Abstract
2016 AM Abstracts
Understanding Multi-allelic Heterozygous Variant Contributions to Dilated Cardiomyopathy
Dilated cardiomyopathy (DCM) is a multivariate disease with poorly understood mechanisms, but recently 30+ different mutations have been suggested to contribute to disease pathology. We have identified the first incidence where a family with high DCM prevalance is caused by the co-segregation of two heterozygous mutations in dissimilar cytoskeletal proteins, i.e. α-tropomyosin (TPM1; +/c.G97A) and vinculin (VCL; +/c.659dupA). To better understand the disease mechanism, we generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from the family cohort carrying the variants and modeled these variants in human embryonic stem cell-derived CMs (hESC-CMs) via RNAi and CRISPR to discern the functional consequences that could induce DCM. Affected CMs contracted with decreased energy compared to non-carrier control CMs. Decreased sodium and potassium channel expression accompanied slowed action potential kinetics in affected vs. non-carrier-derived CMs, which together suggests that VCL and/or TPM1 mutations may cause unique downstream transcriptome regulation that leads to the dysfunction we observed in vitro. To assess the combinatorial regulation by mutations in these dissimilar genes, hESC-CMs were engineered to mirror altered protein expression; these cells exhibited prolonged calcium transients and contractions with decreased energy and irregular timing, suggesting that reduced VCL creates CMs with dysfunctional mechanical properties, resulting in part from prolonged Ca2+ handling, which would likely adversely affect TPM1 mutant phenotypes leading to DCM when the variants co-segregated. Given the lack of disease in single variant carriers, these data provide a unique set of analyses that result in identification of how dissimilar but co-segregating variants can result in disease.
Novel Mechanisms of Non-Coding Genomic Regulation Identified in Cardiac Disease-in-a-dish Models
University of California, San Diego, La Jolla, CA.
Research and Development, BREONICS Inc., Watervliet, NY.
Adult stem cell research group, BioMediTech, University of Tampere, Tampere, FINLAND.
Cell adhesion and cytoskeletal tension are known to regulate the differentiation of mesenchymal stem cells. Cell adhesion is mediated primarily through integrins and focal adhesion proteins, such as focal adhesion kinase (FAK). FAK is interconnected with signaling pathways including extracellular signal-regulated kinase (ERK) and cytoskeleton-related Rho-ROCK (Rho-associated protein kinase) pathway. However, the role and interplay of these mechanisms in the regulation of human adipose stem cell (hASC) differentiation is still unclear. The aim of the present study was to assess the significance of FAK, ERK and ROCK signalling in the stem cell fate decision of hASCs. Specific molecule inhibitors PF-562271, PD98059 and Y-27632 were used to attenuate the signalling mechanisms in FAK, ERK and ROCK pathways, respectively. Differentiation capacity of the hASCs was evaluated under basic, osteogenic and adipogenic media using RT-PCR, and analyses of alkaline phosphatase activity, mineralization and lipid accumulation. Our results indicated that the inhibition of FAK, ERK and ROCK function suppressed both cell proliferation and osteogenic differentiation dose-dependently. ERK inhibition also reduced adipogenesis. FAK suppression decreased the cell density, while adipogenic differentiation of remaining cells was stimulated. ROCK inhibition resulted in increased adipogenesis as shown by less spread morphology and increased formation of lipid droplets. These results suggest that the cell adhesion and morphology modulated by FAK, ERK and ROCK proteins are relevant regulators of differentiation. The functionality of these molecules is essential for the full osteogenic potential of hASCs. The inhibition of the cell attachement mechanism and actin tension supported adipogenic fate of hASCs.
Induced Pluripotent Stem cells (iPSC) are adult skin fibroblasts (sFB) genetically reprogrammed to an embryonic stem cell-like state. Notwithstanding their autologous origin and their potential to differentiate towards cells of all three germ layers, iPSC reprogramming is still affected by low efficiency. We hypothesize that the variability in the sFB reprogramming is due to the sFB used, as sFB derived from different anatomic sites exhibit topographic differentiation and preserve positional memory. Human sFB harvested from five different anatomical sites (neck, breast, arm, abdomen, thigh) were cultured for one week and their morphology, proliferation, and expression of mesenchymal or epithelial markers were evaluated by immunocytochemistry. Additionally, gene expression profile analysis was performed by real-time PCR including genes typically expressed in mesenchymal cells, and involved in cell growth, proliferation, development, and morphogenesis. Intriguingly, while the morphology of sFB derived from different anatomical sites differed only slightly, proliferation rate and expression of distinctive markers varied greatly. Further, different sFB had different genetic program. Interestingly, sFB derived from neck and breast shared genetic signature of Mesenchymal Stem Cells, raising doubts about the existence of two distinct cell population. Since sFB topographic origin defines their genetic program it might remarkably affect the efficiency of reprogramming. Hence, according to our evidence, it is mandatory to carefully select sFB population when planning sFB reprogramming for regenerative medicine purposes.
Tracking and Predicting Cellular Reprogramming from a Biophysical Perspective
Standard reprogramming methods are noisy, laborious, and poorly understood. To address this key bottleneck, here we bring together two innovations in 1) watching and 2) physically-constraining the process of reprogramming. First, watching reprogramming in action elucidates processes occurring in the middle of reprogramming. Compared to the nuclei of starting fibroblasts, nuclei of the endpoint iPSCs are smaller, more circular, and contain different ‘open and closed’ chromatin. How these radical changes in nuclear properties occur is still a mystery. We have developed an innovative micropatterned substrate that enables the live, in situ imaging of nuclei within reprogramming populations. The substrates separate cultures into thousands of small adhesive ‘islands.’ Our image-based “fingerprints” of nuclei can identify cells that have been fully reprogrammed. They also provide a new method to identify so-called “roadblocks” that stall reprogramming progression and to determine in what stage of reprogramming cells reside. Second, by controlling the micropattern geometry of our substrate to physically-constrain the process of reprogramming, we activate mechanotransduction pathways (e.g., YAP/Taz), and directly impact chromatin mobility to promote reprogramming. This work provides new evidence that some aspects of the biophysical microenvironment, using biomaterials of defined properties, can be rationally controlled to promote reprogramming.
Clinical Translation of Dermal Regeneration Matrix for the Treatment of Diabetic Foot Ulcers
Biomaterials based scaffold research has focused on optimizing factors such as the macro- and micro-architecture, biochemistry, pore structure and resorption rate to develop matrices for specific regenerative medicine applications. One such significant area of clinical need is the treatment of chronic, hard to heal diabetic foot ulcers (DFUs) which are wounds that fail to progress through the normal phases of wound healing and typically stall at the inflammatory phase. These wounds are characterized as having high levels of bioburden, elevated levels of inflammatory cytokines and high levels of proteases. The Dermal Regeneration Matrix (DRM) consists of collagen and chondroitin sulfate with the appropriate porosity and mechanical characteristics to produce a matrix that mimics dermal tissue, reduces the levels of pro-inflammatory cytokines, improves the capacity of the matrix for infiltration by cells and for the formation of new vasculature [1]. This technology has been translated to the clinical setting for the treatment of DFUs with the completion of the FOUNDER study which was a multi-center, prospective, randomized, controlled study (307 Patients, 32 Sites) conducted under an Investigation Device Exemption [2]. This study demonstrated that treatment with DRM increased the incidence of wound closure by 59% (P = 0.001), accelerated healing rates by 50% (P = 0.012) and reduced the median time to complete wound healing by 5 weeks. In addition, this study demonstrated significantly improved components of health-related quality of life related to mobility and activities of daily living.
1. J Biomed Mater Res. 1980 Jan;14(1):65–81.
2. Wound Repair Regen. 2015 Nov-Dec;23(6):891–900.
Periodontal Tissue Reconstruction using Decellularized PDL Matrix and 3D-Printed Tooth
Tokyo Medical and Dental University, Tokyo, JAPAN.
One of problem for dental implant is the lack of periodontal ligament (PDL), which can support teeth, prevent infection, and get the feeling such as chewiness. Our objective is to develop the tissue engineered periodontal tissue unit consisting of the mandible bone, PDL and artificial tooth. We here prepared the mouse decellularized mandible bone (DMB) with PDL matrix and combined it with artificial tooth fabricated by 3D printer. The mandible bone with tooth was decellularized by high hydrostatic pressurization and DNase treatment, and the tooth was extracted. The DMB with PDL matrix was obtained. When the DMB with PDL matrix was implanted under the rat renal capsule, the infiltrated cells oriented along the PDL collagen fibers. The extracted molar was demineralized and remineralized, and this modified tooth was used as a model, which could fit the DMB with PDL matrix, and inserted to decellularized PDL matrix on DMB in vitro. After renal capsule implantation, recellularized PDL matrix was combined with modified tooth surface. As a next step, we prepared an artificial tooth by 3D printing. After implantation of the unit of DMB with 3D-printed tooth, PDL matrix was successfully recellularized and combined with 3D-printed tooth, indicating the application possibility to the novel periodontal treatment.
The tissue remodeling response following myocardial infarction (MI) is initially compensatory, but often continues unchecked, leading to sustained collagen production, scar expansion, and eventually diastolic dysfunction and heart failure. A lack of pharmacological and surgical interventions to modulate this response has left a gap in patient care post MI. To improve upon treatments, biomaterials can be used to support the heart wall and encourage healthy remodeling. Adult porcine cardiac extracellular matrix (cECM) has beneficial effects when injected post-MI,1 and fetal-derived cECM has shown regenerative potential in vitro.2 To provide both mechanical support and ECM-based signaling, we have developed tunable, anisotropic silk-based scaffolds containing fetal or adult cECM. Application of this material as a patch in a rodent model of MI demonstrated reduced scar expansion and improved cell infiltration after 11 weeks. To understand the mechanism by which these silk-cECM patches impact host response, we have developed and optimized LCMS-based proteomic techniques. We are able to monitor matrix remodeling, protein expression, and protein signaling in the infarct, border, and remote regions, with initial investigations comparing tissue composition post MI and 3 and 11-weeks post-repair. The use of cellularized and decellularized tissues enables evaluation of the cellular proteasome and matrisome. Results indicate that a silk-cECM patch decreases the progression to heart failure and that the efficacy is impacted by the composition of the incorporated cECM.
1. Wassenaar JW, et al., J Am Col of Cardio. 2016, 67.
2. Williams C, et al., Acta Biomater 2014, 10.
Lipid and Sulfated GAG Accumulation with ECM Source Animal Age Differentially Affects Macrophage Response
Extracellular matrix biomaterials offer regenerative capacity of wounds through immunomodulation and recruitment of regenerative cells. These scaffolds, however, are diverse and dependent on many factors, including donor animal age. Previous studies have shown that increased source animal age impairs macrophage polarization and results in increased fibrosis. This study hypothesized that changes in ECM biomaterial composition would directly affect macrophage polarization. Lipids, advanced glycation end-products (AGEs) and glycosaminoglycans (GAGs) were investigated due to previous studies showing their immunomodulatory properties and changes in content with age. Small intestine submucosa (SIS) from 12, 26 and 52 week old pigs was found to have increases in lipid and sulfated glycosaminoglycan content with age, while AGEs were not found to change via ELISA and immunostaining. Lipids and GAGs were isolated from SIS using chloroform:methanol and urea, respectively. These extracts were used to treat murine bone-marrow derived macrophages and compared to whole ECM digests and digest of extracted ECM. Results indicated that both lipid and GAG extracts had inhibitory effects on iNOS and Arginase surface marker labeling with increased age. Lipid extracts resulted in increases in nitric oxide production phagocytosis with increased age. GAG extracts resulted in decreases in NO and phagocytosis with increased age. SIS remaining after extraction, primarily collagen, promoted increases in NO and decreases in phagocytosis with increased age. Isolation of these different components of ECM can identify relative contributions of immunomodulatory factors that change with age. This can help develop improved processing of ECM from aged donors including cadaveric ECM sources.
