PREY‐displaying MSC‐NVs successfully targeted and prevented the early atherogenic step in mouse, porcine atherogenic models and even in a microfluidic model using human arterial endothelial cells. These promising results suggest our engineered MSC‐NVS as a potential next‐generation platform for translational medicine.
Plant‐inspired pluronic‐gallol micelle: Low critical micelle concentration, high protein affinity, and thermal stability
1Department of Intelligent Precision Healthcare Convergence, Sungkyunkwan University
Polymeric micelles are the most common platform for drug delivery. However, when the micelles are intravenously injected in vivo, versatile biological and physical barrier, such as enzymes and changes in temperature, easily destabilizes the materials. In addition to that, the micelles are rapidly disassembled due to dilution below critical micelle concentration (CMC). Here, we report that the modification of Pluronic micelles with octyl gallate – which is surfactant including gallol moieties widely found in antioxidant plant polyphenols – improves their colloidal stability to heat without any covalent crosslinking. Furthermore, the gallol moieties allowed enzymatic degradation resistance of the micelles by robust affinity to proteins and their low CMC while maintaining hydrophobic cavity. Such low CMC and high biological stability of the gallol‐incorporated micelles would be promising as a drug vehicle with enhanced therapeutic efficacy.
Anti‐inflammatory efficacy of metformin‐encapsulated PLGA
1Department of Biochemistry and Biomimetics, Bowdoin College, 2Department of Physiology, College of Medicine, Gachon University, 3Gachon Advanced Institute For Health Sciences and Technology, GAIHST, Gachon University
Metformin is a synthetic drug that is widely used for type 2 diabetes mellitus, in which the drug effectively prevents the potential inflammatory cascades. Despite the promising efficacy of metformin for the diabetes mellitus, the potential therapeutic use of metformin in arthritis, osteoarthritis (OA), and rheumatoid arthritis (RA), the disease that primarily affects joints with inflammation, was scarcely studied. Metformin exhibits anti‐inflammatory effects by activating the AMPK pathway, and increasing numbers of studies have explored the potential use of metformin in treating inflammatory diseases. However, the type of drug delivery is solely restricted to oral administration. Herein, we formulated the polymeric nanoparticles(NPs) that successfully encapsulated metformin for effective drug delivery.
Metformin‐encapsulated polymeric NPs consists of PLGA nanoparticles, the copolymers of PLA and PGA, poly (lactic acid‐co‐glycolic acid)/poly (lactide‐co‐glycolide); PLGA(Lactel; Cat: B6013‐2P, 50:50 DL‐PLG), and Metformin. The anti‐inflammatory efficacy of NPs was analyzed with fibroblast‐like synoviocytes (FLS) and M1 phenotype (pro‐inflammatory) of macrophages, which play crucial roles in joint destruction by secreting proinflammatory cytokines.
Changes in physicochemical properties confirmed the successful encapsulation of metformin inside the PLGA. The cellular uptakes of NPs showed greater uptake in inflammatory FLS and J774 (macrophage), and particularly, the endocytic pathways of the cells were strongly dependent on caveolae. Finally, the anti‐inflammatory effect was demonstrated by the downregulation of pro‐inflammatory gene expression and the increased protein expression of phosphorylated AMPK.
This study illustrated that metformin‐encapsulated polymeric NP can effectively reduce the pro‐ inflammatory responses by activating AMPK, which could modulate the activity of NF‐ κB signaling.
Acoustic anticancer therapy using nanoparticle
1Gachon Advanced Institute For Health Sciences and Technology, GAIHST, Gachon University,
2Department of Physiology, College of Medicine, Gachon University
Sonodynamic therapies (SDT) are widely used for treating various types of tumors. However, SDT is usually generated by ultrasound of 20 kHz or higher, using soundwaves within the audible frequency has rarely been investigated. Here, we suggested that audible soundwaves trigger aggregation of the nanoparticles and, thus aggregation behavior of the nanoparticles influenced by soundwave can significantly influence intracellular autophagy dynamics. Thus, the amplified autophagy via sound responsive nanodrug stimulated can effectively induce apoptosis in cancer cells. In conclusion, obtained result clearly suggested the evidence of sono‐therapy as an effective anticancer treatment method with noninvasive manner.
Nano‐corona anticancer strategy
1Gachon Advanced Institute For Health Sciences and Technology, GAIHST, Gachon University,
2Department of Physiology, College of Medicine, Gachon University
Novel anticancer strategies using nanodrugs are empowered with immunotherapeutics. The possibility of chemotherapy using nano‐corona, this study propose a strategy using nano‐corona as a nanodrug to enhance the efficacy of immunotherapeutics. It was confirmed that conformational changes, in which the secondary protein structure was altered and thus, the nano‐corona deformed structures of immunoglobulin G (IgG) surrounding gold nanostar (AuS). A small modification in the protein structure caused by a mutual interaction with nanoparticles impacts immune activation in cancer cells. Furthermore, nano‐ corona induced the expression of pro‐inflammatory cytokines and macrophage re‐polarization, thus confirming the therapeutic potential in the tumor environment. This study demonstrated that immunologic responses triggered by nano‐corona led to significant immunotherapeutic efficacy.
PTH and novel PTH analog for osseointegration and bone regeneration in ovariectomized beagle mode
1Ewha Womans University, 2Gachon University College of Medicine
Long‐term anti‐inflammatory effects of injectable celecoxib nanoparticle hydrogels for achilles tendon regeneration
1Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea; Division of Bio‐Medical Science and Technology, KIST School, Korea University of Science and Technology, Seoul, 02792, Republic of Korea, 2Department of Orthopedic Surgery and Rare Diseases Institute, Korea University Medical College, Guro Hospital, Seoul, 08308, Republic of Korea
The treatment of chronic Achilles tendonitis (AT) often requires prolonged therapy and invasive therapeutic methods such as surgery or therapeutic endoscopy. To prevent the progression of chronic AT, excessive inflammation must be alleviated at an early stage. Corticosteroids or nonsteroidal anti‐ inflammatory drugs are generally prescribed to control inflammation; however, the high doses and long therapeutic periods required may lead to serious side effects. Herein, a local injectable poly(organophosphazene) (PPZ) –celecoxib (CXB) nanoparticle (PCNP) hydrogel system with long‐term anti‐inflammatory effects was developed for the treatment of tendonitis. The amphiphilic structure and thermosensitive mechanical properties of PPZ means that the hydrophobic CXB can be easily incorporated into the hydrophobic core to form PCNP at 4 °C. Following the injection of PCNP into the AT, PCNP hydrogel formed at body temperature and induced long‐term local anti‐inflammatory effects via sustained release of the PCNP. The therapeutic effects of the injectable PCNP system can alleviate excessive inflammation during the early stages of tissue damage and boost tissue regeneration. This study suggests that PCNP has significant potential as a long‐term anti‐inflammatory agent through sustained nonsteroidal anti‐inflammatory edrugs (NSAIDs) delivery and tissue regeneration boosting.
Neuroprotective potential of phospholipase A2 against oxidative stress‐ induced toxicity in neuronal cell
1Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Selangor, 2Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Selangor, Malaysia &
Centre of Tissue Engineering and Regenerative Medicine, Faculty of Medicine, The National University of Malaysia (UKM), Cheras, Selangor, Malaysia, 3Monash Venom Group, Department of Pharmacology, Biomedical Discovery Institute, Monash University, Clayton, Victoria, 4Kulliyyah of Pharmacy, International Islamic University Malaysia, Kuantan Campus, Bandar Indera Mahkota, Kuantan, Pahang, 5Centre of Tissue Engineering and Regenerative Medicine, Faculty of Medicine, The National University of Malaysia (UKM), Cheras, Selangor
TIMP1 enhances survival of transplanted adult stem cell spheroids in murine critical limb ischemia mode
1KIST (Korea Institute of Science and Technology), (UST University of Science and Technology)
Stem cell therapy has emerged as a promising regenerative medicine but has been limited by massive clearance of transplanted cells, leading to poor therapeutic outcome. Human adipose‐derived stem cells (hASCs) seeded on maltose binding protein‐fibroblast growth factor 2 (MBP‐FGF2) immobilized matrix form functionally enhanced cell spheroids (FECS‐Ad) that express upregulated levels of tissue inhibitors of metalloproteinases 1 (TIMP1), an anti‐apoptotic factor significantly improve survival of transplanted cells. In this study, the role of TIMP1 as a survival factor was assessed by ELISA, western blot, LIVE/DEAD assay in vitro as well as in vivo. Expression of HIF‐1α and TIMP1 was confirmed to be elevated in FECS‐Ad, compared to HIF‐1α knocked‐down FECS‐Ad. Expression of HIF‐1α in FECS‐Ad was determined to modulate the expression of TIMP1. Altogether, our data suggest that HIF‐1α and TIMP1 have an important role on cell viability, presumably through the anti‐apoptotic signaling pathway via CD63/FAK/Akt/Bcl2 axis. Compared to FECS‐Ad, the expression of HNA decreased and cleaved capspase‐3 increased in critical limb ischemic tissue when HIF‐1α or TIMP1 was knocked down. Stem cell therapy has been limited by extremely poor cell survival of transplanted cells and hence its low therapeutic outcome; here, we propose a novel platform that uses MBP‐FGF2 to form FECS‐Ad which demonstrates enhanced survival both in vitro and in vivo, and hence improved overall therapeutic implications of transplanted cells. Herein, we identified the mechanism for the survival rate of transplanted 3D stem cells and presented scientific evidence for the therapeutic efficacy of stem cell therapy.
Development of recombinant transcription factor proteins for direct conversion of human dermal fibroblasts into osteoblasts
1Department of Biomedical Science, Kangwon National University, Chuncheon‐si, Republic of Korea
Ectopic expression of transcription factors can induce reprogramming of somatic cells into various cell types, such as osteoblasts, without an intermediate pluripotent state. However, virus‐based strategies that cause overexpression of transcription factors have oncogenicity by host cell genomic mutations. According to cell‐penetrating peptide (CPP) capable of delivering various cargo molecules into cells has been widely used for intracellular protein delivery. Previously, the Bombyx mori 30Kc19 protein conjugated transcription factors can be delivered into cells. In addition to intracellular delivery, the 30Kc19 conjugated transcription factor has enhanced stability and soluble expression. Here in, we conjugated transcription factors with 30Kc19, a type of cell‐penetrating protein, to induce direct conversion of human dermal fibroblasts (HDFs) into osteoblasts. Recombinant 30Kc19 conjugated proteins were produced in the Escherichia coli expression system, and HDFs were induced into osteoblasts using osteogenic medium supplemented with 30Kc19 conjugated transcription factor proteins for 24 days. The function of induced osteoblasts (iOBs) was confirmed by Alizarin red S staining quantification and von Kossa staining. Next‐generation sequencing revealed that iOBs had a gene expression pattern similar to human osteoblasts. Microcomputed tomography images showed that iOBs promoted post‐implantation bone regeneration in a calvarial defect model mouse. Consequently, we suggest that our approach using the transcription factor conjugated with 30Kc19 protein could be a means of bone regeneration therapy via osteoblasts direct conversion without DNA integration.
3D spheroids of mesenchymal stem cells attenuate neuropathic pain mediated by chronic constriction injury in mice
1Department of Physiology, School of Medicine, Pusan National University
Chronic pain is caused by dysfunction of peripheral nerve associated with somatosensory system. Mesenchymal stem cells (MSC) draw attention as promising cell therapeutics for chronic pain; however, its clinical application has been hampered by poor in vivo survival and low therapeutic efficacies of transplanted cells. Increasing evidence suggests enhanced therapeutic efficacy of MSC spheroids formed by three‐dimensional culture of MSC. In the present study, we established a neuropathic pain murine model by inducing a chronic constriction injury through ligation of the right sciatic nerve and measured the therapeutic effects and survival efficacy of MSC spheroids. Monolayer‐cultured MSCs and spheroid MSCs were transplanted into gastrocnemius muscle close to the damaged sciatic nerve. Transplantation of MSC spheroids more potently alleviated chronic pain and exhibited more prolonged in vivo survival than monolayer cultured MSCs. Moreover, MSC spheroids significantly reduced infiltration of macrophages into the injured tissues. Interestingly, the expression of mouse‐origin genes associated with inflammatory responses, such as CCL11, IL1A, LTA and TNF, was significantly attenuated by administration of MSC spheroids than monolayer‐cultured MSCs. These results suggest that MSC spheroids exhibit enhanced in vivo survival after cell transplantation and reduced the host inflammatory response as modulating by chronic inflammatory response‐related genes.
Glioblastoma recurrence by neurotransmitters from abnormal neuronal firing via electrical stimulation
1Korea Advanced Institute of Science and Technology (KAIST)
Glioblastoma multiforme (GBM) is the most aggressive diffuse glioma of astrocytic lineage. Its survival rate is below 7 %, and over 90 % of patients with surgical assistance experience recurrence after 6‐9 months. The complexity of GBM in the brain microenvironment remains controversial and is still being actively studied. Herein, we investigate the neuron‐GBM cross‐talk by employing electrical stimulation (ES) to induce normal/abnormal neuronal firing to understand the secretory of neurotransmitters related to GBM recurrence under a co‐culture system in vitro. We demonstrate that hyperstimulation in high frequency (50 Hz) ES expresses abnormal neuronal firing using calcium imaging analysis: the number of signal peaks per cell, starting time point of the first signal, duration of signal, peak periodicity, average width of the peaks, and peak amplitude. Addition, notable secretion of glutamates in abnormal firing exhibited was examined, and cultured at different glutamate concentrations revealed the correlation between glutamate and GBM proliferation. Finally, GBM recurrence was studied using a co‐culture system after chemotherapy using temozolomide (TMZ), which proves hyperstimulation as a key feature of GBM recurrence. Our results help finding a route to improved survival rate with successful clinical treatment that could overcome GBM recurrence. This work was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, and ICT (MSIT) (2021R1C1C2011542).
Identification of mesenchymal stem cell‐specific surface markers
1Department of Otorhinolaryngology‐Head and Neck Surgery, College of Medicine, Ewha Womans University, 1071 Anyangcheon‐ro, Yangcheon‐gu, Seoul 07985, Republic of Korea, 2Graduate Program in System Health Science and Engineering, Ewha Womans University, Seoul 03760, Republic of Korea
It has been recognized that conventional methods for isolating mesenchymal stem cells generate a heterogeneous mixture of cells, in which only a small proportion is likely to be stem cells. Therefore, mesenchymal stem cells (MSCs) have been known to show heterogeneous characteristics with different lineage commitment, which might have a dramatic impact on the effectiveness of stem cell therapy. From this perspective, cell surface markers that enable the purification of stem cells will help to gain better outcomes for cell‐based therapies.
Previous studies have revealed the surface protein profile of various types of MSCs. However, the surface markers profile of only cells with capacity to differentiate into terminally differentiated cells have not been tested.
In this study, forward versus side scatter gating was used to indicate the differentiated cells with lipid droplets and cells without lipid droplets when analyzed by flow cytometry. By high‐throughput screening the expression of 242 cell surface proteins, we identified CD49f and CD146 were specific for bone marrow‐derived MSCs (BMSCs) meanwhile only CD146 was specific for tonsil‐derived MSCs (TMSCs). Subsequent studies revealed the consistent specific expression of CD146 in BMSCs and TMSCs.
Conclusion: This is the first study reporting the different cell surface protein expression between stem cells and niche cells. Further studies are required to confirm the surface protein profile of stem cells and niche cells in stem cell populations from other sources.
Prevention of diet‐induced obesity by adipose tissue browning using extracellular vesicles from stem cells during beige adipogenic differentiation
1Hanyang Univ.
Obesity has emerged as a global severe health concern. There are two main forms of adipose tissues, white and brown. Brown adipocytes containing many mitochondria increase energy expenditure while white adipocytes store a large amount of energy. Therefore, adipose tissue browning has been a target for obesity treatment. Stem cell‐derived extracellular vesicles (EVs) have been suggested as a potential alternative to stem cell therapy with decreased risks. EVs greatly affect the phenotype of recipient cells, cellular proliferation and differentiation in a paracrine manner. We investigated whether EVs secreted from human adipose‐derived stem cells during beige adipogenic differentiation (BD‐EV) may be used to promote adipose tissue browning in diet‐induced obesity (DIO) animal models. EVs were isolated from conditioned media during beige adipogenic differentiation of HASCs by pre‐filtration (0.2 μm syringe filter), followed by tangential flow filtration. EVs were characterized by transmission electron microscopy, dynamic light scattering, and western blotting. To evaluate the capacity of adipose tissue browning, EVs were intraperitoneally injected into high‐fat‐fed mice. The expression levels of mRNAs related to browning were measured by quantitative polymerase chain reactions (qPCR). Histological analysis was performed by hematoxylin & eosin staining and immunohistochemistry. BD‐EV were approximately 80‐120 nm in diameters and expressed exosomal marker proteins, such as CD9, CD63, and ALIX. In vivo studies demonstrated that BD‐EV treatment ameliorated DIO through adipose tissue browning in HFD‐fed mice. In addition, BD‐EV significantly attenuated hepatic steatosis. Our finding suggests that BD‐EV could promote cell reprogramming and provide a new biochemical cue for preventing DIO.
Human neural progenitor cell differentiation into spiral ganglion neurons for sensorineural hearing loss
1Beckman Laser Institute Korea, College of Medicine, Dankook University, Cheonan, South Korea, 21) Beckman Laser Institute Korea, 2) Department of Otorhinolaryngology‐Head & Neck Surgery, College of Medicine, Dankook University, Cheonan, South Korea, 3Dankook University, 4 1) Beckman Laser Institute Korea, 2) Department of Otorhinolaryngology‐Head & Neck Surgery, College of Medicine, Dankook University, Cheonan, South Korea
Our goal is to develop a regenerative therapy for sensorineural hearing loss by targeted delivery of encapsulated progenitor cells and differentiation factors into the modiolus which can then be triggered to release and differentiate using light irradiation. Among the goals of targeted delivery for any regenerative therapy, the most important is to ensure that the delivered package is to function as intended. In this part of the study, we will demonstrate the capability of the encapsulated materials, namely the neural progenitor cells (NPC) and the chosen growth factors, to differentiate into functional spiral ganglion neurons.
3D neurospheres were formed using NPCs (ATCC, ACS‐5004) and induced to further differentiate into mature neurons with the supplementation of either BDNF or NT‐3 or a combination of BDNF and NT‐3. The neurospheres were observed until 4 weeks and evaluated for immunofluorescence (IF), RNA sequencing and electrophysiological analyses.
The resulting neurospheres from BNDF only and BDNF+NT3 were able to generate cells with visible neurite protrusions. IF staining confirmed the presence of TUJ1 and MAP2 expression indicating mature neurons. Voltage‐gated sodium channels were also observed using NaV1.2 and NaV1.6 staining and supported by electrophysiological recordings. RNA sequencing analysis showed the upregulation of several DEGs associated with neuronal development as well as auditory cell fate specification.
The confirmation of NPCs ability to successfully differentiate into functional neurons in now ready for succeeding encapsulation and targeted delivery to regenerate the damaged structure and function of spiral ganglion neurons.
Three‐dimensional environment improves efficiency of chemically‐ induced direct cardiac reprogramming
1Department of Physiology, Yonsei University Medical College, 2Department of Physiology, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine
Chemically driven direct cardiac reprogramming, involving no genetic manipulation may provide a promising approach to provide safe cell sources for cardiac regenerative therapies. This method may be more desirable than genetic reprogramming involving delivery of transcription factors and/or microRNAs for clinical applications, as it avoids safety concerns associated with genetic manipulations. However, current culture systems require further improvement in the conversion efficiency and cardiac maturation. Here, we report that cardiomyocyte‐like cells can be generated from primary fibroblasts with a combination of small molecules. These chemically induced cardiomyocytes (CiCMs) were encapsulated with 3D extracellular matrix‐based hydrogel to provide in vivo‐like microenvironment. The 3D culture resulted in increased cardiomyocyte‐specific markers (Cardiac troponin T, α‐actinin) expression, sarcomeric organization compared to 2D culture. For Metabolic purification, we cultured CiCMs with glucose‐depleted and lactate‐abundant conditions. Our platform expands the range of potential applications for purified CiCMs and can facilitate the use of CiCMs for cardiac regenerative medicine. This work was supported by the Brain Korea 21 Project for Medical Science, Yonsei University. This work was also supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF)& funded by the Korean government (MSIT) (No.2020M3A9I403845513).
Blood outgrowth endothelial cells (BOECs) in re‐endothelialization of human saphenous veins (hSV): An ex vivo model
1UKM, 2PPUKM
Mesenchymal stem cells can promote the healing of the ocular surface by corneal epithelial cell regeneration in the alkali burn model of the rabbit
1Department of Ophthalmology, Asan Medical Center, University of Ulsan College of Medicine
Ocular chemical burn presents an acute emergency and requires immediate medical care. Here, we found that subconjunctival injection of phosphate‐buffered saline (PBS) containing 2 × 10^6 mesenchymal stem cells (MSCs) from umbilical cord blood promoted the wound healing of the ocular surface and regeneration of corneal epithelial cells in the rabbit alkali burn model. MSCs have the potential ability to differentiate into various cell types such as adipose tissue, heart tissue, and epithelial tissue. In the present study, we aimed to investigate the therapeutic effect of subconjunctival injection of the MSCs in the alkali burn model of the rabbit. Alkali burn injuries were caused by applying an 8‐mm diameter paper soaked in
0.5 N NaOH on the cornea for 60 seconds, followed by washing with the balanced salt solution for 60 seconds. Compared with eyes without any treatment, those with subconjunctival injection of MSCs showed significantly better recovery seven days after subconjunctival injection of the MSCs. It is also accompanied by improved fluorescein staining score of the ocular surface, immunohistochemistry (IHC) staining (alpha‐SMA, P63, CK12) of corneal and conjunctival tissue, and mRNA expression of inflammatory cytokines (IL‐8, IL‐1alpha, IL‐6, TNF‐alpha) through real‐time PCR. We found that eyes with subconjunctival injection of MSCs showed faster re‐epithelization by anti‐inflammation effect. Our finding suggests that subconjunctival injection of the MSCs can be a good approach to treating the alkali burn.