Peripheral Nerve-Specific Extracellular Matrix Hydrogel Supports Repair after Peripheral Nerve Injury
University of Pittsburgh, Pittsburgh, PA.
Peripheral nerve injury commonly results in loss of neuromuscular function, often resulting in significant impact upon both quality of life and cost of care for patients. One promising target for improving patient outcomes is the use of a peripheral nerve specific extracellular matrix hydrogel (PNS-ECM). PNS-ECM provides a tissue-specific microenvironment which is conducive to nerve repair, including: nerve specific growth factors that are chemotactic signals for Schwann cells, promote neurite outgrown, as well as factors that modulate the macrophage inflammatory response to injury. PNS-ECM was created by decellularizing porcine sciatic nerve by a previously described method. The PNS-ECM was validated for removal of immunogenic cellular components, retention of beneficial nerve specific proteins, and tested for bioactivity in a series of in vitro and in vivo experiments. We found that PNS-ECM significantly enhanced Schwann cell migration and axon extension in vitro and in vivo using a critical length nerve defect rodent model. We also confirmed that exposure to PNS-ECM generates a unique macrophage phenotype similar to an M2, anti-inflammatory phenotype and a greater M2:M1 ratio than negative controls. Rodents treated with PNS-ECM were also seen to experience about 15% less muscle atrophy and similarly significant improvement in function locomotion return. As an easily injectable material in an off the shelf formulation which promotes recruitment of alternately activated, M2 macrophages, Schwann cell migration, and axon extension, we believe that PNS-ECM would significantly improve quality of life for affected patients and result in a significant reduction of cost associated with their chronic care.
Saitama Medical University, Saitama prefecture, JAPAN.
In recent years, engineered muscles that mimic functions of real muscle at much smaller scale (about um to mm) have been developed for a drug screening test platform. We have established 3D engineered muscles that have a fascicle-like shape, high volumetric density, and optimal alignment in 3D. However, the engineered muscle has less performance in force compared to a real muscle. Also, most of current platforms for engineered muscles focus on RNA/protein expression rather than mechanical properties, even though they significantly affect muscle development and force generation. Therefore, we developed a novel training protocol that can enhance muscle performance, and investigated changes in both active and passive mechanical properties, such as contractile force, sarcomere length, and compliance. We trained the muscle tissues by coordinated electrical and mechanical stimuli, called co-stimulation, only for 3 minutes to minimize RNA/protein level changes. To give suitable co-stimulation, frequency and phase shift between the two stimuli were optimized based on the experiments. As a result, co-stimulation synergistically enhanced the performance in force up to 20% in 3 minutes, when the two kinds of stimulation were out-of-phase. From the results of measuring a sarcomere length and compliance, this improvement in a short time is probably because the adequate co-stimulation made the myosin-actin overlap and compliance appropriate to produce higher contractile force. We also computationally tested remodeling of the extracellular matrix by the training through a simulation. Our results advance the potential of high-performance engineered muscles for a drug testing platform and an actuator of bio-robots.
Localized SDF-1α Delivery Promotes Endogenous Cell Recruitment to the Supraspinatus Muscle After Severe Rotator Cuff Injury
W.H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA.
Degeneration of the supraspinatus muscle after severe rotator cuff tear correlates with increased re-tear rates.1,2 Thus, methods to promote muscle healing may significantly improve clinical outcomes. The hypothesis of this study was that stromal cell-derived factor-1α (SDF-1α) delivered via heparin-based microparticles to the supraspinatus muscle after severe rotator cuff injury would significantly enhance the recruitment of pro-regenerative cell populations to the injured muscle.
Rotator cuff injury was induced in rats through transection of the rotator cuff tendons and the suprascapular nerve. Microparticles were fabricated via free radical polymerization with 10 wt% N-desulfated heparin methacrylamide and 90 wt% poly (ethylene glycol) diacrylate and subsequently loaded with 60 ng SDF-1α. Immediately following injury, the supraspinatus muscle was injected with no microparticles (injury-only), unloaded microparticles, or SDF-1α-loaded microparticles. 7 days later, flow cytometry was used to quantify cell types recruited to the muscle (N = 6).
Fatty infiltration was identified in the injured supraspinatus 42 days post-injury indicating that muscle degeneration was induced in our model. Furthermore, compared to unloaded microparticles and injury-only controls, significantly more total leukocytes (CD11b+, 5.75 ± 1.14 fold change over uninjured contralateral), total macrophages (CD11b +CD68+, 6.8 ± 0.23), anti-inflammatory macrophages (M2, CD11b+ CD68+CD163+, 5.79 ± 0.67), and MSCs (CD29+CD44+CD90+, 4.78 ± 0.84) were recruited to the supraspinatus muscles containing SDF-1α-loaded microparticles 7 days post-injury.
This study indicates that SDF-1α-loaded microparticles are able to recruit significantly more pro-regenerative cells, specifically M2 macrophages and MSCs, to the supraspinatus muscle after severe rotator cuff injury, which may lead to enhanced muscle regeneration and improved clinical outcomes.
1. Yamamoto. J. Shoulder & Elbow Surgery 2010.
2. Gladstone J. Shoulder & Elbow Surgery 2007.
Development of a bioengineered skeletal muscle tissue construct can be a promising solution to achieve the functional recovery of volumetric muscle injuries. However, the conventional fabrication methods in tissue engineering are limited to building volumetric tissue constructs with functional cellular organization. More importantly, bioengineered muscle tissues need to be integrated with the host nervous system following implantation, as failure of innervation results in muscle tissue atrophy. In this study, we utilized 3D bioprinting strategy to fabricate volumetric skeletal muscle constructs that mimic native skeletal muscle organization. To facilitate long-term tissue survival and accelerate neuromuscular junction (NMJ) formation, human neural stem cells (hNSCs) were combined with human muscle progenitor cells (hMPCs) in the 3D bioprinted muscle constructs. Introduction of hNSCs enhanced the cell viability and tissue maturation, which includes highly aligned myotube formation in vitro. The implanted bioprinted muscle constructs developed highly oriented myofibers with integration of host vascular and nerve tissues, and muscle mass and muscle function were increased in a rat tibialis anterior (TA) excisional model. Our results demonstrate that creation of innervated volumetric engineered muscle tissue constructs using the 3D bioprinting system is feasible, and that the functional muscle construct can contribute to restoration of muscle functions.
Addition of Adipose Precursor Cells to a Poloxamer-Filled PCL Nerve Conduit Results in Enhanced Functional Nerve Regeneration In A Rat Model of Peroneal Nerve Ablation
University of Virginia, Charlottesville, VA.
1. Nature Biotechnology 32, 773–785 (2014).
2. Skardal, A. Acta Biomaterialia. 2015. In Press.
Rapid Generation of 3D Microchannels using a Scanning Spot fs Laser System for Structured Tissue Vascularization
Biomedical Engineering, Florida Institute of Technology, Melbourne, FL.
One of the main limitations for three-dimensional engineered tissues is the lack of microvasculature that can provide nutrient delivery. Soft lithography and additive processing techniques have been used to provide template channels for vascularization. However, capillary-sized channels (<50 μm) requires a more time consuming 3D printing technique such as multiphoton excited photochemistry. In this study, we demonstrate the use of femtosecond laser-based 3D-subtractive printing for microchannel construction. The system used a galvanometer-based scanner capable of moving the optical system's focal position three-dimensionally at 2.5 mm/ms to generate channels in cell-containing polyethylene glycol diacrylate (PEGDA)/collagen composite hydrogels. This system was able to process a 1.8 ml volume with 1μm spatial feature placement without the need for sample translation. Hydrogels were produced with PEGDA/collagen ratios from 100–25% v/v to determine the limits of this approach and to create a material that can be processed by the laser, allows cell attachment, and is structurally suitable. Hydrogel blends were produced in DMEM with Irgacure 2959 and then crosslinked with a 100 W 365 nm UV lamp (6 min, 8.5 cm distance). After hydrogel characterization, NIH3T3 fibroblasts were incorporated (500,000 cells/mL) and their viability/morphology assessed after 1–3 days. Preliminary data demonstrated that increasing collagen percentage increased both yield strength (p < 0.004, 25 vs 100%) and swelling ratio (up to 50% collagen, p < 0.041; n = 4). All conditions tested with ≥25% PEGDA were sufficiently transparent to allow microchannel generation. Importantly, cells exhibited live and cytoskeletal-staining both without and with channel construction. Overall, we demonstrated that this technique can rapidly generate complex capillary-sized structures within hydrogels.
Canary Center at Stanford for Cancer Early Detection, Stanford University, Stanford, CA.
Cell behavior and function expressed in bi-dimensional (2D) monolayer culture can significantly differ from those in the three-dimensional (3D) native environment. These differences currently affect the relevance of 2D models in preclinical and clinical research and have fostered the development of 3D cell culture methods able to recapitulate the in vivo environment. Here, we report a method based on magnetic levitation for 3D cell assembly. In this method, we apply the principles of magnetic levitation to rapidly form viable and functional spheroids with controlled geometry and cellular organization. The reported approach allows real time imaging and does not require the use of engineered scaffolds or magnetic nanoparticles. Moreover, this method is broadly applicable to various cell types, including endothelial cells, stem cells and cancer cells. Immunofluorescence staining and gene expression profiling were performed and compared to 2D to evaluate differences in protein expression and extracellular matrix (ECM). Overall, the results indicate that magnetically assembled spheroids mimic the in vivo cell-cell and cell-ECM interactions and organization better than 2D cultures, and could improve biological and clinical relevance of in vitro models for cell culture research and drug screening.
Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA.
(1) Alexander P.G, et al. Exper Biol Med. 2014, 239: 1080–1095.
(2) Lin H. et al. Mol Pharm. 2014, 11(7): 2203–12.
(3) Lozito, T.P. et al. Stem Cell Res & Ther 2013, 4(Suppl 1):S6.
Engineering mature tissues requires a guided assembly of cells into organized three-dimensional (3D) structures with multiple cell types. Guidance is usually achieved by microtopographical scaffold cues or by cell-gel compaction. The assembly of individual units into functional 3D tissues is often time-consuming, relying on cell ingrowth and matrix remodeling, whereas disassembly requires an invasive method that includes either matrix dissolution or mechanical cutting. We invented hook-in tissue, a bio-scaffold with a microfabricated hook and loop system. The assembly of hook-in tissue preserved the guided cell alignment realized by the topographical features in the 2D scaffold mesh and allowed for the instant establishment of coculture conditions by spatially defined stacking of cardiac cell layers or through endothelial cell coating. The assembled cardiac 3D tissue constructs were immediately functional as measured by their ability to contract in response to electrical field stimulation. Facile, on-demand tissue disassembly was demonstrated while preserving the structure, physical integrity, and beating function of individual layers.