Designing engineered stem cells hybrid spheroids for inflammatory disease
1Department of Precision Medicine, School of Medicine, Sungkyunkwan University, 2Department of Pharmaceutical Science, College of Pharmacy, Keimyung University
Immunotherapy is promising approach to inflammatory diseases using Mesenchymal stem cells (MSCs) because of having diverse immunomodulatory properties via secreting potential secretomes as well as cell to cell adhesion. However, 3D stem cells or 3D hybrids stem cells are still unexplored except single MSCs for treating sepsis. Therefore, we engineered MSCs to enhance their immunomodulatory properties via hybrid spheroids especially for Sepsis model. Metformin‐loaded poly (lactic‐co‐glycolic acid) microspheres (Met‐MS) were prepared using double emulsion method, exhibiting the particle size of 2.2 ± 1.3 μm and loading capacity of 2.81 ± 0.03 %. Further, scanning electron microscope (SEM) and Fourier‐transform infrared spectroscopy (FTIR) illustrated the spherical shape and encapsulation of metformin inside microspheres. Initially, metformin was released approximately 20% as burst release and sustainably release more than 25 days. Then, 1.1 x 106 MSCs cells or 1.1 x 106 MSCs with 0.5, 1, and 2 mg Met‐MS were kept in AggreWell for spheroids or hybrid spheroids formation, respectively. After 6 h, uniformly metformin distributed hybrid spheroids were obtained which was confirmed via coumarin‐6 loaded microsphere in hybrid spheroids. The average size of hybrid spheroids was 185 ± 5 μm which is slightly increased after loading of Met‐MS. In addition, metformin from MSCs hybrid spheroids also showed significant antioxidant activity and non‐toxic to MSCs. In conclusion, synthesized MSCs hybrid spheroids can synergistically enhance their activity as opposed to an individual. Therefore, it can be successfully applied to inflammatory models such as sepsis or colitis in the future.
Alginate encapsulation of 3D cultured mesenchymal stem cell spheroids for intraperitoneal injection in DSS‐induced murine chronic colitis
1Sungkyunkwan University, 2Keimyung University
Donor‐dependent skeletal muscle differentiation mechanism of tonsil‐ derived mesenchymal stem cells
1Department of Otorhinolaryngology‐Head and Neck Surgery, College of Medicine, Ewha Womans University, 1071 Anyangcheon‐ro, Yangcheon‐gu, Seoul 07985, Republic of Korea, 2Department of Biochemistry, College of Medicine, Ewha Womans University, Seoul 07804, Republic of Korea, 3Graduate Program in System Health Science and Engineering, Ewha Womans University, Seoul 07804, Republic of Korea
It is becoming important to understand the heterogeneity between the many donors that can potentially be used for clinical applications of MSCs. Stem cells' differentiation potency are important considerations for clinical applications. TMSCs has been previously demonstrated to be able to differentiate into various cells including endocrine organs, schwann cells and skeletal muscle cells. In this study, the differentiation variation from two different donors (#001 and #002) into skeletal muscle were analyzed.
TMSCs obtained from two donors were differentiated into myocyte (TMSC‐myocyte). The expression levels of muscle markers such as PAX7, Myogenin and Dystrophin were compared in cells derived from two donors, respectively. Morphological changed at each passage of differentiation were also analyzed. To compare the mechanisms of autophagy acting during muscle differentiation, the expression of LC3II, P62, Atg7, and Atg12 were evaluated.
The morphological differences were distinguishable between TMSC‐myocyte #001 and #002. Also, #002 did not increase Myogenin and Dystrophin in TMSC‐myocytes compared to undifferentiated. Immunofluorescence analysis was used to confirm higher expression of LC3II in #001, where autophagy was active. Conversely, #002 confirmed once again that LC3II did not differentiate into skeletal muscle cells as evidenced by reduced expression.
In addition, we reconfirmed whether autophagy was activated in each donor cell by treatment with 5‐ azacytidine and bafilomycin A1. Finally, our study suggests that the action of autophagy different between TMSC‐myocyte#001 and #002 and this difference influences skeletal muscle differentiation.
Generation of functional airway epithelial cells from human tonsil‐ derived mesenchymal stem cells by mimicking stepwise differentiation
1Ewha Womans University
The respiratory tracts are the air passages from the nasal passages to the trachea, and defects occur as a result of tumor resection, trauma, and infection. The key to treating respiratory defects is the subsequent functional restoration of the respiratory epithelium. Autologous epithelial cells or progenitor cells are not suitable for treatment, because they are difficult to expand and differentiate. Here, we developed a stepwise differentiation protocol to induce human tonsil‐derived mesenchymal stem cells (TMSCs) into epithelial cells as an alternative for regeneration of the respiratory epithelium. TMSCs were differentiated into epithelial cells through several steps along with embryonic development. The processes were divided into four steps, and each step was applied to TMSCs with a combination of various chemicals to generate airway epithelial cells. We found that activin A, which activates the Nodal/ TGF‐β signaling pathway, induces TMSCs towards the definitive endoderm (DE) at low concentrations. DE‐induced TMSCs were differentiated into lung progenitor cells using a precise combination of growth factors regulating BMP, TGF‐β, and WNT signaling, as evidenced by increased gene expression of NKX2‐1, an early lung progenitor marker. Furthermore, TMSCs differentiated into lung progenitor cells generated airway epithelial cells, including basal and ciliated cells, after air‐liquid interface culture. Our results demonstrate that TMSCs‐derived epithelial cells can provide an alternative for the reconstruction of respiratory defects.
Optimization of in vitro culture conditions to maintain hepatic stellate cell in quiescence
1University of Tokyo, Department of Bioengineering, 2University of Tokyo, Institute for Quantitative Biosciences
Hepatic stellate cell (HSC) plays a critical role during liver fibrosis by transforming from a quiescent to an activated state and secreting excessive extracellular matrix (ECM) under chronic inflammation. However, the detailed mechanism is still unknown. To solve this mystery, in vitro liver models with HSC has been developed. But it remains challenging because HSC gets spontaneously activated in vitro. Therefore, in this work, we investigated the stiffness and different ECM to optimize in vitro culture conditions that can maintain HSC in quiescence.
First, HSC were differentiated from human iPSC and cultured for 5 days. Since Type‐I collagen is the ECM secreted by active HSC, and Matrigel has previously been reported to inhibit HSC activation, they were used as different ECM to culture HSC respectively. Each ECM was used as a thin coat, as a gel layer and as an embedment to culture the iPSC‐derived HSC. TGFβ, which is known to activate HSC, was supplemented to make positive controls. Immunostaining and RT‐qPCR were done for evaluation.
Results showed that softer environment is more advantageous to maintain HSC quiescence. ECM type also has some effect but not as much as the stiffness. In the embedment conditions, there is no significant difference in the expression of activation marker between Type‐I collagen and Matrigel. Meanwhile, it is observed that TGFβ can significantly activate the iPSC‐derived HSC in every condition, indicating a potential that such HSC can be stimulated from healthy to diseased state when incorporated to develop in vitro liver models.
Salivary gland organoids as therapeutic models for radiation‐induced xerostomia
1Department of Microbiology, CHA University School of Medicine, Seongnam, Republic of Korea., 2ORGANOIDSCIENCES Co., Ltd., 406, 560, Dunchon‐daero, Jungwon‐gu, Seongnam‐si, Gyeonggi‐do, Republic of Korea, 3Department of Otorhinolaryngology‐Head and Neck Surgery, the Research Institute, Konkuk University School of Medicine, Seoul, Republic of Korea
Xerostomia is a symptom in which a patient's mouth is dry all the time due to a lack of saliva, lowering their quality of life. Pilocarpine is a parasympathomimetic agent that has resulted in significant improvement in symptoms of dry mouth. However, it has only a minor effect in patients who have lost all their salivary gland function. Adult stem cells have already been shown to have regenerative effects in organs such as the intestine and the heart, but their effect on the salivary glands is unknown. Here, we prove the potential effect of tissue regeneration capacity of the salivary gland (organoids SGOs) in the mouse model of radiation‐induced xerostomia. The SGOs were able to self‐renew and differentiate into major salivary lineages. Immunofluorescence staining analysis showing the expression of acinar cell marker AQP5, duct cell marker CK19. Furthermore, stimulation with the carbachol on SGOs increased the intracellular calcium gradient which is known to promote salivation. To assess SGOs function in vivo, we transplanted the GFP‐expressing SGOs into the salivary gland of a mouse model of radiation‐induced xerostomia. Engrafted GFP‐expressing SGOs were observed in mouse salivary glands after 12weeks. SGOs have restored saliva production and significantly enhanced the regenerative potential of irradiated salivary glands. Together, this paper illustrates the potential of SGOs to regenerate the salivary gland and will pave the way for the regenerative therapy of salivary gland dysfunction.
Synthesis of polystyrene nanoplastics degraded forms and their effect on stem cell
1School of Chemical Engineering, Sungkyunkwan University, Suwon 16419, Republic of Korea
There are many studies about the effects of micro‐ and nanoplastics on cells, microbes, and the environment. However, only a few studies have focused on their effects—especially, those of their reduced cohesiveness—on cell viability and physiology. We synthesized surfactant‐free amine‐ functionalized polystyrene (PS) nanoparticles (NPs) and PS‐NPs with decreased crosslinking density (DPS‐NPs) with general properties, such as size, shape, and zeta potential and examined their effects on cell viability and physiology. PS‐ and DPS‐NPs upregulated GPX3 expression and downregulated HSP70 (ROS‐related gene) and XBP1 (endoplasmic reticulum stress‐related gene) expression in human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) and it supports their reactive oxygen species (ROS) scavenging activity. Additionally, they enhanced mitochondrial fusion in hBM‐MSCs by upregulating MFN2 (mitochondrial fusion related gene) expression and downregulating FIS1 (mitochondrial fission related gene) expression. Cell‐cycle analysis revealed that PS‐ and DPS‐NPs increased the proportion of cells in the S phase, indicating that they promoted cell proliferation and, specifically, the adipogenic differentiation of hBM‐MSCs. However, the cytotoxicity of DPS‐NPs against hBM‐MSCs was higher than that of PS‐NPs after long‐term treatment under adipogenic conditions.
Development of a stem cell spheroid‐laden patch with high retention at skin wound site
1School of Chemical Engineering, Sungkyunkwan University
Mesenchymal stem cells such as human adipose tissue‐derived stem cells (hADSCs) have been used as a representative therapeutic agent for tissue regeneration because of their high proliferation and paracrine factor‐secreting abilities. However, certain points regarding conventional ADSC delivery systems, such as low cell density, secreted cytokine levels, and cell viability, still need to be addressed for treating severe wounds. In this study, we developed a three‐dimensional (3D) cavity‐structured stem cell‐laden system for overdense delivery of cells into severe wound sites. Our system includes a hydrophobic surface and cavities that can enhance the efficiency of cell delivery to the wound site. In particular, the cavities in the system facilitate hADSC spheroid formation, increasing therapeutic growth factor expression compared with 2D cultured cells. Our hADSC spheroid‐loaded patch exhibited remarkably improved cell localization at the wound site and dramatic therapeutic efficacy compared to the conventional cell injection method. Taken together, the hADSC spheroid delivery system focused on cell delivery, and stem cell homing effect at the wound site showed a significantly enhanced wound healing effect. By overcoming the limitations of conventional cell delivery methods, our overdense cell delivery system can contribute to biomedical and clinical applications.