3D Dynamic Blood Brain Barrier Model
The utilization of human cells and tissues for in vitro physiological platforms has become a growing area of interest to bridge this gap and more accurately predict human responses for drugs under development. With growing interest in developing selective and potent inhibitors for the treatment of central nervous system (CNS) diseases, there is an urgent need to understand the challenging aspect of crossing the BBB and good physiological models of the BBB are germane to the success of those studies. In this work we have developed a dynamic microfluidic 3D human cell culture platform to more accurately investigate compound permeability from the bloodstream to the CNS using a second on-chip platform we have also developed. Our BBB model mimics the microenvironment of brain capillaries, with dynamic flow subjecting endothelial cells grown on a hollow fiber to uniform shear stress. The human endothelial cells and primary human astrocytes have been viably co-cultured in the device for up to 34 days under static conditions and under flow conditions for 2 weeks. Staining for tight junctions and testing permeability to dextrans of various sizes confirmed barrier function. The model was validated using various compounds of known permeability and barrier modulation was assessed both chemically (histamine) and mechanically (shear stress). Current work is focused on reproducing this model without a synthetic fiber using a direct ink write system to bioprint the BBB. To date, using custom made co-axial needles we have demonstrated perfusable vascular channels lined with human brain endothelial cells.
Engineered Tubular Mesenchymal Condensations with Controlled Dual Growth Factor Presentation for Endochondral Bone Formation
Biomedical Engineering, Case Western Reserve University, Cleveland, OH.
Biomimetic tissue engineering aims to recapitulate embryonic development through cellular self-organization and targeted morphogen presentation. To date, no cellular self-assembly system mimicking long bone diaphyseal architecture has been reported to examine the regenerative capacity of this geometry. Here, we describe scaffold-free tubular mesenchymal condensations featuring controlled growth factor release from incorporated microparticles, and assess in-vitro differentiation and bone formation at ectopic and orthotopic sites. Mesenchymal condensations were engineered by seeding hMSCs with microparticles delivering TGF-β1 (early) and BMP-2 (sustained) in custom agarose wells to form self-assembled rings followed by stacking to fuse into tubes. Subcutaneous implantation was performed in 9-wk-old NCr-nude mice. Treatment groups were: 1) TGF-β1-, 2) BMP-2-, or 3) BMP-2+TGF-β1-loaded. 8-mm femoral defects were created in 12-wk-old RNU rats, stabilized with axially-compliant fixators, and implanted with BMP-2+TGF-β1-loaded tubes or randomly-oriented condensate-sheets. Bone formation was evaluated by biochemical, μCT, and histological analyses. BMP-2+TGF-β1 presentation significantly enhanced mineralization in subcutaneous explants; μCT revealed significantly increased bone volume vs. TGF-β1- or BMP-2-groups at 6 wks (p < 0.05). Histology showed new bone following the tubular architecture and more prominent cartilage vs. TGF-β1- or BMP-2-loading, mimetic of endochondral ossification. Longitudinal μCT revealed progressively increasing bone volume in femoral defects with >60% bridged by 12wks with tubes; a trend toward enhanced regeneration in tubes vs. sheets was observed. Data suggests that: 1) geometry of scaffold-free mesenchymal condensations guided 3D ossification, 2) BMP-2+TGF-β1 presentation augmented ectopic bone formation vs. TGF-β1- or BMP-2-loading via endochondral ossification, 3) tubular architecture promoted femoral defect regeneration vs. random organization. Funding: NIH(1F32DE0 24712, R01AR063194); ICTSI(UL1TR001108).
Tissue regeneration in critical-size bone defects often requires supraphysiological dosages of recombinant bone morphogenetic protein-2 (BMP-2), potentially eliciting bone overgrowth and other undesirable consequences. Alternative to delivering BMP-2 protein, as well as to improve control of BMP-2 levels in target cells, we hypothesize that a light-activatable adeno-associated virus (AAV) gene delivery platform may provide a facile, robust approach for externally fine-tuning BMP-2 administration. We previously developed a light-activatable AAV platform leveraging red/far red light responsive proteins, Phytochrome B and Phytochrome Interacting Factor 6 [1]. We employed this engineered vector to deliver the BMP-2 transgene to cells in vitro. Briefly, a transgene cassette (BMP-2 fused to green fluorescent protein (GFP)) generated by standard molecular cloning was packaged into AAV serotype 2 wild-type and and light-responsive variants. qPCR was used to determine virus titers, while Western Blot confirmed virus capsid formation. After introducing BMP2-GFP into human embryonic kidney (HEK) 293T cells, flow cytometry revealed up to 90% transduction efficiencies. qPCR showed increased BMP-2 expression in transduced cells versus untransduced controls, while ELISA detected BMP-2 protein in the supernatants collected from transduced cells. Red/far red light intensities modulated GFP in transduced HEK 293T and human mesenchymal stem cells, and we are now characterizing ratiometric tunability of BMP-2. This platform is minimally invasive compared to UV light/chemical inducer-controlled systems and utilizes wavelengths within tissue penetration range, holding promise for in vivo applications.
1. Gomez EJ et al. ACS Nano 10(1), 225–237, 2016.
3D Tissue Engineered Blood Vessels To Model Progeria Using iPS-derived Cell Sources
Biomedical Engineering, Duke University, Durham, NC.
The development of in vitro vascular constructs that recapitulate the biological structure and function of those found in vivo are of great interest for disease modeling and drug testing. Induced pluripotent stem cells (iPSCs) are attractive for these constructs due to the ability to easily culture iPSCs prior to differentiation. iPSCs also allow for patient specific disease models due to their ability to maintain a disease phenotype post-differentiation, however, their function is not always comparable to primary cell sources. We have developed tissue engineered blood vessels containing human iPS-derived smooth muscle cells (iSMCs) from healthy or Progeria patients or mesenchymal stem cells (MSCs) embedded in a dense collagen matrix. These vessels are endothelialized with human cord blood-derived endothelial progenitor cells to mimic small diameter blood vessels. iSMC TEBVs show reduced vasoactivity in response to 1 μM phenylephrine and 1 μM acetylcholine compared to MSC TEBVs. Immunostaining shows reduced contractile protein expression in iSMC TEBVs compared to MSC TEBVs. Histology shows Progeria disease markers such as calcification and apoptosis at higher levels in TEBVs derived from HGPS iSMCs compared to TEBVs derived from normal iSMCs or MSCs. Combined treatment with 20 μM isoxazole and 10 ug/mL doxycycline recovers vasoactivity to comparable levels of TEBVs fabricated from MSCs. These results suggest the potential for our TEBVs containing human iPS-derived cells to serve as a test bed for disease modeling and drug testing. Results also indicate the ability to modulate iSMCs at the 3D level to create a more biologically relevant tissue phenotype.
Biomedical Engineering, University of Arizona, BME GIDP, Tucson, AZ.
The efficacy of cell-based regenerative therapy is contingent upon factors related to donor tissue availability, processes used for cellular extraction, extracted cell sample quality, and host-response. With the intent of improving the therapeutic potential and reproducibility of cell-based treatments using adipose-derived stem cells (ASCs), this study developed a novel technique for selectively enriching viable cells with greater regenerative capacity in tissue-extracted cell samples. Adherent cultures of stromal vascular fractions (SVFs) extracted from human adipose tissues were exposed to nutrient deficient conditions - eliciting a bi-modal cellular response and creating two dissimilar cell culture subpopulations. The isolated cell subpopulations were evaluated by assessing their characteristic morphology, metabolic activity, ability to undergo multi-lineage differentiation, and expression of mesenchymal stem cell associated biomarkers. The SVF subpopulation which demonstrated sensitivity to the nutrient deficient conditions expressed typical morphological expression of adherent cell cultures, elevated metabolic activity, and the ability to differentiate along adipogenic, chondrogenic, and osteogenic lineages. The SVF subpopulation which demonstrated resistance to the nutrient deficient conditions, however, expressed atypical morphologies, impaired metabolic activity, and did not survive culture with differentiation growth media. Stem cell associated biomarkers were also much more highly expressed in the treatment sensitive SVF subpopulation than control SVF cultures when evaluated using PCR and flow cytometry. Based on our measurements, the “treatment-sensitive” SVF subpopulation exhibited characteristics that demonstrated a significantly greater regenerative capacity than the “treatment-resistant” subpopulation. This was also true when compared to the original overall SVF.
Culturing cells on thermoresponsive polymers enables cells to be harvested as an intact cell sheet without disrupting the extracellular matrix. In this study MC cell sheets characterized in vitro and in vivo for differentiation potential. Cell viability and proliferation were analyzed using LIVE/DEAD® staining and PicoGreen assay. hASCs in cell sheets remained viable for 21 days and proliferated until confluence in vitro. In order to demonstrate osteogenic potential in vitro, MC cell sheets were cultured in stromal and osteogenic medium for a 21-day study. Cell sheets, cultured in osteogenic conditions, exhibited upregulation of alkaline phosphatase (ALP) at day 7, as well as calcium deposition at 21 days when stained with Alizarin Red. Additionally, expression of osteocalcin (OCN), a late-stage marker of osteogenesis, was quantified at days 14 and 21 using RT-PCR. OCN was upregulated in MC cell sheets at day 14. Extension of this work to a mouse calvarial defect model has shown MC cell are capable of driving human ASC osteogenic differentiation in vivo. Bone formation was observed 8 weeks after the surgery using micro-CT and H&E staining. These results indicate that hASCs formed into cell sheets commit to an osteogenic lineage when cultured in osteogenic conditions. Furthermore, cell sheet stacking enables fabrication of quasi-three-dimensional tissues with limited or no integration of synthetic materials while allowing cell-cell junctions to remain intact. Cell sheets may provide an informative model system for studying cell interactions and spatial organization in a controlled microenvironment.
Control of Bone Regeneration by Spatiotemporal Morphogen Presentation within Engineered Mesenchymal Condensations
Biomedical Engineering, Case Western Reserve University, Cleveland, OH.
Developmental engineering comprises the design of regenerative strategies guided by principles of developmental biology, namely, recapitulating cellular self-organization and morphogenetic pathway activation. Here, we describe mesenchymal condensations featuring spatiotemporally-controlled growth factor release from microparticles embedded within cell constructs for functional regeneration of critically-sized rat femoral defects, and assess the ability to control the ossification mode through defined morphogen presentation. Mesenchymal condensations were engineered by mixing hMSCs with growth factor-loaded microparticles delivering TGF-β1 (early) and BMP-2 (sustained). 8-mm segmental femoral defects were created in 12-wk-old male RNU rats stabilized with stiff or axially-compliant fixators. Treatment groups were: 1) without growth factor (control), 2) BMP-2-, or 3) BMP-2+TGF-β1-loaded. Bone regeneration was evaluated by in-vivo μCT, with post-mortem analysis at 12 wks by torsional testing and histology. Longitudinal in-vivo μCT revealed minimal bone formation in controls over 12 wks irrespective of mechanical cues. BMP-2-delivery increased bone formation at 8 & 12 wks vs. controls but defects remained largely unbridged. BMP-2+TGF-β1 presentation significantly augmented bone volume vs. control and BMP-2-loaded groups at 8 & 12 wks (p < 0.05; highest values with compliant fixators). Torsional stiffness/maximum torque were also significantly enhanced (p < 0.05), achieving functional properties comparable to intact femurs. Histology exhibited fibrous tissue in controls, punctate bone with BMP-2-delivery, and distinct cartilage bands embedded in new bone partially resembling the native growth plate with BMP-2 + TGF-β1-loading. Data showed that: 1) mesenchymal cell condensation alone was insufficient for bone regeneration, 2) sustained BMP-2 presentation stimulated defect healing, which was promoted by early TGF-β1-delivery recapitulating endochondral ossification, 3) mechanical loading had potentiating effects across groups. Funding: NIH(1F32DE024712, R01AR063194); ICTSI(UL1TR001108).