Study for region specific differentiation programme through distinct transcriptome of mouse small intestinal epithelial stem cells
1Department of Microbiology, CHA University School of Medicine, Seongnam 13488, 2Department of Microbiology, CHA University School of Medicine, Seongnam 13488, Korea
Crypt‐villus axis of small intestine consist of various epithelial cells such as absorptive enterocytes, Paneth, goblet, enteroendocrine or M cells. And small intestine is classified according to region specific functional specialization such as different nutrients absorptive and digestive functions. Lgr5+ stem cells are obtained from same individual's small intestine, but gene expression patterns between duodenum, ileum are different. But, there is a lack of understanding of the clear differentiation program. Here we performed microarray analysis to select region specific expressed gene in Duodenum, Ileum and each crypt of tissues. By using the Lgr5+ stem cell derived organoid technique, we established each region specific small intestinal organoids and characterized small intestine and each tissue location‐derived organoid. Furthermore, we proceeded lentiviral transduction to understand region specific gene expression. Each selected transcription factor transduced organoid samples were differentiated through repeated passage processes, and when confirmed through RT‐PCR and immunofluorescence staining, it was indicated the oppositive region‐specific gene expression signals increased. These data show that location specific gene expression differences are caused by selected transcription factor in differentiation program.
Regulation of head and neck squamous cell carcinoma migration and invasion behaviors by mild reduction of cell surface
1Soonchunhyang Institute of Medi‐Bio Science (SIMS), Soonchunhyang University, 2Department of Otorhinolaryngology‐Head and Neck Surgery, College of Medicine, Soonchunhyang University, Cheonan
Head and neck squamous cell carcinoma (HNSCC) is one of the common malignant cancers and it has shown to exhibit a highly metastatic characteristics and a high mortality rate. Here, we aim to elucidate the role of cell surface free thiol groups in regulation of head and neck squamous cell carcinoma behaviors through changes in the formation of focal adhesion (FA) complexes and their subsequent contribution to the cancer cell migration and invasion. Our initial finding revealed that mild reduction of cell surface proteins using a tris(2‐carboxyethyl) phosphine hydrochloride (TCEP) could generate free thiol groups on head and neck cancer cells and lead to the increased FA expression of the cells. Furthermore, cell surface thiol groups newly generated by TCEP treatment could change the cell morphology from cuboidal or epithelial to elongated mesenchymal‐like cell shapes, which could be achieved by the upregulation of integrin‐mediated phospho‐FAK signaling pathway, and lead to the activation of mesenchymal to epithelial transition (MET), which consequently could suppress MET markers in both mRNA and protein levels as well as cell migration. These results were further corroborated by evaluating the mechanotransduction behaviors of head and neck cancer cells cultured on physiological condition (2.55 kPa) and tumor microenvironment (49.4 kPa) through traction force microscopy (TFM) and intracellular force microscopy (IFM). The results described in this study provide a proof‐of‐principle that cell surface free thiol groups can be a novel therapeutic target to inhibit cancer metastasis or reduce the aggressive properties of HNSCC.
Evaluation of the efficacy of SDF‐1‐based novel polypeptides by structure‐based drug design in an acute myocardial infarction model
1Department of Biomedical Sciences, College of Medicine, Korea University, Seoul 02841, Korea, 2R&D center, Scholar Foxtrot, Seoul 02796, Korea, 3Korea university, 4Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea
Stromal cell‐derived factor‐1 alpha (SDF‐1α, CXCL12) stimulates the migration of circulating cells to desired sites for tissue development, homeostasis, and regeneration, and may play a role in the promotion of cardiac regeneration by recruiting stem cells. However, the use of SDF‐1α in the injured heart necessitates not only higher binding affinity to its receptor, CXCR4+, but also better robustness against enzymatic degradation than other SDF‐1 isoforms. In this study, we describe a screening platform of SDF‐1α analog peptides designed using structure‐based drug design (SBDD), a type of computer‐aided drug design (CADD). Using in vitro and in vivo methods, we have evaluated the effect of peptides on the migration of human mesenchymal stem cells (hMSCs) and cardiac regeneration in acute myocardial infarction (AMI)‐induced animals. We demonstrate that one type of SDF‐1α analog peptide, SDP‐4, among the 4 analog peptides preselected by SBDD, is more potent than native SDF‐1α for cardiac regeneration following AMI. An interesting observation is that the migration effects of SDP‐4 and native SDF‐1α determined by a wound healing assay, Transwell assay, and 2D migration assay are comparable. Consequently, the results suggest that not only in vitro, but also in vivo testing of the analog peptides designed by SBDD is necessary for developing regenerative medicine. In this paper, we present an example of a screening platform that can be used to evaluate novel therapeutic compounds for the treatment of heart regeneration.
PINK1 deficiency induces adipogenic differentiation and suppresses osteogenic differentiation in mouse mesenchymal stem cells
1Cha University
Numerous genes are involved in the regulation of osteoblast and adipocyte differentiation; however, little is known about factors associated with adipose tissue formation and osteoporotic bone loss. Here, I investigated the role of PINK1 in adipogenesis and osteogenesis using PINK1 knockout (PINK1−/−) mice. Results demonstrated that both bone loss and adipose tissue formation were increased in old mice. Using ALP, ARS, Oil Red O staining, and RT‐PCR analysis, I confirmed that PINK1 deficiency suppressed osteogenic differentiation and promoted adipogenic differentiation in mouse embryonic calvaria‐derived wild type (PINK+/+) and PINK1−/− cells. I also verified that pre‐adipocyte 3T3‐L1 cells transfected with PINK1 siRNA (si‐PINK1) had increased adipogenic differentiation as demonstrated by Oil Red O staining and RT‐PCR. RNA sequencing analysis showed that the inflammatory response pathway was upregulated in si‐PINK1‐transfected cells. Results indicate that PINK1 deficiency induces adipogenic differentiation, suggesting that the levels of PINK1 expression could regulate osteogenic and adipocyte differentiation, and, therefore, determine stem cell fate.
Cartilage repair in temporomandibular joint osteoarthritis mediated by inflammatory cytokines‐stimulated human umbilical cord stem cells via immunomodulating activation of M2 macrophages
1Biomedical Engineering Research Center, Asan Institute for Life Sciences, 2Asan Medical Institute of Convergence Science and Technology, 3Department of Oral and Maxillofacial Surgery, Asan Medical Center
Osteoarthritis (OA) is a degenerative condition of the temporomandibular joint (TMJ) characterized by chronic inflammation and damage to joint structures. Despite the diverse etiology and pathogenesis of OA, increasing evidence suggests that macrophages can play a significant role in modulating joint inflammation, and thus OA severity, via various secreted mediators. This study aims to evaluate the inflammatory cytokines stimulated human umbilical cord stem cells for repairing OA‐induced cartilage lesions. Our in vitro data show that stimulated MSC induces M2 polarization of macrophages, and activates macrophages to express growth factors, which greatly improves the microenvironment around chondrocytes to produce type II collagen and glycoaminoglycan. The in vitro effects of stimulated MSC are further confirmed in vivo. In rat model of TMJ OA, stimulate MSCs ameliorate inflammation, increase the ratio of M2 macrophages and elevate the levels of growth factors in chondrocytes, and inhibits MMP13 production. Our findings show that inflammatory cytokines‐stimulated MSCs immunomodulate M2 activation of macrophages to skew the local OA microenvironment towards a pro‐ chondrogenic atmosphere, and promotes cartilage repair under inflammatory condition. It shows inflammatory cytokines stimulated human umbilical cord MSCs may be an effective treatment option for the TMJ‐OA.
Engineering hair follicle organoids through microenvironmental reprograming
1Kanagawa Institute and Industrial Science and Technology, 2Yokohama National University
In tissue morphogenesis, different types of cells self‐organize in a pre‐programmed manner using messenger systems such as epithelial‐mesenchymal interactions. Hair follicle morphogenesis is also triggered by reciprocal interactions between epithelial and mesenchymal cells. Here, we revealed that microenvironmental reprogramming by regulating these interactions facilitated hair follicle neogenesis in vitro. Supplements of low concentration extracellular matrices (ECMs) modulated spatial distributions of epithelial and mesenchymal cells from dumbbell‐shape to core‐shell configuration through spontaneous organization. The newly formed hair follicles with typical morphological features emerged in the core‐ shell‐shape aggregates (termed hair follicle organoids) and hair shafts sprouted with near 100% efficiencies in vitro and measuring up to 5 mm in length after 30 days of culture. The global gene expression analysis showed the activation of ECMs remodeling in the core‐shell‐shape aggregates, suggesting that ECMs remodeling to form microenvironments during spontaneous tissue formation may be critical for hair follicle regeneration in vitro. We further examined the feasibility of hair follicle organoids for drug screening and regenerative medicine. Incorporating melanocyte‐stimulating hormones and hair growth promoting drugs into the culture medium significantly improved the hair pigmentation and growth of the hair‐like fibers, respectively. Furthermore, transplantation of hair follicle organoids enabled the efficient hair follicle regeneration with repeating hair cycles. The present study of hair follicle organoids may be useful for screening hair drug candidates, regenerating hairs, and understanding hair follicle development.
Zika virus infection accelerates Alzheimer's disease phenotypes in brain organoids
1Seoul National University
Alzheimer's disease (AD) is one of the progressive neurodegenerative diseases characterized by β‐ amyloid (Aβ) production and phosphorylated‐Tau (p‐Tau) protein in the cerebral cortex. The precise mechanisms of the cause, responsible for disease pathology and progression, are not well understood because there are multiple risk factors associated with the disease. Viral infection is one of the risk factors for AD, and we demonstrated that zika virus (ZIKV) infection in brain organoids could trigger AD pathological features, including Aβ and p‐Tau expression. AD‐related phenotypes in brain organoids were upregulated via endoplasmic reticulum (ER) stress and unfolded protein response (UPR) after ZIKV infection in brain organoids. Under persistent ER stress, activated‐double stranded RNA‐dependent protein kinase‐like ER‐resident (PERK) triggered the phosphorylation of Eukaryotic initiation factor 2 (eIF2α) and then BACE, and GSK3α/β related to AD. Furthermore, we demonstrated that pharmacological inhibitors of PERK attenuated Aβ and p‐Tau in brain organoids after ZIKV infection.
Development of in vitro 3D unidirectional cerebral region circuit analytic platform by controlling the growth rate of neurites
1KIST, 2Yonsei University
In vitro 3D neural circuit models can be actively used to identify disease pathogenesis and therapeutic drug screening. Especially, the hydrogel‐based 3D neural circuit can align the hydrogel related to cell behavior and cell signaling and can simulate the 3D structure and environment of the body. Therefore, it is necessary to develop and functionally analyze an in vitro unidirectional neural circuit similar to the three‐dimensional neural circuit connection in the body. However, due to the randomly directional growth of neurites in the hydrogel, it is difficult to form a unidirectional connection in the body. In addition, 3D neural circuit fluorescence imaging using immunostaining is difficult to perform functional analysis due to the limitation of microscopic resolution. So, we developed the in vitro 3D unidirectional cerebral region circuits by physical dissection of the cerebral organoids and alignment of collagen. First, the physical stimulus is applied to the neurites firmly bound in the organoid, the bonds of the inner neurites are broken, and the regrowth of the neurites to the outside is promoted. Secondly, the three‐dimensional environment of the neural circuit was formed based on the collagen scaffold, and the alignment of collagen fibers through shear stress in the pathway included the growth direction of the neurites. Finally, in vitro 3D unidirectional cerebral region circuits were measured functional activity using the 3D neural probe. Therefore, in vitro 3D unidirectional cerebral region circuit analytic platform can be used to analyze the mechanism of pathogenesis and develop treatment methods for mental disorders.