Eastern Virginia Medical School, Norfolk, VA.
PDGF-Metronidazole Functionally Graded Membrane for Alveolar Ridge Regeneration
Human Placenta Derived Extracellular Matrix Sponges for Osteochondral Tissue Engineering
Rameshbabu A.P. et al. Journal of Materials Chemistry B. 4, 613, 2016.
Gelatin Microspheres Modulate Endothelial Sprouting in Fibrin Hydrogels Containing Co-cultures of HUVEC-ASC or HUVEC-NHLF
Engineering Gradient Hydrogel Scaffolds for Vascularized Tissue Formation
Biomedical Engineering, Illinois Institute of Technology, Chicago, IL.
Neovascularization is highly dependent on gradients of soluble (chemotactic) and immobilized (haptotactic) extracellular matrix signals as well as gradients of physical structure and mechanical properties (durotactic). The development of polymerization strategies that allow for controlled spatial and temporal gradients of these signals in 3D biomaterials holds great promise for rapid and stable vascularization of engineered tissues. We have developed a photopolymerization technique (ascending-frontal-photopolymerization (AFP)) to generate combined as well as individual gradients of chemotactic, haptotactic and durotactic signals in synthetic hydrogel scaffolds. AFP was used to create MMP-sensitive poly(ethylene) glycol (PEG) hydrogels with individual gradients of 1) the immobilized cell adhesive RGD peptide (350 ± 240–865 ± 295 μM) and with uniform elastic modulus (1174 ± 80 Pa), 2) elastic modulus (528 ± 94–1587 ± 77 Pa) and with uniform RGD concentration (1400 ± 51 μM) and 3) fluorescently labeled hydrogel nanoparticle carriers (fluorescent intensity range 7.1 ± 1.9–06 ± 0.4 a.u) that promote sustained release of proangiogenic factors. A 3D neovascularization in vitro assay was used to investigate the effect of the RGD gradient on vascular sprout invasion. Preliminary data indicate that the invasion area in RGD gradient scaffolds increases 4.2–4.9-fold over 7-days, but is not significantly different as compared to control scaffolds with uniform RGD content. Current efforts are focused on quantifying in vitro response to durotactic and chemotactic cues as well as to variations in gradient magnitude and slope.
One challenge common to large tissue engineered constructs is the need for vascularization in vivo and in vitro to facilitate oxygen delivery. Pre-vascularization is particularly challenging in the kidney due to the organ's size and unique vascular architecture. Biofabrication and microfluidics techniques can produce simple branching vessel-like structures, however they do not replicate the vascular organization seen in native tissues. In addition, these methods are often complex and expensive. We propose a simple, cost-effective and novel method for the fabrication of biomimetic microvascular scaffolds for pre-vascularization of constructs based on vascular corrosion castings of kidneys. Vascular corrosion casts are capable of capturing the entire vascular network of an organ, including glomerular capillaries. Starting with these corrosion casts as a template, we are able to mold microvascular scaffolds that reflect the native anatomy. The microvascular scaffolds can support endothelialization and incorporation into hydrogel-based renal constructs. Human renal cells in 3D culture around the vascular scaffold self-assemble into tubule-like structures, which anatomically and functionally resemble segments of the nephron. We believe that these pre-vascularized renal constructs will be capable of integration after implantation and may be used to augment the function of diseased or damaged kidneys.
The Role of the Perivascular Niche in Regulating Cancer Stem Cells In Glioblastoma Multiforme
Glioblastoma Multiforme (GBM), the most common and lethal primary brain tumor in adults, is hallmarked by vascular dysfunction, abnormal extracellular matrix (ECM) turnover, and the presence of cancer stem-like cells (CSCs). CSCs localize near the perivascular space in the brain that is rich in collagen type I and basement membrane proteins and respond to changes in ECM physicochemical properties. However, the functional relationship between the perivascular niche, ECM, and CSCs remains relatively unclear. Here, we embedded endothelial cells and CSCs into microfabricated collagen type I/Matrigel hydrogels whose microarchitecture was controlled by adjusting the gelation conditions. Through combining confocal microscopy, biochemical assays, and loss-of-function studies we evaluated interactions between endothelial and CSCs in these models. Our results suggest that ECM composition and structure control microvascular network assembly and that these differences are linked to altered IL-8 secretion by endothelial cells. Moreover, endothelial cell-secreted IL-8 induces CSC localization to microvascular structures and plays a direct role in modulating subsequent ECM remodeling and endothelial cell behavior. Based on these results, we propose that the specific ECM characteristics of the brain perivascular niche control GBM tumorigenesis by regulating the crosstalk between CSCs and endothelial cells as a function of altered IL-8 signaling. Indeed, based on Oncomine analysis IL-8 is upregulated in human brain cancers and we are currently evaluating whether this finding is linked to altered ECM perivascular niche characteristics in vivo. Engineered tumor-mimetic culture models will be critical to further interrogate the mechanisms contributing to our observations and disease progression.
Design of Micro and Mesoscale Human Vascularized Models for the Study of Cancer Metastases
IRCCS Istituto Ortopedico Galeazzi, Milano, ITALY.
1 Valastyan, Weinberg. Cell147, 275, 2011.
2 Jeon et al. PNAS112, 214, 2015.
Usami et al. Ann Biomed Eng. 1993, 21:77–83.
Biomimetic Anisotropic Polymeric Particles with Naturally Derived Cell Membranes for Enhanced Drug Delivery
There has been growing interest in the use of naturally derived cell membranes supported by polymeric particles for a multitude of applications in drug delivery.1 Through fusion of vesicles derived from red blood cells, platelets, and cancer cell membranes, these particles have shown tremendous therapeutic promise.2 Despite this potential, all therapeutics to date have been synthesized with spherical particles and thus lack an important aspect of cellular biomimicry: shape. Here we have synthesized particles that mimic the shape, size, and membrane content of red blood cells and platelets for enhanced drug delivery. Nonspherical acid terminated platelet shaped particles (PSPs) or red blood cell shaped particles (RBCSPs) were synthesized by emulsion methods followed by thin film stretching in two dimensions. Red blood cells and platelets were processed into 200 nm sized vesicles through sonication and extrusion. The subsequent vesicles were coated on the anisotropic particles under sonication. Through fusion of a fluorophore lipid mediated by a PEGylated accessory lipid, we were able to visualize the membranes on both the spherical and ellipsoidal microparticles under confocal microscopy. There was a clear enrichment of the membrane signal on the surface of the anisotropic RBCSPs and PSPs indicating successful coating. Furthermore fusion of synthetic lipids can be used to conjugate any desired protein or small molecule to the membrane surface. Taken together, this platform provides a modular and versatile technology for enhancing drug delivery.
1 Meyer RA et al., Trends in Biotechnology 2015.
2 Hu CMJ et al., Nature 2015.
In Vivo Nanoparticle-Mediated RNAi in Bone Marrow Enhances Hematopoietic Stem Cell Mobilization and Harvesting
IL4 Eluting Nanolayered Coating Promotes Better Implant Integration into the Host Tissue via Macrophage Modulation
Recent studies have implicated that an inadequate host biomaterial response, driven predominately by pro-inflammatory (M1) macrophages and downstream formation of fibrotic capsule, is a major cause of the severe mesh-related complications in prolapsed patients. We have hypothesized that shifting this macrophage response towards an anti-inflammatory (M2) phenotype will lead to a better implant integration into the host tissue. Therefore, a nanolayered coating for controlled delivery of IL-4 (M2 cytokine) has been developed. Polypropylene mesh was plasma irradiated and coated using layer-by-layer (LbL) deposition with chitosan and dermatan sulfate (previously incubated with IL-4). A uniform coating was assessed using XPS, FTIR and Alcian Blue staining. ELISA assays showed that the amount and release of IL-4 were dependent on the number of coating layers. IL-4 bioactivity after coating was demonstrated by M2 macrophage polarization using an in-vitro culture. Histologic studies in a mouse implantation model showed an increased glycosaminoglycan deposition in mouse implanted with IL-4 loaded meshes, but also an increased the percentage of M2 macrophages and decreased the percentage of M1 macrophages compared to untreated meshes. Interestingly, rtPCR analysis from tissue surrounding mesh detected higher expression levels in M2-related genes, MMPs but lower Col1A1 expression in mice implanted with IL-4 loaded meshes, compared to untreated meshes. These shifts in macrophage polarization led to decreased fibrotic capsule formation surrounding mesh fibers and increased alignment of collagen fibers surrounding the mesh in the long term, suggesting improved mesh integration into the host tissue and the potential for a reduction in downstream complications.
Controlled Angiogenic Peptide Delivery from Hydrogel Nanoparticles for Therapeutic Neovascularization
Therapeutic neovascularization involving proangiogenic growth factor delivery has shown promise for restoring perfusion in ischemic tissues; however, the clinical efficacy of growth factors has been limited by their associated immunogenicity and structural instability in vivo. A promising alternative uses growth factor-mimetic peptide sequences to interact with receptors for regulating neovascularization while providing
Novel PEG-OES Nano-fibers For Local Immunomodulation of Conformal Coated Pancreatic Islet Grafts for Type-1 Diabetes
Diabetes Research Institute, University of Miami, Miami, FL.
Patients with type-1diabetes (T1D) are dependent on exogenous insulin injections and are susceptible to life-threatening complications.
Strategies to improve tissue regeneration require modulation of dynamic cellular processes (Burdick, Nature Communications, 2012). Growth factor delivery is one strategy to modulate these processes, but preventing denaturation and temporally controlling delivery of growth factors remains challenging. Thus, we developed core-shell heparin-poly(ethylene-glycol) (PEG) microparticles with tunable shell degradation, allowing temporally modulated release of a protein-laden heparin core to enhance protein stability and achieve user-defined timing of protein delivery.
Core-shell microparticles were formed via water-in-oil emulsion. For microparticles with a degradable shell, dithiothreitol was integrated into the PEG-diacrylate network. Microparticles were loaded with 1.5 μg BMP-2/mg heparin at 4°C, and release was conducted at 37°C. Microparticles were added directly to C2C12 cells for three days and ALP activity, produced by C2C12 cells in response to BMP-2, was normalized to DNA content.
Core-shell microparticles degraded and released the heparin core in six days. Degradable and non-degradable core-shell microparticles released only 5.8 ± 3.2% or 7.9 ± 4.3% of BMP-2, respectively, over seven days, indicating most BMP-2 remained bound to the heparin core. Degradable core-shell microparticles induced similar C2C12 ALP activity as the soluble control after shell degradation, while non-degradable core-shell microparticles induced significantly lower activity after three days (85.4 ± 19% vs. 9.0 ± 4.8% of soluble control, respectively).
Core-shell microparticles were engineered to degrade and release a protein-laden, bioactive heparin core that initiated a cell response only after shell degradation. By tuning degradation rate of the PEG shell, user-controlled delivery of bioactive therapeutic proteins can be achieved, providing a novel tool to temporally control protein presentation for tissue regeneration.