Physiomimetic bioprinting of stem cell‐derived human pancreatic islet‐ like cellular aggregates‐vascular platform for studies of diabetic diseases
1POSTECH
Pancreatic islets are dense packages of hormonal cells surrounded by vascular networks and extracellular matrix (ECM). Recent innovations in 3D bioprinting technology enabled the fabrication of pancreatic tissue constructs mimicking the spheroidal morphology of the human islets. Currently, the application of stem cell‐derived pancreatic islets in in vitro models is limited by the immaturity of insulin‐producing beta cells. To promote the functional maturation of beta cells, we fabricated a bioprinted human pancreatic islet‐like cellular aggregates (HICAs)‐vascular platform that can simultaneously provide not only vascular niches but tissue‐specific extracellular cues. As the main material to build the platform, we developed pancreatic tissue‐derived ECM‐based peri‐islet niche‐like (PINE) bioink by supplementing additional basement membrane proteins to recreate optimal microenvironments for stem cell‐derived islets in vitro. Stem cell‐derived islets encapsulated in the PINE bioink exhibited improved functionality over 7 days compared to islets encapsulated in the other types of bioinks, suggesting that beta cell‐ bespoke bioink could enhance insulin secretion capacity. Using PINE bioink, the supporting bath was stacked in a planar shape, HICAs were printed as a point‐shape with high cell density and a linear blood vessel was printed close to HICAs. Physiomimetic patterning of vascularized human pancreatic tissue enabled self‐assembly of islets and vasculatures, recapitulating the microarchitecture of native pancreatic tissue. Furthermore, we verified whether the bioprinted platform would reproduce in vivo‐like responses of diabetic tissue. This strategy may facilitate the application of the stem cell‐based pancreatic tissue model for the studies of diabetic diseases, including various types of diabetic complications.
Aging of the blood‐brain barrier (BBB) via reactive oxygen species (ROS) stimulation
1KIST
Reactive Oxygen Species (ROS) is a chemical of oxygen produced in living things, and unlike general oxygen, it has strong power that attacks tissue and damages cells. Healthy cells can remove free radicals, but aging cells lower the ability to remove oxygen. Accumulation of ROS in cells damages cell structures through oxidative damage and induces intracellular inflammatory responses. In addition, ROS is also a significant cause of cellular senescence. Therefore, we created an aged blood‐brain barrier by applying ROS stimulation to a platform that mimics the three‐dimensional blood‐brain barrier. According to the concentration of ROS, the morphological and functional changes of blood vessels were confirmed under a confocal microscope, and senescence‐associated beta‐galactosidase expression was also increased. Our model can be used as an advanced environment for researching three‐dimensional disease models, such as cancer, Alzheimer's disease, and hypertension, representing aging‐related diseases. It is also expected to help build an aging environment closer to the reality of the existing aging disease model.
Microrheological system for hepatic function enhancement of human liver organoids
1University of Ulsan , 2Asan Medical Center
A microphysiological system is an advanced tool for drug toxicity testing by generating hepatic organoids with maturated liver function. Researchers have attempted to develop reliable hepatic models to mimic the liver microenvironment and analyze liver functions using hepatocytes cultured in the developed systems. However, mimicking of the liver microenvironment in vitro remains a great challenge owing to the lack of perfusable vascular networks in the model systems and the limitation in maintaining hepatocyte function over time. In this study, we established a microphysiological system using a passive micropump that supplies nutrients and oxygen with fresh medium to liver organoids as well as removes waste products secreted from the cells, imitating the function of blood vessels. Our results emphasize the critical role of the biochemical microenvironment in regulating hepatic differentiation and enhancing overall liver function.
3D microfluidic meningeal lymphatic vascular system to study age‐ related pathological effects of cerebrospinal fluid
1UNIST
Cerebrospinal fluid (CSF), a watery fluid that fills the meninges surrounding the brain parenchyma and spinal cord, provides mechanical and immunological protection to the brain inside the skull. CSF normally flushes through the meningeal lymphatic vessels (mLVs) as a waste removal mechanism in our brain. Impaired drainage function of mLVs leads to accumulation of toxic metabolites in the brain and increases the risk of developing age‐related neurodegenerative disease such as Alzheimer's disease (AD). Considering that the composition of CSF is relevant to the brain aging and AD, we questioned if composition CSF could directly affect the functionality of human mLV system. To demonstrate the impact of CSF on the mLVs, we built a human mLV microphysiological system (MPS) where lymphatic endothelial cells are cultured on the engineered basement membrane under the slow and pulsatile physiological flow. Confocal microscopic analysis and permeability assay of a mLV MPS revealed the expression of mLV markers and lymphatic barrier formation. Finally, CSF obtained from young, old, and AD mice was treated to the lumen of human mLV, and the transcriptomic and functional changes of mLV were monitored in the MPS device. Notably, it displayed a strong influence of CSF composition in the cellular behavior of mLV, highlighting the mLV as a therapeutic target to treat age‐related mLV regression.
A microphysiological system reproducing obesity‐associated adipose tissue inflammation
1UNIST
Obesity causes complex alterations in adipose tissue, which include abnormal genetic regulations, polarization of macrophages, excessive accumulation of extracellular matrix (ECM) and dysfunction of the secretion system. These changes in adipose tissue cause metabolic disorders, leading to or exacerbating various diseases such as diabetes, cardiovascular disease and cancer. Although 2D adipocyte cell cultures have contributed in the understanding the physiology of adipose tissue in obesity, they do not recapitulate the complex and dynamic multicellular microenvironment in adipose tissue. Here we introduce an adipose tissue microphysiological system (MPS)― a 3D adipose tissue construct composed of primary adipocytes embedded within the decellularized extracellular (dECM)‐based hydrogel interfaced with microvessels in the microfluidic device. In order to mimic the obese adipose tissue, dECM scaffold was generated from the adipose tissue of obese mice showing distinctive features of ECM compositions in obesity. Primary adipocytes maintained their functions over a week without dedifferentiation and the adipose tissue‐derived endothelial cells co‐cultured in the device showed higher expression of genes encoding pro‐inflammatory cytokine (TNF‐α) and adhesion molecules (ICAM, VCAM and SELE), mimicking the increased migration of breast cancer cells and the recruitment of immune cells in obese adipose tissue. Our MPS, therefore, provides a new approach for studying molecular alterations by providing integrated information of cell‐cell and cell‐ECM interactions in obese adipose tissue.
Analysis of metastatic organotropism in breast cancer cells using a microphysiological systems
1UNIST
Distant recurrence, also called metastatic breast cancer, is much more serious than local recurrence showing a poor prognosis and low 5‐year survival rate. Recently, it has been suggested that metastatic tumor cells possess intrinsic cellular properties creating non‐random distribution of sites for distant relapse, known as metastatic organotropism. However, due to the significant diversity among patients and within each patient's cancer cells, it is difficult to predict the metastatic organotropism of breast cancer cells. Here, we describe a new organ‐on‐a‐chip approach to estimating the organ‐specific metastatic properties of breast cancer cells. To recapitulate the breast cancer metastases to the brain, lung, and liver, we used two‐channel microfluidic devices containing an organ‐specific vascular layer and decellularized extracellular matrix (ECM) hydrogel, given that exit of cancer cells from the circulation and successful infiltration into organ ECM are critical for metastatic organotropism. First, a great contribution of organ‐ specific ECM environment in the selection of organs was demonstrated by observing the distinct invasion patterns of multiple breast cancer cell lines into organ‐specific decellularized ECM (dECM) hydrogel, which was matched with their computationally analyzed organotropism shown in a previous report. We further assessed the invasion of breast cancer cells on the microfluidic device containing dECM hydrogel with a controlled fluid flow to mimic the dynamic adhesion of circulating cancer cells, demonstrating the potential of our platform for the prediction of the metastatic organotropism.
Generation of 3D innervated skeletal muscle mode
1Yonsei Univ.
Neuromuscular junction (NMJ) is a synaptic connection between a motor neuron axon and a skeletal muscle fiber, which translate the biochemical cues and control physical activity. Several models have been reported that co‐cultured skeletal muscle and nerve cell to form NMJ, but conventional 2D co‐ culture system does not reflect the architecture of native tissue and does not represent the actual function and physiological complexity of NMJ. Here, we developed a 3D neuromuscular junction model composed of induced skeletal muscle generated by direct reprogramming and primary motor neurons. 3D skeletal muscle constructs integrated with motor neurons showed enhanced myotube maturation and contractile activity. We confirmed that myotube and motor neurons were co‐localized and the neurite outgrowth from the motor neurons wrapped along the myotube. In addition, 3D innervated skeletal muscle responded to glutamate, which activates the motor neuron. Thus, we confirmed that a functional NMJ was formed between the muscle fiber and motor neuron. The results suggest that this 3D engineered NMJ model could be used to treat extensive volumetry muscle loss or to study NMJ‐associated diseases.
Acknowledgement: This work was supported by Samsung Research Funding & Incubation Center of Samsung Electronics under Project Number SRFC‐TC2003‐03.
Cortical‐blood vessel assembloids exhibit Alzheimer's disease pathologies by activating glia after SARS‐CoV‐2 infection
1Seoul National University, 2Konkuk University
A correlation between COVID‐19 and Alzheimer's disease (AD) has been proposed recently. Although the number of case reports on neuroinflammation in COVID‐19 patients has increased, studies of SARS‐ CoV‐2 neurotrophic pathology using brain organoids have restricted recapitulation of those phenotypes due to insufficiency of immune cells and absence of vasculature. To overcome these limitations, we developed fused cortical‐blood vessel organoids to provide blood vessels to brain organoids and obtained the characteristics of increased expression of glial cells and blood‐brain barrier‐like structures in brain organoids. Furthermore, we observed AD pathologies, including Aβ plaques, which were affected by the inflammatory response from SARS‐CoV‐2 infection. These findings provide an advanced platform to investigate human neurotrophic diseases, including COVID‐19, and suggest that neuroinflammation caused by viral infection facilitates AD pathology.