Highly Flexible And Resilient Elastin Hybrid Cryogels With Shape Memory, Injectability, Conductivity And Magnetic Responsive Properties
Development of conducting or magnetic responsive hydrogels, requires high resilience, flexibility, injectability, responsive sensitivities for the applications of biosensors or bioelectronics. In this paper, an ultra-flexible elastin-peptide based hybrid (elastin-gelatin-CNT, EGC) cryogel was fabricated as scaffold to load large amount of rigid functional components, including carbon nanotube (CNT), polypyrrole (PPY) and/or iron oxide magnetic nanoparticles (IONP), for combining excellent conductivity or magnetic responsive property with high elasticity, flexibility, shape memory property and injectable property together. EGC loaded with dispersed PPY aggregates showed highly flexible and injectable property with a moderate conductivity. However, when PPY formed a second rigid network on this soft scaffold, the bicontinuous network exhibited an extraordinarily high resilience enduring 97.5% compressive strain and an outstanding conductivity of 50.1 ± 2.9 S/cm at 90% strain together, as well as excellent bulk pressure sensitive conductivity. Also, the magnetic EGC-IONP-PPY could merge high flexibility, conductivity and magnetic property together, showing the potential as remotely controlled robots or bioactuators.
Controlled Delivery of Platelet-Derived Proteins Accelerates Porcine Wound Healing
Bioengineering, University of Pittsburgh, Pittsburgh, PA.
Platelet-rich plasma (PRP) is a therapy used widely in the clinic for many indications including wound healing due to the presence of therapeutic proteins such as vascular endothelial growth factor and platelet-derived growth factor. However, the short half-life of these proteins requires multiple large doses, and the efficacy of this treatment is highly debated among researchers. Here we report a study on protecting these proteins and releasing them in a controlled manner using our heparin-based coacervate delivery vehicle to improve wound healing in a porcine model. Platelet-derived proteins incorporated into the coacervate were protected and slowly released over 3 weeks in vitro. In an in vivo porcine model, PRP coacervate significantly accelerated the healing response over 10 days. Treatment with PRP coacervate increased the reepithelialization rate by 35% compared to control. Additionally, this treatment doubled the rate of wound contraction compared to all other treatments, including that of naked PRP proteins. This accelerated wound closure reduces the risk of infection due to restoration of the skin's barrier function. Wounds treated with PRP coacervate exhibited increased deposition of aligned collagen within the wound and an advanced state of vascularity compared to control treatments. PRP is easy to obtain, which explains its wide usage clinically despite the ongoing debate over its efficacy. This study suggests that PRP is ineffective as a therapy in healing cutaneous wounds without a controlled release vehicle. The coacervate delivery vehicle is a simple and effective tool to improve the therapeutic efficacy of platelet-derived proteins for wound healing.
Chemical Engineering, University of Michigan, Ann Arbor, MI.
Therapeutic potential of allogeneic transplanted cells to replace damaged tissue function depends critically on immune evasion for transplant survival. Currently, immune rejection is limited via broad immunosuppression, but approaches incorporating antigen-loaded nanoparticles were recently reported to establish antigen-specific tolerance.1 Herein, we examined immune cell interactions with poly(lactide-co-glycolide) (PLG) nanoparticles carrying chemically-conjugated antigen to identify tolerance mechanisms triggered. Antigen-presenting cells (APCs) were obtained from murine bone marrow differentiated in vitro into macrophages or dendritic cells (DCs). To measure internalization kinetics, fluorescent nanoparticles were administered to lipopolysaccharide-activated APCs. Internalization occurred within hours, as confirmed through quenching of extracellular fluorescence. Subsequently, APCs were treated for 24h with Eα52-68-loaded nanoparticles (PLG-Eα) and analyzed using cognate Y-Ae antibody that specifically detects major histocompatibility complex (MHC)-restricted Eα52-68. At nanoparticle concentrations of 1, 10, and 100 μg/mL, antigen presentation was detected on 1.3%, 1.9%, and 8.2% of macrophages and 1.9%, 4.8%, and 32.9% of DCs. Increasing nanoparticles from 1 to 100 μg/mL reduced APC expression of positive co-stimulatory molecules (CD86, CD80, CD40) while expression of the negative co-stimulatory molecule, PD-L1, remained high. T cells harvested from mice immunized against PLP139-151 were co-cultured with APCs receiving either antigen-specific PLP139-151, or nonspecific, OVA323-339-loaded nanoparticles. T cells co-cultured with macrophages receiving PLG-PLP had decreased IFNγ, increased IL-10, and minimal proliferation relative to PLG-OVA. When co-cultured with DCs, T cells proliferated, but expressed higher levels of phosphatidylserine, indicating apoptosis. These observations suggest collectively, different APCs incorporate multiple mechanisms to induce antigen-specific tolerance.
1 Bryant J, et al. 2014. Biomaterials. 35, 31, 8887–94.
Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA.
Mesenchymal stem cells (MSCs) can enhance angiogenesis and reduce inflammation through the secretion of cytokines such as vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2). MSC therapies are limited by rapid death and poor engraftment when delivered to ischemic conditions. The formation of MSCs into spheroids enhances their survival, proangiogenic and anti-inflammatory potential. However, effective strategies to optimize spheroid function in this application are lacking. In this study, we determined conditions for forming MSC spheroids that simultaneously enhance their proangiogenic and anti-inflammatory potential with an eye toward wound healing.
Using Design-of-Experiments (DOE) multivariable analysis, we determined the interaction between three input variables (oxygen tension, cells/spheroid, and the inflammatory stimulus, Pam3CSK4) on MSC spheroids by quantifying secretion of VEGF and PGE2. We validated our results by stimulating endothelial cells or macrophages with conditioned media from spheroids predicted to have superior (Sph1) or lesser (Sph2) wound healing potential.
VEGF secretion increased as a function of decreasing oxygen tension and increasing density of cells in the spheroid, while PGE2 secretion exhibited a positive correlation with increasing cells/spheroid and inflammatory stimulus. Conditioned media from Sph1 was significantly more proangiogenic than that from Sph2, as evidenced by forming longer tubes with more branch points. In stimulated macrophages, conditioned media from Sph1 decreased TNFα expression and increased IL-10 expression, suggesting that the macrophages were polarized from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. These data demonstrate that DOE analysis can determine culture conditions that enhance the proangiogenic and anti-inflammatory potential of MSC spheroids.
Hypoxic Response Of Oral Mucosa Fibroblasts In A 3D Collagen Lattice
Oral and Maxillofacial Surgery, University School of Dentistry, Niigata, JAPAN.
Department of Biomedical Engineering, University of Michigan, ANN ARBOR, MI.
Integra LifeSciences Corporation, Plainsboro, NJ.
The objective of this study was to assess the feasibility and efficacy of a Flowable Wound Matrix (FWM) mixed with Platelet Rich Plasma (PRP) for the treatment of full thickness wounds (FTWs). FTWs heal with scars and contracture due to inadequate tissue regeneration, which can be improved using growth factors (GFs) released from PRP. However, the traditional method of PRP activation using thrombin releases GFs rapidly, limiting its efficacy. Controlled GF release from PRP may provide sustained signaling to improve healing outcomes. In this study, we hypothesized that FWM, made of collagen and chondroitin sulfate, can be used for controlled GF release from PRP, and can improve FTW healing. Released supernatant was collected from PRP mixed with FWM and controls (FWM+saline and thrombin activated PRP) at 1, 4, 8 and 24 hours. PDGF-BB, EGF, TGF-β1 and VEGF were quantified using standard ELISAs. In vitro efficacy of the released supernatant was assessed using scratch assay. In vivo efficacy of FWM+PRP was tested in a porcine FTW model. GF release from FWM+PRP was controlled and sustained for 24 hours compared to thrombin activated PRP, which had rapid GF release. Correspondingly, greater fibroblast proliferation and migration was observed for FWM+ PRP group in scratch assay. In vivo, decreased wound contraction was observed in FWM+PRP group compared to PRP, suggesting improved FTW healing. These results suggest that FWM may be used synergistically with PRP to provide controlled GF release, which may translate into an improved quality of tissue healing in FTWs.
Biotecnologia, Instituto Nacional de Rehabilitacion, Mexico, MEXICO.
Our goal was to analyze cytocompatibility of human adipose-derived mesenchymal stem cells (hADMSCs) cultured onto radiosterilized pig skin (RPS) and radiosterilized human amnion (RHA) evaluating cell adhesion, proliferation, and migration.
Wake Forest University Health Science, Winston Salem, NC.
School of Life and Environmental Sciences, University of Sydney, Sydney, AUSTRALIA.
Mechanical Engineering, The University of Tokyo, Tokyo, JAPAN.
In this work, we sought to develop a tissue engineered periosteum to improve bone regeneration through the controlled release of multiple growth factors. A co-electrospinning setup was used to fabricate a crosslinked gelatin graft with tunable growth factor release. Altering the degree of crosslinking gelatin using two distinct mechanisms was used to tune gelatin degradation rate, and thus, growth factor release kinetics. In-line blending with a hexamethylene diisocyanate crosslinker was used to both crosslink the gelatin and anchor the growth factors directly to the gelatin through reaction with the lysine residues. Methacrylation of the gelatin was achieved by reacting the gelatin with 2-isocyanatoethyl methacrylate prior to electrospinning and photocuring with UV light during spinning. In contrast to the in-situ crosslinked gelatin, growth factor release from the photocrosslinked gelatin-methacrylate occurs upon fiber swelling. SEM analysis confirmed that fiber diameter was unchanged in both of these systems. Initial results demonstrated that in-situ crosslinked gelatin resulted in 39% release of model growth factor, FITC-albumin, over 7 days whereas the photocrosslinked gelatin resulted in 86% release over 4 hours. We have also demonstrated successful co-electrospinning to generate two distinct fiber populations in a single mesh without affecting fiber morphology. Current studies are focused on evaluating bioactivity retention of growth factors and directing osteogenic differentiation of hMSCs. This research highlights the potential of this biomaterial platform to generate an engineered periosteum with independent control of growth factor release kinetics achieved by modulating crosslinking of gelatin.
Bone Engineering Potential of Cow And Human Derived Decellularized Bone Scaffolds
The New York Stem Cell Foundation Research Institute, New York, NY.
A common trend in tissue engineering is the use of decellularized tissue matrices as scaffolding materials, since they provide the cells with architectural, topographic and chemical cues typical of the native tissue. We recently demonstrated that viable and functional bone grafts could be engineered by combining mesodermal progenitors derived from human induced pluripotent stem cells (iPSC-MPs) with cow-derived decellularized bone scaffolds. However, it is unclear whether interspecies differences in bone parameters influence the quality of bone grafts engineered from human stem cells. In this study, we have compared decellularized scaffolds derived from cow and human bone, and assessed in vitro their bone engineering potential using human iPSC-MPs. Decellularized bone scaffolds (4 mm Ø × 4 mm height, mineral densities range between: 0.333- 0.858 mg/mm3) were prepared from cow (Green Village Packing) and human (LifeNet_Health®) trabecular bone, and characterized for chemical composition, porosity, and mechanical properties. Human iPSC-MPs (line 1013A) were expanded, seeded onto the scaffolds and cultured for 5 weeks in osteogenic medium in perfusion bioreactors. Cell attachment, viability, proliferation and osteoblastic differentiation, tissue formation and mineralization were assessed via microscopy, molecular biology assays, histological techniques and medical imaging procedures. Results demonstrate that, despite differences in composition and architecture, cow and human derived scaffolds equally support viability, proliferation, and osteoblastic differentiation of human iPSC-MPs, resulting in the formation of mineralized bone-like tissue. In conclusion, cow bone represents a more economical and easily accessible source material to engineer bone substitutes for basic and applied research, and potential future clinical applications.
Biomedical Engineering, Northwestern University, Evanston, IL.
Type 1 diabetes mellitus is a chronic condition characterized by the autoimmune-mediated destruction of pancreatic β-cells. Islets transplantation in the liver often results in limited islets survival presumably due to uneven islet distribution, abnormal blood flow dynamics and acute inflammatory response. In this study, we demonstrated the ability of using an antioxidant biodegradable citrate-based thermoresponsive hydrogel poly(polyethyleneglycol citrate-co-N-isopropylacrylamide) (PPCN) to support the viability and function of transplanted islets in an extra-hepatic location. The use of PPCN hydrogel preserved the normal islet morphology, viability and reduced oxidative damage during both in vitro culture and in vivo transplantation. The antioxidant property and hydrophilicity distribution within PPCN as well as its biocompatibility (low to no inflammation) likely created an environment that supports the viability and function of the islets, allowing successful extrahepatic islet transplantation in this mouse model. The observed restoration of euglycemia and glucose tolerance response is superior to what has been reported for other hydrogels when taking into account the number of islets required to achieve euglycemia and the time it took to reach the euglycemic state. These findings confirm that a novel citrate-based thermoresponsive hydrogel can serve as a platform vehicle for extrahepatic islet transplantation.
Angiograft, LLC, Amherst, NY.
1. M. T. Koobatian et al. Biomaterials
2. R. J. Smith, Jr. et al. Biomaterials
3. M. T. Koobatian et al. J Vis Exp
B2OA UMR CNRS 7052, Université Paris Diderot, Paris, FRANCE.
In the context of cell-based regenerative medicine, exogenously administered mesenchymal stromal cells (MSCs) exhibited a poor survival rate. This issue can be overcome by in situ supplying glucose that acts as the main metabolic fuel for MSCs in severe hypoxia and significantly enhances their survival (Deschepper et al.). The objective of the present study is to engineer a tissue-construct that provides sufficient level of glucose to MSCs and enhances their survival in vivo. To this aim, fibrin hydrogels containing a polysaccharide (i.e. starch) and supplemented with free alpha-amyloglucosidase (AMG/ an enzyme that cleaves starch to yield glucose)) or AMG-containing PLGA nanoparticles were designed. Fibrin/starch hydrogels were injectable, self-supported and released glucose amounts in accordance with that required by hMSCs for their survival. In vitro, under near anoxia, MSCs loaded in fibrin/starch hydrogels exhibited a survival rate 115 times higher than the one loaded in fibrin hydrogels, after 14 days. Moreover, when ectopically implanted in nude mice, hMSCs loaded in fibrin/starch hydrogels showed a significant improvement of their viability (×10 after 14 days) in comparison to hMSCs loaded in fibrin gels as demonstrated by bioluminescence imaging and immunochemistry. In conclusion, this work establishes the proof of concept that fibrin/starch hydrogel containing AMG is able to deliver glucose over time and enhance MSC survival.
Deschepper M., et al. J Cell Mol Med. 2011 and Deschepper M., et al. Stem Cells. 2013.
Enhancing Translatability of Expanded Chondrocytes to Engineer Native-like Neocartilage
BME, UC Davis, Davis, CA.
The intractable problem of osteoarthritis necessitates tissue-repairing solutions. Therefore, the goal of this study was to use expanded chondrocytes to engineer neocartilage with compressive properties reaching those of native cartilage. It was hypothesized that 1) increasing chondrocyte purity via ammonium-chloride-potassium (ACK) buffer, 2) applying novel redifferentiation methods, and 3) enhancing chondrocyte activity via cytoskeleton-modifying agents would yield neocartilage with a compressive modulus on par with native tissue. Chondrocytes were isolated from fetal sheep stifles, as fetal cells represent a highly-clinically relevant cell type for tissue engineering. First, ACK buffer treatment of primary (P0) chondrocytes decreased red blood cell contamination by 60% and increased neocartilage aggregate modulus (1.8-fold), shear modulus (1.3-fold), and tensile modulus (0.8-fold). Subsequently, seeding density optimization of expanded/redifferentiated (P3R) chondrocytes to 2 million cells/construct increased aggregate modulus (1.0-fold) and shear modulus (1.1-fold) further. Lastly, cytochalasin D treatment further increased neocartilage aggregate modulus to 400kPa, on par with native cartilage, 9.6-fold over the untreated P0 control. ACK buffer- and cytochalasin D-treated P3R cells notably yielded neocartilage with compressive properties beyond that of P0 neocartilage and akin to native cartilage. These sequential studies allowed 4000-times fewer primary cells to be used to engineer robust neocartilage, specifically using 1000 primary cells per P3R construct versus 4,000,000 per P0 construct, greatly enhancing the clinical translatability of expanded chondrocytes for tissue engineering.
Mechanical stimulation has been demonstrated to be one of the strategies to optimize tissue engineering construct. However, it is challenging to rationally determine the optimal mechanical stimulation to control the tissue differentiation in a construct. This leads to propose the energy dissipation when a construct is submitted to a load as a new mechanobiological variable. The aim of this study is to evaluate if dissipation could be correlated to a specific tissue differentiation when a seeded scaffold is mechanically loaded. HEMA-based scaffolds have been developed having different levels of dissipation when subject to the same loading in terms of frequency and amplitude of deformation. The levels of dissipation were chosen to match the level of dissipation of healthy and degenerated cartilages. Scaffolds were seeded with epiphyseal chondro-progenitor cells at 3 mio cells/scaffold. Cell-scaffold constructs of the different groups were subject to mechanical stimulation. Dynamic compression of 1 Hz and 10% strain (with 10% pre-strain) was applied to scaffolds 2 h/day during 4 consecutive days. After the 4th day of testing, immediately after mechanical stimulation, gene expressions of TGF-beta, Sox9, Col2a and Aggrecan were analyzed for the loading group and the free-swelling group used as control. As a result, in general upregulation for all genes was observed with dissipation close to the dissipation of healthy cartilage, and downregulation was observed for dissipation close to degenerated cartilage. This study highlights that there is a sensitivity of chondrogenic expression to dissipation. Dissipation could then be a variable to be considered in cartilage mechanobiology.
Osteogenic Differentiation Of iPSC-MSCs On Novel Nanofibrous Spongy Microspheres
Biomedical Engineering, University of Michigan, Ann Arbor, MI.
Limitations in the available treatments for critical size bone defects have led to the development of the field of bone tissue engineering. While research in this field relies frequently on adult bone marrow stromal cells as a multipotent stem cell source, these cells have limited capacity for proliferation and self-renewal. To address one of the major limitations in autologous bone grafts, a renewable and plentiful source of patient-derived tissue is needed. Thus, we propose the use of mesenchymal stem cells derived from induced pluripotent stem cells (iPSC-MSCs) as a cell source that can be used in bone regeneration and treatment of critical size defects. Injectable biomaterials offer the advantage of reducing the invasiveness of treatments and matching any defect geometry. However, many injectable materials are hydrogels, which can reduce the ability of cells to migrate, attach, and proliferate. We propose the use a novel and injectable nanofibrous spongy microsphere (NF-SMS) synthesized from star-shaped poly(l-lactic acid) (PLLA). These NF-SMS possess a fibrous nanostructure that mimics natural extracellular matrix as well as an interconnected, porous microstructure that facilitate in-growth, attachment, proliferation, and differentiation of iPSC-MSCs. We will demonstrate the in vitro and in vivo osteogenic potential of iPSC-MSCs on NF-SMS using 3D cell culture followed by a mouse calvarial defect model. This project will be the first to combine iPSC-MSCs with a NF-SMS cell carrier. It would be an important step toward building a clinically relevant treatment of critical size defects and would further elucidate the potential of iPSC-MSCs in regenerative medicine.
Illinois Institute of Technology, Chicago, IL.
Bone defects can result from trauma, congenital abnormalities, tumor excision and infection. Generation of vascularized bone tissue of clinical volume remains a challenge in tissue engineering. Recently, we have developed a scaffold with platelet-derived growth factor BB (PDGF-BB) gradient that stimulates vascularization in vivo.1 In this study we examined degradable gradient scaffolds with ceramic in a clinically translatable periosteum guided large animal model. Degradable gradient scaffolds with ceramic (10 mm height) were generated using a particulate leaching method1. Hydrogel structure was composed of polyethylene glycol diacrylate (PEG-DA) and PEG-poly(l-lactic acid)-DA. Degradable structure, PDGF-BB gradient and ceramic effects were tested. PDGF-BB (200 ng) and ceramic materials (hydroxyapatite to tri-calcium phosphate, 70:30%) were incorporated into hydrogel structure to enhance bone regeneration. Ceramic hydrogel composites exhibited 1.2 times longer degradation times than controls in vivo. Presence of ceramic did not affect mechanical properties of the scaffolds. Compressive modules of the scaffolds were 18 kPa and 20 kPa for ceramic loaded and controls, respectively. Regenerated bone volume was quantified based on micro-computed tomography images for 4 weeks and 8 weeks. Tissue structure was evaluated with histological stains. The maximum bone regeneration (45%) was seen within ceramic supplemented degradable gradient scaffold at 8 weeks. Gradient scaffolds with ceramic showed a significant promise to engineer vascularized bone tissue in clinical size.
Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA.
(1) Mannella GA. J Polymer Sci B: Polymer Physic. 2014, 52(14): 979–983.
(2) Mannella GA et al. Mater Letts. 2015, 160: 31–33.
(3) Lozito TP et al. Stem Cell Res Ther 2013, 4(Suppl 1):S6.
(4) Lin H et al. Mol Pharmaceut. 2014, 11(7): 2203–12.
Diabetes mellitus (DM) is a metabolic disease frequently associated with impaired bone healing, but despite its increasing prevalence worldwide, the molecular etiology of DM-linked skeletal complications remains poorly defined. Using advanced stem cell characterization techniques including parabiosis, we comprehensively analyzed the intrinsic and extrinsic determinants of mouse skeletal stem cell (mSSC) function in diabetic (Db) mice to identify specific mSSC niche-related abnormalities that could impair skeletal repair. We discovered that high serum levels of Tumor necrosis factor-alpha in Db mice directly repress the expression of Indian hedgehog (Ihh) in mSSCs and their downstream skeletogenic progenitors. When hedgehog signaling is inhibited during fracture repair, injury-activated mSSC expansion and differentiation is suppressed, resulting in impaired healing. We also find that the same Ihh deficiency is evident in skeletal progenitors isolated from diabetic patients relative to normal non-diabetic patients. This deficiency could be reversed, however, by precise delivery of purified Ihh to the fracture site using a specially formulated slow-release hydrogel. In the presence of exogenously applied Ihh, the injury-induced expansion and osteogenic potential of mSSCs in Db mice is restored, culminating in the rescue of diabetic bone healing. Our results demonstrate a feasible strategy for precise molecular diagnosis and treatment of molecular aberrations in specific stem and progenitor cell populations to restore skeletal manifestations of systemic disease.
Craniofacial Bone Regeneration Guided by 3D Printed Architecture
Guided bone regeneration has been a technique utilized in ridge augmentation in preparation for dental implant placement. This technique utilizes a membrane to resist soft tissue ingrowth while localizing material placed in the site to promote bone growth.1 This technique is not sufficient for other bone defects as it cannot withstand loading. Moreover, a membrane is not capable of providing complex shape guidance necessary in other craniofacial applications. In this study, we tested the effect of guided bone regeneration in a mandibular bone defect of rabbits with a soft tissue resistant architecture paired with an internal architecture designed to facilitate bone ingrowth. Bone scaffolds were made of a composition of poly(ɛ-caprolactone) and β-tricalcium phosphate (PCL/TCP) and fabricated on the Integrated Organ and Tissue Printing (ITOP) system.2 We examined bone regeneration in a critically sized defect at 4 and 8 weeks with a dual architecture bone scaffold and inhibited bone regeneration in a defect treated with a scaffold without the soft tissue resistant architecture. The results showed the increased bone density and volume and new bone formation and maturation with time. We demonstrated that the printed bone scaffolds were able to organize into mature tissues of their specific characteristics in vitro and in vivo.