Wnt‐activating human skin organoid model of atopic dermatitis Induced by Staphylococcus aureus and its protective effects by Cutibacterium acne
1Seoul National University
A recently developed human pluripotent stem cell (hPSC)‐derived skin organoid model has opened up new avenues for investigating skin development, disease, and regeneration. However, the developed skin organoids are unwanted off‐target formation of hyaline cartilage in the tail region, therefore, the current skin organoid culture method limit their applications. To overcome those, we first showed that the activation of Wnt signaling increased size without off‐target formation of cartilage in the long‐term culture of skin organoids. In this study, we developed an organoid model for atopic dermatitis (AD) induced by Staphlyococcus aureus (S. aureus). S. aureus infection of air‐liquid interface (ALI)‐skin organoids led to a disrupted skin barrier and increased production of epidermal and dermal origin inflammatory cytokines, similar to human AD patient skin. To find protective skin bacteria in S. aureus induced AD organoid model, we found that pre‐treatment with Cutibacterium acnes (C. acnes), a commensal skin bacterium in healthy adults, had a protective effects on S. aureus induced ALI‐skin organoids. Thus, Wnt‐activating human skin organoid may provide a valuable tool to model human skin diseases and test the efficacy of novel therapeutics.
Endometrium organoid as an in vitro model for female reproductive diseases
1Yonsei University College of Medicine, 2Department of Physiology, Yonsei University College of Medicine
Infertility and associated female reproductive diseases, such as endometriosis and Asherman's syndrome (AS) are closely related to the function of the endometrium. However, due to the limitation of the current in vitro models, understanding of the implantation process and initial development of embryos in the endometrium is inadequate. Here, we investigated endometrial organoids (EO) culture to construct an in vitro model for embryonic implantation and development study. We optimized protocols for culturing adult stem cell‐derived EOs. EOs harvested from uterine horns proliferated and grew stably in 3D extracellular matrix‐based hydrogel. These EOs displayed endometrium‐specific markers and showed physiological response to sex hormones as in vivo. Together, our results show that EOs significantly mimic the endometrium, and EOs can be used as an in vitro model for studying implantation and development of embryos. Further, by creating an environment that can cultivate EOs close to in vivo endometrial cells, EOs may be applied as cell therapy for endometrium‐related diseases, including AS. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF‐2021R1C1C2009131).
Development of functional hepatic organoids with liver‐specific microenvironments
1Yonsei University
Liver organoid models derived stem cells have attracted attention for their potential applications in drug screening, disease modeling, and tissue regeneration. However, conventional liver organoid models developed so far lack cellular and extracellular components essential for constituting the human liver microenvironments. Therefore, for more precise disease modeling and drug testing, liver microenvironment‐integrated organoid model should be developed in vitro. Herein, we established primary stem cell‐ or pluripotent stem cell‐derived liver organoid models through co‐culture of endothelial cells, hepatic stellate cells, and Kupffer cells in liver extracellular environments to increase complexity and functionality. Organoids incorporating liver microenvironmental cues showed increased hepatic functions, such as urea synthesis and albumin secretion, compared to conventional organoids. Expression of hepatic differentiation markers was also highly upregulated in microenvironment‐ integrated liver organoids. Our study demonstrates that organoids with liver‐specific cellular and extracellular microenvironments enable efficient in vitro 3D modeling of various liver diseases and screening of candidate drugs.
Acknowledgment: This study was supported by a grant (2018M3A9H1021382) from the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT), Republic of Korea.
Human stomach microphysiological system for modelling Helicobacter Pylori pathogenesis
1Ulsan National Institute of Science and Technology, 2Seoul National University College of Medicine
Helicobacter pylori (H. pylori) infection is a major cause of gastric adenocarcinoma and chronic gastritis. The prevalence of H. pylori remains nearly 50% of the global populations. However, the pathogenic mechanism of H. pylori‐associated gastric diseases is still unclear because of lack of reliable in vitro human gastric models that closely recapitulate the gastric mucosa in antral units where H. pylori can colonize. So far, although human gastric organoids have been reported a promising culture technology, the functional maturation is still insuffient. In addition, these drawbacks lead to inability to fully recapitulate interaction between host defense system and pathogens. Here, we established a physiologically relevant human gastric microphysiological system (hsMPS) for studying H. pylori pathogenesis. To mimic the multiple cell types of antral units, we established the human antral organoids (hAOs) and gastric mesenchymal stromal cells (gMSCs) from human gastric tissue and reconstituted the antrum in a microfluidic device. By differentiation of hAOs interfaced with gMSCs under flow condition, the matured gastric epithelium, including maintenance of self‐renewal properties, mesh‐like mucus layer, secretion of mucin‐associated peptides (Trefoil factor 1, TFF1), was successfully generated. Furthermore, the level of proinflammatory cytokines induced by the H. pylori infection was observed in the H. pylori‐ infected hsMPS. Moreover, recruitment of immune cells was validated by co‐culturing with PBMC in the infected hsMPS. Finally, hsMPS was suggested as a promising platform to assist the understanding of the pathogenic mechanism of H. pylori and development of strategies for the treatment H. pylori infection.
Differentiation of vascularized functional liver organoids using transcription factors in iPSCs
1Department of Biomedical Science, Kangwon National University, Chuncheon, Gangwon‐do, 24341, Republic of Korea.
The generation of vascularized liver organoids derived from human induced pluripotent stem cells (iPSCs) promotes an understanding of disease modeling and provides a drug screening platform. Highly developed microvascular structures in the liver are important for maintaining normal liver function (detoxification, hormone metabolism, carbohydrate, and protein metabolism). Therefore, a highly developed vascular structure is essential to forming organoids functionally similar to the human liver. However, the development of advanced microvascular systems for liver organoids has technical limitations. In the previous study, vascularized organoids were produced by culturing HUVEC with hepatocytes or controlling the microenvironment using extracellular matrix (ECM). In this study, in the process of differentiating iPSC into liver organoids, the vascular formation was induced initially to form mature liver organoids. We delivered a transcription factor that induces vascular endothelial cell differentiation into iPSCs to form an enhanced vascular structure. In our proposed method, effective vasculature was observed in the low concentration signaling protein environment. It was confirmed that the increased vascular structure improved the function of liver organoids. This study was supported by the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (NRF‐ 2020R1A4A1016093) and (NRF‐2021R1A2C1010865).
Engineered heterochronic parabiosis in 3D microphysiological system
1Emory University
Skeletal muscle possesses a remarkable ability to regenerate following injury due to the presence of resident muscle satellite cells (MuSCs) but this capacity declines with aging. Previous studies using heterochronic parabiosis, in which young and aged animals are surgically attached to share blood circulation, revealed that systemic factors in the blood of young animals can rejuvenate the regenerative capacity of aged muscle. However, due to the complexity of in vivo parabiosis, underlying mechanisms and the identity of putative rejuvenation factors remains elusive. To address this challenge, we engineered a 3D muscle microphysiological system that recapitulates key characteristics of the native MuSC microenvironment. This system enabled mechanistic studies of cellular dynamics and molecular interactions in response to conditional extrinsic stimuli of local and systemic factors. Then, we integrated young and old muscle stem cell niche with blood circulation to mimic heterochronic parabiosis by co‐ culturing cells/sera from young and aged animals. Hence, we observed the restored myogenic function of the aged MuSCs and identified vascular endothelial growth factor (VEGF) as one of the potential factors driving muscle rejuvenation. Furthermore, in subsequent studies, we are integrating bio‐orthogonal proteome labeling in conjunction with tamoxifen‐inducible, cell specific Cre‐LoxP system to spatiotemporal identify novel youthful factors responsible for parabiosis effects. All in all, our in vitro parabiosis system shows great potential to serve as a pre‐clinical testing tool that can facilitate our understanding of the dynamic regulation of circulating humoral factors and potential drug discovery. More importantly, our approach can be translated into human parabiosis model.
3D tumor angiogenesis models for effective anti‐cancer treatment
1KIST
The development of an experimental model system that implements the angiogenic tumor microenvironment is a prerequisite for the study of the efficacy and mechanism of action (MOA) of antineoplastic agents. In 2D culture, there were some restrictions to verify the effectiveness of anticancer drugs with various biological analyses. Therefore, we created an organoid‐on‐a‐chip using polydimethylsiloxane (PDMS) and co‐cultured with multiple human cells to develop a cancer microenvironment‐like system to induce angiogenesis. Angiogenesis is known to be closely related to the growth of solid tumors. We will verify the efficacy of nanovesicular anticancer drugs on a biochip with angiogenesis to reduce the survival of solid tumors. In addition, for the development of an improved tumor vascular model system, specific drug selectivity using a patient‐derived primary cancer cell will provide an alternative that can overcome the disadvantages of chemotherapy of cancer.
3D bioprinting‐based tissue assembly to generate multi‐axially contracting engineered heart tissue
1POSTECH
Various types (e.g., strip, ring, and chamber‐like) of tissue have been developed for the in vitro study of the human heart. The strip and ring types of EHT could reproduce contractility and electrophysiology of the heart, however, these models have limited pump‐like cardiac functions due to the lack of structural complexity. The advancement of biofabrication enabled the generation of chamber‐like models that reproduce volume‐pressure dynamics. However, the level is still poor and needs improvement. In this study, we suggest a 3D bioprinting‐based tissue assembly as a strategy to achieve myocardial fiber orientation, a critical architectural feature of a cardiac chamber for maximizing the chamber contraction. Tissue assembly is a method that creates larger or more complex constructs based on functional tissue units. The strip EHT was generated and functional validations regarding contractility and electrophysiology and drug responsiveness. Then the pin‐hole‐based assembly platform was developed to perform tissue assembly, and the assembly process was established. Based on the assembly platform, the EHTs were assembled, and assembled EHT exhibited synchronized contractile and electrophysiological functions. Simultaneously, the 3D bioprinting‐based tissue assembly proved to be capable of controlling fiber orientation. Subsequently, strip and ring types of EHT, showing longitudinal and radial contraction, respectively, were generated. Then, two types of EHTs were assembled to generate multi‐axially contracting EHT. In conclusion, we proposed a 3D bioprinting‐based tissue assembly as a method of fabricating complex contractile tissue, which will further be advanced to build a cardiac chamber having myocardial fiber orientation.
Integrating the endosteal and perivascular compartments of the bone marrow niche in a microfluidic device
1Institute for Tissue Engineering and Regenerative Medicine, The Chinese University of Hong Kong, 2Biomedical Manufacturing Commonwealth Scientific and Industrial Research Organisation (CSIRO), Melbourne, Australia; Australian Regenerative Medicine Institute, Monash University, Melbourne, Australia
The bone marrow (BM) niche is comprised of an endosteal and a perivascular compartment and is crucial for proper hematopoietic stem cell (HSC) maintenance and function throughout life. It is also involved in the progress of various hematological diseases. Current approaches of HSC culture and study ex vivo are met with limited success, as HSC behavior strongly depends on their microenvironment. Therefore, engineering the BM niche in vitro can provide the means to study physiological and pathophysiological processes within the BM as well as be utilized as a drug screening device.
Using a microfluidics system, we engineered a BM niche that comprised both the endosteal and perivascular compartments. For this we established a methodology based on the polyelectrolyte‐driven assembly of extracellular matrix (ECM) to produce a human ECM‐rich bone‐like tissue surface with mature osteoblasts and resembling the endosteal compartment. We also utilized human endothelial cells, which formed a microvascular network (MVN) that closely emulated physiologically relevant MVNs in vivo and could be stabilized for up to 10 days by macromolecular crowding (MMC).
Both compartments were successfully integrated in a microfluidic device enabling the close recapitulation of the BM niche. Incorporated HSC demonstrated good cell survival and proliferation, while retaining the expression of HSC markers, repopulating the bone marrow niche. Future studies will focus on further characterizing the function of the cultured HSC, their interaction with various components of the BM niche, as well as the modelling of physiological processes such as HSC homing and mobilization, and pathophysiologies on‐a‐chip.
Bone marrow on‐a‐chip for in vitro bone disease modeling with Osteo‐ Vascular biphasic niche
1Korea National University of Transportation
Since bone marrow models are challenging to create in vitro, especially for disease models using on‐a‐ chip systems, the majority of contemporary bone disease research is done in vivo models. Many animal models, on the other hand, have difficulties fully replicating human pathological and physiological conditions. Therefore, in order to avoid using animal models and generate functional responses at the individual Human organ or tissue level, bone marrow on a‐chip technology is required. The construction of bone marrow on‐a‐chip in vitro using a biphasic osteo‐vascular biomimetic scaffold underflow is described. The biphasic scaffold consists of a relatively rigid backbone structure and cell‐laden soft tissue matrix. This complex enables the rapid formation of bone environment and mimics native bone more accurately. Controlling various factors such as base materials, cell culture environment, and medium flow condition. We attempted to validate vascular and bone formation. The creation of the bone imitating niche is verified using confocal microscopy and Micro‐CT analysis as well as histology for further disease model application. Following that, we will examine and optimize bone marrow formation via flowing marrow cells. Developing bone marrow on‐a‐chip offers a new approach to building the bone disease model and platform for drug responses in vitro. This research may also help enhance the process of developing new drugs for personal use.
Spatial restriction of diffuse gastric cancer cells promotes cell softening and filopodia formation
1Department of Bio and Brain Engineering, KAIST, 2Department of Surgery, Severance Hospital, Yonsei University College of Medicine
Cancer cells are known to invade through the dense extracellular matrix during metastasis. In order to understand cell behavior under such mechanical stress, many studies have utilized micro‐channels to simulate cell migration through confined spaces. However, microchannel systems can only observe bidirectional cell movement, limiting the observation of innate cell behavior under confinement in vivo. In this study, spatial restriction is applied to gastric cancer (GC) cells using a sandwich hydrogel platform of a collagen hydrogel gel bed with collagen and agarose overlays, which offers spatial restriction with or without additional adhesion to the upper interface, respectively. With initial restriction in all directions, GC cells displayed limited movement, and over time, they proliferated in clusters in their original seeded locations unlike their unrestricted counterparts. Interestingly, intestinal and diffuse gastric cancer cell types showed different activation of the mechano‐sensor Yes‐associated‐protein (YAP) in response to both types of spatial restriction. Diffuse GC cells showed increased YAP phosphorylation under mechanical restriction with limited motility compared to the 2D control group, suggesting a cell softening response to adapt to mechanical stress. Furthermore, the collagen overlay with added adhesion and restriction increased the filopodia of GC cells, indicating a switch to a more mesenchymal phenotype beneficial for metastasis. Overall, this study may offer insight on the poor survival for diffuse GC patients and establish a novel, viable method for observing cellular response to mechanical stress.
Development of drug screening platform to mimic pancreas tumor microenvironment using decellularized extracellular matrix and pancreas organoids
1Asan Medical Center
Pancreatic cancer organoids are three‐dimensional cell aggregates which their cell composition and structure are similar to original pancreatic tumor tissues. Generally, organoids have been cultured in Matrigel, a material obtained from mouse sarcoma cells. Matrigel has a cell adhesion structure to cancer cells, and easily forms hydrogel at the body temperature to serve suitable extracellular matrix (ECM) for organoid formation. However, it does not exactly reflect the tumor microenvironment. In this study, we developed a new ECM‐based matrix that may substitute Matrigel in pancreas organoids culture. In the proteomics analysis of decellularized human pancreas tissue, we found that periostin is a main ECM component in tumor tissues compared to normal pancreas. Periostin is an ECM secreted from cancer‐ associated fibroblasts in tumor tissues and is an extracellular matrix protein involved in drug resistance, immune response, and metastasis of cancer tissues. Through analysis of Human Protein Atlas database, patients with high periostin had a poorer overall survival in the whole population. To prepare periostin ECM platform, periostin were extracted from periostin overexpressing cells. Compared to the existing Matrigel‐based evaluation platform, periostin‐based culture plated form showed drug resistance and increased EMT markers in vitro, which can mimic tumor tissues in vivo.
Modeling pancreatic cancer with patient‐derived organoids integrating cancer‐associated fibroblasts
1Department of Microbiology, CHA University School of Medicine, Seongnam 13488, Korea, 2Department of Pathology, Ajou University School of Medicine, Suwon 16499, Korea, 3Department of Otolaryngologyu2014Head and Neck Surgery, Seoul St. Maryu2019s Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea, 4R&D Institute, ORGANOIDSCIENCES Ltd., Seongnam 13488, Korea, 5Department of Surgery, CHA Bundang Medical Center, CHA University, Seongnam 13496, Korea, 6R&D Institute, ORGANOIDSCIENCES LTD, Seongnam 13488, Korea, 7Pancreatobiliary Cancer Clinic, Department of Surgery, Gangnam Severance Hospital, Yonsei
University College of Medicine, Seoul 06273, Korea
Pancreatic cancer is a devastating disease and is highly resistant to anti‐cancer drugs because of its complex microenvironment. Cancer‐associated fibroblasts (CAFs) are an important source of extracellular matrix (ECM) components, which alter the physical and chemical properties of pancreatic tissue, thus impairing effective intratumoral drug delivery and resulting in resistance to conventional chemotherapy. The objective of this study was to develop a new cancer organoid model including a fibrous tumor microenvironment (TME) using CAFs. The CAF‐integrated pancreatic cancer organoid (CIPCO) model developed in this study histologically mimicked human pancreatic cancer and included ECM production by CAFs. The cancer cell‐CAF interaction in the CIPCO promoted epithelial‐ mesenchymal transition of cancer cells, which was reversed by CAF inhibition using all‐trans retinoic acid. Deposition of newly synthesized collagen I in the CIPCO disturbed the delivery of gemcitabine to cancer cells, and treatment with collagenase increased the cytotoxic effect of gemcitabine. This model may lead to the development of next‐generation cancer organoid models recapitulating the fibrous TME.
Differentiation of human hair follicle stem cells into a vascularized hair bearing skin organoids
1ORG Corp., 2Korea Institute of Toxicology
Reconstructed human skin equivalents have been widely used as alternatives to animals for skin irritation and toxicity tests, however the absences of appendages (hair follicles and glands), blood vessels and immune cells limits their applications for drug screening and long‐term follow‐up studies. Recently a differentiation protocol of human pluripotent stem cell toward hair‐bearing skin organoid was reported, but it is still difficult to generate a vascularized hair‐bearing skin organoid with immune cells. In this study we aimed to generate a patient‐specific complex skin organoid through hair follicle stem cell (HFSC) differentiation.
HFSCs were isolated from patient's skin biopsy and cultured in DMEM containing B27, ascorbic acid and 3D culture medium (ORG Co.). Expanded HFSCs transformed to skin organoids, detached from the culture dish by itself, and maintained over 1 year in vitro. Hair shafts observed inside of the organoids were grew outside and the number of hairs were increased over time. Stratified epidermis and emitted oil drops from the organoid were evident at 4‐month. At 6‐month, skin organoids over 1milimeter were cleared with an organoid one‐step clearing solution (ORG Co.) and subjected for immunofluorescence staining. HFSC‐derived skin organoids (HSO) were self‐organized and composed with (epidermis keratin 10+/keratin14+) and dermis (extracellular matrix with vimentin+ fibroblasts) including vessels (a network of PECAM1+ cells), nervous system (a network of TUBB3+ cells) and immune cells (CD34+ monocytes).
This data suggests HFSCs and HSO as a novel therapeutic cell source for skin regeneration and a drug discovery platform, respectively.
Enhancing maturation of human vascularized cardiac organoids using a magnetic torque stimulation (MTS) system
1Korea Univ
Cardiac organoid is an ideal tool to recapitulate the cardio‐genesis in vitro which is affected by the chemical as well as physical factors. Treatment of chemical factors such as cytokines and growth factors generated the complex structure of cardiac tissues. Recently, physical forces such as compression, stretching, and shear stress play an important role for developing and maturating functional cardiac tissues and cardiac organoids.
Here, we established magnetic torque stimulation (MTS) system which is consists of an external rotating magnetic field and engineered magnetic particles with cardiac organoids that could induce the spin of the magnetic particles. In this process, cardiac organoids were direct force transmission physically stimulated by the generated torsional force.
Cardiac organoids matured by the MTS system are characterized by well‐organized vascularization and compartmentalization of the atrial and ventricle compared to the control group. Immunofluorescence staining analysis demonstrated that MTS system increasing vasculo‐genesis by CD31 and more compartmentalization of the ventricles and atria by MLC‐2a/v.
These results have significant implications that torque force elicits the compartmentalization of ventricles and atrial as well as vasculo‐genesis, which is a key step in cardiac development and maturation.
Effect of direct oxygenation and coculture on primary hepatocytes & intestine epithelial cells cultured in stirrer‐based microphysiological system (MPS) device
1Department of Chemical System Engineering, Graduate School of Engineering, University of Tokyo, Tokyo, Japan, 2School of Life Science and Technology, Tokyo Institute of Technology, Kanagawa, Japan, 3PhoenixBio Co. Ltd., Higashi‐Hiroshima, Hiroshima, Japan, 4Sumitomo Bakelite Co. Ltd., Tokyo, Japan, 5Faculty of Pharmacy Institute of Medical, Pharmaceutical and Health Science, Kanazawa University, Kanazawa, Japan, 6Department of Mechanical Engineering, Tokai University, Kanagawa, Japan
Conventional preclinical models used for prediction of new drug candidates have limited predictability. For liver in vitro models using primary human hepatocytes, such low predictability could be caused by decrease of cell function as consequence of separation of hepatocytes from their native environment. Furthermore, adverse effect may rise not only from the liver but also other organ such as intestines, especially for orally administered drugs. However, multi‐organ in vitro models are still limited due to their complexity, low throughput, etc.
To address such problems, increasing number of microphysiological systems (MPS) are being developed. MPS can be defined as cell culture system that goes beyond the conventional 2D and include several aspects such as 3D structure, exposed to media flow, or having multi‐cellular/organ environments. In this study, we cocultured human iPSc‐derived intestinal epithelial cells and primary human hepatocytes harvested from chimeric mouse (PXB cells) in a novel MPS with on‐chip pumping mechanism using a micro‐stirrer and direct oxygenation using oxygen permeable membrane. We evaluated the functions and interactions of both cells. Direct oxygenation improved hepatocytes function as indicated by albumin secretion and gene expression of drug metabolizing enzymes. On the other hand, coculture greatly modulate gene expression of hepatocytes, indicating potential effect of crosstalk between liver and small intestine. This liver‐intestine coculture system using MPS shows potential for new drug screening tools that include organ interactions effect.