1. Jovanovic, S. A. et al. Bone reconstruction following implantation of rhBMP-2 and guided bone regeneration in canine alveolar ridge defects. Clin. Oral Implants Res. 2007;18:224–230.
2. Kang, H.-W. et al. A 3D bioprinting system to produce human-scale tissue constructs with structural integrity. Nat. Biotechnol. 2016;34:312.
Tissue Engineering of Tri-Layered Auricular Cartilage Implants for External Ear Reconstruction
1. R. Giardini-Rosa et al. Development of scaffold-free elastic cartilaginous constructs with structural similarities to auricular cartilage. Tissue Eng. Part A. 20, 1012–26 (2014).
Human Salivary Stem/Progenitor Cells Assemble and Grow in a HA-based Hydrogel with MMP-Degradable Substrates and Integrin-binding Sites
BioSciences, Rice University, Houston, TX.
A suitable valve scaffold prosthesis is necessary in pulmonary artery reconstructive surgery of congenital heart disease (CHD), especially for children. For clinical and long-term success of valve scaffold substitute, this achievement should be taken into consideration together with biomechanics, biocompatibility, functionality and the growth potential. In this study, we implemented a thermally induced phase separation (TIPS)-based strategy, combining 3D printing mold, to rapidly fabricate poly(L-lactic acid)/poly(L-lactide-co-ɛ-caprolactone) (PLLA/PLCL) integrated valve scaffolds with bionic structure. The novel integrated valve scaffolds exhibiting favorable mechanical properties, in comparison to the physiological pulmonary artery of porcine. It had satisfied functional performance in the pulsating flow field, as shown by computational hemodynamic simulation analysis. Moreover, excellent biocompatibility and vascularization for regeneration of the valve scaffolds has been proved in vivo. Additionally, fibers morphology and the production of collagen from host cells supported that this original strategy was an efficient method to fabricating integrated valve scaffolds with potential value for clinical application of complex CHD.
Domain V of Perlecan Covalently Immobilized on Silk Biomaterials Modulates Endothelial Cell and Platelet Interactions for Vascular Applications
Cardiovascular disease is the leading cause of mortality worldwide, with coronary artery disease the largest contributor to this epidemic. In the absence of a suitable native vessel, synthetic polymer grafts are materials of choice in surgical vascular bypass surgery but they characterized by high re-occlusion rates in small diameter vessels. Modulation of endothelial cell and platelet interactions is an essential feature of vascular materials. The objective of this work was to assess the utility of silk biomaterials functionalized with human perlecan in modulating endothelial cell and platelet interactions for vascular applications. Perlecan is a large, conserved extracellular matrix proteoglycan that plays a major role in the vascular niche through its α2β1 integrin binding site and heparan sulfate chain-mediated growth factor signaling. The C-terminal domain V of perlecan is of particular interest in vascular research as contains the endothelial cell α2β1 integrin binding site and the only glycosaminoglycan binding site outside domain I. Domain V of human perlecan was recombinantly expressed as a proteoglycan decorated with heparan sulfate and chondroitin sulfate glycosaminoglycan chains. Silk biomaterials were functionalized with recombinant domain V using passive adsorption or covalent cross-linking via carbodiimide chemistry. Covalent cross-linking modulated domain V orientation on the silk surface, presenting cell-interactive domains and glycosaminoglycan chains in a different conformation to passive adsorption. Silk biomaterials covalently functionalized with domain V supported endothelial cell adhesion, spreading and proliferation and were anti-adhesive for platelets, making them promising surfaces for the development of the next-generation of vascular grafts.
Myocardial Matrix Hydrogel As A Platform For Controlled Delivery Of Therapeutic Exosomes
Bioengineering, UCSD, La Jolla, CA.
National Cerebral and Cardiovascular Center Research Institute, Osaka, JAPAN.
Lifelong dosing of anticoagulants such as warfarin is inevitable after the implantation of a mechanical artificial heart valve, which leads to decreased quality of life. Here, we have been developing and evaluating a polymer-based blood compatible mechanical valve, which requires less dose of anticoagulants. A bileaflet mechanical valve was made of poly(ether-ether-ketone) (PEEK), which shows good workability and endurance. The PEEK has a benzophenone-like structure in its unit, where radicals are generated by photoirradiations.1) Thus, antithrombogenic 2-methacryloyloxyethyl phosphorylcholine (MPC) was directly graft-polymerized on the surface of the PEEK valve by a 27-mW/cm2 UV irradiation at 60°C for 30 min. The existence of poly(MPC) on the PEEK surface was confirmed by strong peaks of nitrogen and phosphorus on X-ray photoelectron spectra. Antithrombogenic effects of the poly(MPC)-grafted PEEK was evaluated by indwelling poly(MPC)-grafted PEEK fibers in blood vessels of miniature pigs for one hour. Scanning electron microscopic observation showed that blood clots were attached onto unmodified PEEK fibers. In contrast, poly(MPC)-grafted PEEK surface was smooth and no clot was observed. Short-term evaluation of poly(MPC)-grafted PEEK valve was conducted using an aortic valve replacement on miniature pigs, where no anticoagulants were used after the operation. Blood clots were formed on the surface of an unmodified PEEK valve within two hours post-implantation, while these clots were not observed on poly(MPC)-grafted PEEK valves harvested at 20–24 hours post-implantation. Long-term antithrombogenicity of the poly(MPC)-grafted PEEK valve is now being investigated.
1) Kyomoto M, Ishihara K. ACS Appl Mater Interfaces 1, 537, 2009.
Highly Elastic and Moldable Polyester for Cardiac Tissue Engineering Applications
Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, CANADA.
Polyester biomaterials are used in tissue engineering as scaffolds for implantation of tissues developed in vitro. An ideal biodegradable elastomer for cardiac tissue engineering exhibits a relatively low Young's modulus, with high elongation and tensile strength. Here we describe a novel polyester biomaterial that exhibits improved elastic properties for cardiac tissue engineering applications. We synthesized poly(octamethylene maleate (anhydride) 1,2,4-butanetricarboxylate) (124 polymer) pre-polymer gel in a one-step polycondensation reaction. The pre-polymer was then molded as desired and exposed to ultraviolet (UV) light to produce a crosslinked elastomer. 124 polymer exhibited highly elastic properties under aqueous conditions that were tunable according to the UV light exposure, monomer composition, and porosity of the cured elastomer. Its elastomeric properties fell within the range of adult heart myocardium, but they could also be optimized for higher elasticity for weaker immature constructs. The polymer showed relatively stable degradation characteristics, both hydrolytically and in a cellular environment, suggesting maintenance of material properties as a scaffold support for potential tissue implants. When assessed for cell interaction, this polymer supported rat cardiac cell attachment in vitro as well as comparable acute in vivo host response when compared to poly(L-lactic acid) control. This suggests the potential applicability of this material as an elastomer for cardiac tissue engineered constructs.
Increasing healthcare costs require the development of new cost-effective biomaterials for a broad spectrum of clinical applications. Human placenta represents a feasible source for different biomaterials. The aim of this study was to isolate collagen type 1 and 3 (COL1/3) and laminin-111 (Lm-111) from placenta and test their potential in 2D and 3D in vitro cultures. COL1/3 was isolated using pepsin digestion; Lm-111 was non-enzymatically isolated. The biomaterials were characterized by HOECHST DNA staining, polyacrylamide gel electrophoresis, dot blot and western blotting. 2D and 3D in vitro assays were performed using HUVECs, primary hepatocytes, and Schwann cells. COL1/3 and Lm-111 yields were 550 ± 71 and 175 ± 35 mg/100 g wet weight basal tissue, respectively. DNA content of COL1/3 and Lm-111 were 0.36 ± 0.11 and 4.18 ± 2.15 μg per mg dry weight (mean±SD, n = 5). Dot and western blot identified collagen 1, 3, and laminin-111. Significant higher cell viability of rat hepatocytes on COL1/3 compared to gelatin was assessed using MTT tests. Standardized sprouting assays using COL1/3 were performed with no significant difference in the number of sprouts, compared with rat tail collagen. No significant differences of Schwann cell viability on Lm-111-coated wells compared to commercially available laminin was assessed using MTT. We have developed efficient, rapid and cost-effective methods to isolate biomaterials from placenta. These materials effectively support adhesion, growth and differentiation of different cell types in vitro. Human placenta represents a cost-efficient source for fully allogenic clinical products. Supported by FFG grant 849755.
Nidogen-1 Significantly Increases Heart Function Post-Acute Myocardial Infarction
Engineering Pre-Vascularized Skeletal Muscle for Treatment of Volumetric Muscle Loss
Stanford University, Stanford, CA.
Unfortunately, many drugs have progressed through preclinical and clinical trials, after which they existed on the market for years in some cases, before being recalled by the FDA for toxicity in humans. We have developed a 6-tissue type body-on-a-chip (
Statin Responses within a Human Vascular Microphysiological System under Inflammatory Conditions
Biomedical Engineering, Duke University, Durham, NC.
Endothelialized human tissue engineered blood vessels (TEBVs) that elicit physiological responses to biological and pharmaceutical compounds demonstrate potential to create microphysiological systems to study drug responses and disease conditions. Endothelial progenitor cells from coronary artery disease patients (CAD EPCs) were compared to those of healthy adult volunteers (HV EPCs) as the endothelial source within a TEBV comprised of dense collagen gels embedded with human neonatal dermal fibroblasts cultured under physiological fluid flow [1]. The effect of statins on the endothelium-dependent vasodilation of TEBVs cultured with CAD EPCs or HV EPCs was evaluated in the presence of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). TEBV vasoactivity was measured non-invasively using a stereoscope and video display. Exposure to 1 μM phenylephrine caused endothelium-independent vasoconstriction, and 1 μM acetylcholine caused endothelium-dependent relaxation. Vessels with healthy endothelium dilate in response to acetylcholine, while those with injured endothelium constrict. Mature CAD EPC TEBVs exposed to 200 U/mL TNF-α for 4.5 hours constricted in response to 1 μM acetylcholine, indicating endothelial activation; however, exposure to 1 μM lovastatin, atorvastatin, or rosuvastatin for 3 days prior to TNF-α exposure prevented acetylcholine-induced constriction. Mature HV EPC TEBVs exposed to 1 μM rosuvastatin for 3 days prior to activation with 500 U/mL TNF-α for 4.5 hours also dilated with acetylcholine, suggesting protection from endothelial injury. This suggests that microphysiological systems may be utilized to characterize the behavior of cell sources from various donor populations under healthy and inflammatory conditions.
1. Fernandez CE, et al. Scientific Reports 6:21579, 2016.
Fischell Department of Bioengineering, University of Maryland, College Park, College Park, MD.