3D multicellular cancer microenvironment platform supporting survival of acute myeloid leukemia (AML) in cultures
1Tissue Engineering Laboratory, Department of Mechanical Engineering, The University of Hong Kong; Advanced Biomedical Instrumentation Centre, Hong Kong Science Park, 2L8‐16, Lab block, Department of Medicine, LKS Faculty of Medicine, The University of Hong Kong, 3Tissue Engineering Laboratory, Department of Mechanical Engineering, The University of Hong Kong
Acute myeloid leukemia (AML) is the most lethal hematopoietic cancer and one of the most common acute leukemia. The current standard treatment for AML has been used for 40 years, but only 50% overall survival is achieved. The poor therapeutic outcome could be explained by the highly heterogeneous nature of AML and the difficulty in drug selection especially for frail patients. This leads to an emerging shift towards personalized treatment. However, an obstacle for AML drug screening is their spontaneous apoptosis in ex vivo cultures. We hypothesized the bone marrow microenvironment mimicked by osteolineage cells encapsulated in extracellular matrix (ECM) would support the survival of primary AML cells, and the presence of 3D cancer microenvironment would provide more accurate screening results. Studies have shown that stromal cells and ECM provides a pro‐tumoral environment and chemoresistance to AML cells. Therefore, we aimed to develop a high‐throughput drug screening platform which consists of osteogenic differentiated (OD) mesenchymal stem cells that are encapsulated in collagen microspheres being co‐cultured with primary AML cells. Our laboratory has been developing a 3D microencapsulation platform using naturally occurring ECM to fabricate physiologically relevant and ECM‐based 3D microtissues. Our results showed that the viability of AML cells was around 20‐40% when being cultured alone but significantly higher (80%) when being co‐cultured with OD microspheres, which could be explained by the increase in GRO‐α, OPG and IGFBP2 levels detected in the collected conditioned medium. Further experiments are underway to demonstrate the application of the platform for drug screening purposes.
Generation of functional porcine and human thyroid organoid
1Department of Microbiology, CHA University School of Medicine, Seongnam 13488, Korea
Thyroid hormones play an essential role in the growth of various tissues such as the brain, skeletal muscle, and bones. Thyroid hormones also play an important role in maintaining various homeostasis in the body. Cells that play a major role in the secretion of thyroid hormones in the thyroid gland are thyroid follicular cells, which make up the colloid and absorb the iodine to release thyroid hormones. Studies have been conducted using thyroid follicular cells, which play a major role in the thyroid gland, to screen certain drugs on the thyroid gland when such thyroid gland is damaged, but thyroid follicular cells are known to grow and regenerate slowly. Here, we describe the establishment of a 3D organoid culture system for porcine and human thyroid gland that retain morphological features and gene expression of original the tissue.
Developing a novel strategy to support in vitro self‐renewal of patient‐ derived head and neck squamous carcinoma cells
1Soonchunhyang Institute of Medi‐bio Science (SIMS), Soonchunhyang University, Republic of Korea,
2Department of Otolaryngology, Soonchunhyang University, Republic of Korea
Although head and neck squamous cancers (HNSCs), including tongue, vocal cord, and buccal cancers, are one of the most common cancers worldwide with more than 40% mortality, there have been no solid standard biomarkers for effectively diagnosing HNSCs at the early stages and no effective protocols available to support in vitro expansion of HNSC cells. Here, to overcome these limitations, we aim to elucidate the role of tumor microenvironments such as neighboring cells, soluble cues, and cell surface properties in regulation the self‐renewal of patient‐derived head and neck squamous cancer cells. Our initial findings revealed that tongue cancer epithelial cells isolated from the patient's tumor could be expandable for multiple times without losing their phenotypes in the presence of both with mouse embryonic fibroblasts (MEFs) as feeder cells and Rho kinase inhibitor (Y‐27632), evident by robust expression of both cancer proliferation markers (Ki67) and HNSC stemness marker (CD44) in mRNA and protein levels. Furthermore, mild reduction of cell surface proteins using a tris(2‐carboxyethyl) phosphine hydrochloride (TCEP) as a reducing agent could generate free thiol groups on cells, resulting in the decreased migratory and invasive capacity of patient‐derived tongue cancer cells. On the other hand, when TCEP‐treated cells were co‐cultured with a cancer‐associated fibroblast‐conditioned medium their inhibited migratory and invasive capacity were recovered. Taken together, these results highlight the synergetic contributions of MEFs and Rho kinase inhibitor to the self‐renewal of patient‐derived HNSC cells.
Evaluation of the efficacy of perfusion culture for the construction of three‐dimensional endometrial‐like tissue
1Waseda University, 2Tokyo Women's Medical University
The purpose of this study is to elucidate the causes of implantation failure by observing and evaluating the process of fertilized egg implantation by constructing endometrium‐like tissue in vitro.
In this experiment, cell sheet engineering and a perfusion bioreactor were applied in the culture process. A single sheet has only a two‐dimensional extent of several tens of μm in thickness, but by stacking sheets, a three‐dimensional tissue can be constructed. Since the diameter of a fertilized rat egg is approximately 70 μm, we aimed to maintain a thickness of 100 μm or more in this experiment to observe the implantation process after infiltration.
The uterine horns are removed from 3‐week‐old female rats, and endometrial cells are isolated by trypsin treatment. The isolated cells were separated into epithelial cells and stromal cells, and each cell was seeded onto UpCellTM. The endometrial‐like tissue was placed on a collagen gel with microfluidic channels and cultured by perfusion for 3 to 6 days, the period of implantation.
The three‐dimensional endometrium‐like tissue of epithelium and stroma was cultured at 0.3 mL/min on collagen gel with microfluidic channels and maintained a thickness of more than 100 μm for 3‐6 days. Histological evaluation of the cells showed that the two‐layered epithelial‐stromal structure was maintained, and that they had endometrial gland structure bordered by epithelial cells. Furthermore, in an experiment in which a fertilized egg was implanted in this three‐dimensional endometrium‐like tissue, adhesion to the endometrium‐like tissue was confirmed after 7 days of culture.
In vitro modeling of atherosclerosis in human blood vessel organoids
1Seoul National University
As modeling of atherosclerosis needs recapitulation of complex interactions with vasculature and immune cells, previous in vitro models have limitation that lack of atherosclerotic phenotypes due to their insufficient vascular structure or cell types. To overcome this, we developed atherosclerotic blood vessel organoids (BVOs) through mimicking the environment by co‐culture with monocytes in spinning culture condition. Treatment of enzyme‐modified low‐density‐lipoprotein (eLDL) with TNFα in culture media induced atherosclerosis in BVOs. Since BVO contains multiple cell types including endothelial cells, vascular smooth muscle cells and monocytes/macrophages self‐assembled in blood vessel structure, we could observe representative atherosclerotic phenotypes including endothelial dysfunction, inflammatory responses, monocyte accumulation, foam cell formation and fibrous plaque formation in BVOs. Furthermore, we evaluated the inhibitory effects of HMG‐CoA reductase inhibitor lovastatin in atherosclerosis BVOs. As the results, lovastatin‐treated groups exhibited repressed atherosclerotic phenotypes compared to vehicle‐treated atherosclerotic BVOs. These results suggest that atherosclerotic BVO is an advanced in vitro model which is suitable for large scale drug discovery and further elucidation of mechanisms.
Nucleic acids-functionalized nanomaterials for healthcare
1Korea Institute of Science and Technology
Early diagnosis is able to provide properly timed treatment resulting in successful therapeutic effect. Since various diseases are complex system, detection of multiple biomarkers which are correlated with disease development mechanism should be required to increase the accuracy. In this regard, sensor system has been developed to improve the sensitivity and specificity of the detection method. However, many previous approaches were designed without considering the surrounding disease environment, which results in fail to apply in clinical system. Manipulating the biomimetic disease model platform with novel biomolecule monitoring system can provide key knowledge for diagnosis and treatment. Here, I will introduce new diagnosis tools using biomolecules-functionalized metal nanoparticles on biomimetic platforms. Additionally, I will explain new therapeutic gene editing system based on bioinspired nanoparticle carrier. Finally, I will discuss how the nano materials can lead us to new breakthroughs in biomedical technologies. These approaches will be able to realize the smart health care system for overall patient health to help cure and treat.
Nanomaterial‐reinforced alginate bioinks for potential bone tissue engineering
1Gwangju Institute of Science and Technology
Alginate has been widely used as bioink materials due to their biocompatibility and ease in crosslinking. However, their mechanical properties and lack of biological activity remained drawbacks in support the growth and differentiation of the printed cells. To produce functional alginate‐based bioinks, we have elaborated to examine the effects of molecular weights of alginate, alginate derivatization, and formulation with various nanomaterials on cell printing. In particular, nanomaterials (e.g., graphene oxide, PLGA nanoparticles, or polydopamine nanoparticles) were found to significantly improve the printability and osteoinductivity of mesenchymal stem cells. Overall, we envision that the nanomaterial‐ reinforced alginate bioinks enable the cell printing for tissue engineering applications.
U.S. Patents - How many examples you need to disclose in your life science patent applications
1K&L Gates, LLP
The patent system is to protect inventions in return for the disclosure of inventions. While all countries require disclosure of inventions, the disclosure requirements differ from country to country. The United States requires written description and enablement, which is one of the most stringent among other countries. In comparison, Korea does not require something similar to the written description or enablement of the U.S. patent law. For life science inventions, generally more examples are needed for broad protection because of the unpredictable nature of the technology. However, sometimes life science inventors have only one or a few examples, and may not have resources to pursue additiona l examples. Due to the differences of disclosure requirement among countries, the same disclosure with one or a few examples in a life science patent application may be sufficient in one country and may be inadequate in another country even for the same claims. This presentation will discuss the disclosure requirements for patent protection and consider issues and unfortunate scenarios when life science patent applications with only one or a few examples are pursued in multiple jurisdictions.
The Role of Chemokine Receptor CXCR6 Expression on Differentiated Cells from Human Adipose Tissue‐Derived Mesenchymal Stem Cells
1Dongguk University
hADMSCs secrete growth factors, cytokines, and chemokines, and also express various receptors that are important in cell differentiation or migration to injured tissue. The expression level of chemokine receptor CXCR6 was significantly increased in adipogenic differentiated cells, but not osteogenic differentiated cells. Increased expression of CXCR6 on Ad was mediated by both receptor recycling, which was in turn regulated by secretion of CXCL16, and by newly synthesized protein. The level of soluble CXCL16 was highly increased in both Ad and, in particular Os, which inversely correlates with the expression on transmembrane‐bound form of CXCL16. Furthermore, interaction with macrophages was examined to find the role of CXCR6 expressed on adipocytes. Adipocytes interact with various immune cells, especially adipose tissue macrophages (ATMs), which exist as a form of M2 macrophages in healthy adipose tissue and are polarized into M1 macrophages when adipocytes are stimulated by oxidative stress. Here, human monocytes that can be polarized into M1 or M2 macrophages by LPS or IL‐4 and IL‐13, were used to evaluate the interaction with differentiated adipocytes. As results, the gene expression levels of the M1 polarization‐inducing marker were significantly decreased, while those of the M2 polarization‐ inducing marker were significantly increased, in differentiated adipocytes when THP‐1 cells were co‐ cultured and polarized into M1 or M2 macrophages with additional CXCL16 treatment. This suggests that adipocytes that highly express CXCR6 interact with CXCL16 and then modulate M1 or M2 polarization by expressing pro‐inflammatory or anti‐inflammatory factors via inflammatory pathways in a co‐culture with THP‐1 cells.