The placenta serves as a protective barrier, exchanging nutrients and waste between mother and fetus. However, potentially harmful substances can traverse the barrier to the fetus resulting in abnormal development. Our objective is to better understand this process by developing a tissue-engineered model of the blood-placenta barrier (BPB), the interface between maternal and fetal blood, consisting of cytotrophoblasts, syncytiotrophoblast, fetal endothelial cells, and extracellular matrix (ECM). We hypothesize that our tissue-engineered model will determine the directional permeability of physiologic and harmful agents through each BPB layer, as well as their influence on barrier tightness. In our model, BeWo cells, a choriocarcinoma cell line, were encapsulated within a gelatin methacrylate hydrogel at 1,000,000 cells/mL, and then seeded on top of the hydrogel at 100,000 cells/cm2 and fused, mimicking the cytotrophoblasts and syncytiotrophoblast, respectively. Monolayer formation was determined through transepithelial electrical resistance (TEER) testing and immunostaining for ZO-1, a tight junction protein. Cell fusion was demonstrated by fluorescent staining of nuclei, actin, and E-cadherin. TEER values reached nearly 80 Ω·cm2, comparable to literature values. The presence of ZO-1 indicated tight junction formation between cells. Fused cells had more disperse actin and loss of cadherin between nuclei compared to non-fused cells, indicating two distinct cell populations. These data suggest that we can successfully recapitulate the major features of the BPB. Future studies will evaluate glucose transport using a 3D printed perfused system with this model. Broadly, this model will be useful in studying materno-fetal transport of normal physiologic and potentially harmful agents.
An Optogenetic 3D Model of Human Neuromuscula Junctions
Biomedical Engineering, Columbia University, New York, NY.
Neuromuscular junctions (NMJ) are synapses formed between motor neurons and skeletal muscle fibers, and are targets of several motoneuron diseases. Since repeated visualization and manipulation of human NMJs is not feasible, it is necessary to develop in vitro models for the study of NMJ formation and evolution under physiological or pathological conditions.
Here we present a novel microfluidic system for compartmentalized 3D culture of human skeletal muscle cells and hiPSC-derived motoneurons. The muscle chamber features two compliant pillars for myotube attachment and force sensing, and connects to the motoneuron chamber via a channel for axon outgrowth.
For controlled testing of neuromuscular connectivity, hiPSCs were transduced with a lentivirus containing the channelrhodopsin-2 protein (Boyden et al., 2005), a cation channel that opens in response to blue light. Following myotube differentiation (Guo et al., 2014), motoneurons derived from the channelrhodopsin-expressing hiPSCs (Maury et al., 2015) are seeded in the adjacent chamber, and NMJ function is confirmed by muscle contraction in response to motoneuron activation with blue light.
We observe the first signs of NMJ formation as early as 5 days after motoneuron seeding, with improvement in NMJ functionality over the following 2 weeks. Furthermore, our system enables NMJ maturation through repeated optical stimulation, as evidenced by increased muscle force and structural changes in the NMJ.
The platform described here can be easily used for high-throughput drug testing and disease modeling. In particular, the use of hiPSCs enables the use of patient-specific cell lines for the study of genetic diseases and personalized medicine.
Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN.
When metastatic tumors establish in bone, patients experience bone pain, increased risk of fractures, and reduction in mobility. Unfortunately, it is not possible to predict which tumors will induce bone disease or how tumors will respond to therapeutics. The objective of this study is to develop an in vitro bioreactor model of tumor establishment in bone to predict the course of disease progression and response to drug treatments in individual patients. Using microCT imaging in tandem with 3D inkjet printing technology, we have designed 3D Tissue Engineered Constructs (TECs) that recapitulate the mechanical and topological properties of trabecular bone. MDA-MB-231 breast cancer cells are cultured on the TECs to assess the effects of scaffold properties on tumor cell gene expression in response to drug treatment. MicroCT images of three anatomical sites were used to 3D-print wax templates. TECs fabricated from these templates showed no significant differences in bone morphometric parameters (BV/TV, SMI) or elastic modulus compared to the human bone templates. When MDA-MB-231 cells were treated with the integrin inhibitor Cilengitide or the TGF-β receptor kinase inhibitor SD208 in 2D culture, expression of the bone-metastatic factors Gli2 and PTHrP decreased two-fold, however this effect was not apparent when cultured on the 3D TECs. In contrast, treatment with the Gli2 inhibitor GANT58 reduced both Gli2 and PTHrP expression three-fold in both 3D and 2D. In vitro 3D TEC models with bone-like mechanical and topological properties may be an effective high-throughput approach for predicting the tumor cell drug response.
Orthopaedic Trauma Institute, University of California San Francisco, San Francisco, CA.
Approximately 2.2 million orthopaedic procedures worldwide are done annually to resolve complex bone injuries, spinal damage, and skeletal diseases using bone grafts. Though these grafts stimulate direct bone growth, endochondral ossification drives normal fracture repair through an indirect mechanism. A hypertrophic cartilage callus develops to bridge the fracture gap, and serves as a potent regulator of angiogenesis and osteogenesis - two important events in restoring the functional bone. Previously we have shown that cartilage grafts transform into bone when implanted in orthotopic murine models; this study suggests cartilage is an alternative graft to drive regeneration. In order to develop clinically viable cartilage grafts to treat or prevent bone defects, a source of cartilage capable of inducing bone formation is required. Here, we compared the molecular and biochemical composition of cartilage tissues derived from a variety of sources in order to assess their potential angiogenic and osteogenic capacity. We hypothesize that various cartilages present biomolecular cues by coordinating matrix composition and cellular phenotype to control regeneration. This bioactivity is characterized by histology, immunohistochemistry, qPCR, biochemical assays, and spectroscopy (Fourier Transform Infrared Spectroscopy and Mass Spectroscopy). Results reveal striking differences between articular cartilage, costal cartilage, growth plate cartilage, and fracture callus cartilage. Interestingly, two different regions of the costal cartilage also exhibit distinguishable molecular and biochemical compositions, and enhanced osteogenic markers in vitro and in vivo. Together, this work reveals strong translational relevance of different cartilages defined by their potential to drive endochondral ossification, a critical evaluation for developing tissue-engineered grafts.
BME, University of California, Irvine, Irvine, CA.
Fibrin is a major component of the provisional extracellular matrix following injury, and plays a role in both homeostasis and wound healing. Macrophages are dynamic regulators of wound healing, advancing and resolving inflammation. Fibrinogen, the soluble precursor of fibrin, is largely thought to increase inflammatory activation of macrophages through binding to toll-like receptor 4 (TLR4). However, little is known about how the insoluble fibrin matrix regulates macrophage activation. We examined the effects of fibrin and fibrinogen on bone marrow derived macrophage activation. Macrophages were cultured on fibrin gels that were fabricated by mixing fibrinogen with thrombin. Supernatants were collected at 42 hours post seeding and assessed for tumor necrosis factor alpha (TNF-a, an inflammatory cytokine) and interleukin-10 (IL-10, an anti-inflammatory cytokine) secretion levels by enzyme-linked immunosorbent assay (ELISA). We observed that the stimulation of macrophages by soluble fibrinogen significantly enhanced their secretion of TNF-a and moderately enhanced IL-10, confirming previous reports. In contrast, cells cultured on a fibrin matrix elicited very little TNF-a secretion and high levels of IL-10. When cells were cultured on fibrin and further stimulated with fibrinogen, they maintained low TNF-a and high IL-10 secretion. Together, these data suggest that the presentation of fibrin(open) regulates a switch between inflammatory and anti-inflammatory behavior.
Each year, 5.7 million Americans suffer from volumetric muscle loss (VML) from combat injury, trauma or reconstructive surgeries. VML results when severe musculoskeletal damage limits functional recovery. These injuries damage the physical and biochemical cues that direct tissue regeneration. Autologous tissue transfer is the current treatment for VML, but non-functional scar tissue limits functional recovery. There is a need for an off-the-shelf treatment for VML capable of enhancing skeletal muscle regeneration. One promising approach is an implantable scaffold capable of promoting in situ regeneration through host cell recruitment. Our laboratory developed novel fibrin microthreads, a bioactive scaffold resembling the structure of a native muscle fiber. To regenerate highly aligned myotubes, anisotropic surface topography is used to provide cells with contact guidance. We devised a novel strategy to impart aligned surface topography on fibrin microthreads, and hypothesized that these features will promote alignment, outgrowth, and differentiation of cells seeded on the scaffold. MES buffer with pH ranging from 5.0 to 6.0 was used to etch the surface of fibrin microthreads. Threads etched in MES buffer created repeatable, grooved topographical features along the long axis of the thread. Etched threads showed tensile strengths and strains at failure that were comparable to control threads. Mouse myoblasts seeded on etched threads demonstrated initial cellular orientation and evidence myotube alignment. The results of these studies suggest that fibrin microthreads with anisotropic surface topography are a promising scaffold for promoting the formation of aligned myotubes and enhanced skeletal muscle regeneration for treating VML.
A Robust 3D Organotypic Model of Human Mammary Ducts
Chemistry, University of Wisconsin - Madison, Madison, WI.
We've developed a microfluidic mammary duct model of estrogen-receptor positive (ER(+)) breast cancer that attempts to balance the goal of mimicking in vivo conditions with the needs of a robust toxicant screening model. Epithelial cells of the human mammary duct receive signals from a complex mixture of extracellular matrix (ECM) proteins found in its microenvironment. We've modeled the mammary duct using natural (collagen I, collagen IV, fibronectin, or laminin) and synthetic (poly(ethylene glycol) (PEG) hydrogels) ECM materials. Preliminary results in the 3D model suggest that stiffer natural matrices generally improve adherence, confluence, and morphology of mammary epithelial cells lining the ductal geometry. We hypothesized that synthetic PEG hydrogels that present adhesion-promoting peptide sequences are able to mimic the function of natural ECM fibers and reduce inherent variability in the system. A screen of normal and tumorigenic cell lines (MCF12a and MCF7) cultured on 152 PEG hydrogel compositions identified several candidate substrates for use in our 3D ductal model. In 2D, PEG hydrogels that incorporated matrix metalloproteinase-cleavable cross-linkers showed improved adherence compared to PEG hydrogels with a non-cleavable cross-linker, regardless of adhesion-promoting peptide sequence presented. 3D mammary duct models using candidate PEG hydrogel compositions were assessed for the following properties: confluence of epithelial cell monolayer, f-actin organization, and viability. Results from the 3D synthetic model suggest that cleavable cross-linkers are important for recapitulating the in vivo mammary duct microenvironment. These results support our hypothesis that synthetic hydrogels mimic the function of natural ECM fibers and reduce inherent variability in the system.
Orthopaedic Surgery, Stanford University, Stanford, CA.
Hydrogels have been widely employed as artificial matrices to enhance stem cell-based therapy. However, cells are often restricted by the nano-sized meshes of chemical hydrogel networks and cannot change their morphology or reorganize the matrix, which hinders desirable cellular fates and tissue formation. To address these challenges, the goal of this study is to develop a sliding hydrogel with mobile crosslinks and biochemical ligands as a 3D stem-cell niche. The sliding hydrogel exhibits stability comparable to that of chemical hydrogels, but it displays molecular mobility due to its sliding/ mobile crosslinks and biochemical ligands. We successfully synthesized the sliding hydrogels as stem cell niche, with high cell survival. We have confirmed the mobility, indicated by the reversible distribution and the dynamic movement of αCDs tethered with ligands and crosslinks to the sliding hydrogel network. This mobility allows cells to reorganize ligands and to rearrange crosslinks to change the network structure of the hydrogel, which in turn allows them to change their morphology and to form protrusions, which cannot be achieved using chemical hydrogels. Importantly, without changing matrix stiffness, sliding hydrogels support efficient stem-cell differentiation toward multiple lineages including adipogenesis, chondrogenesis and osteogenesis. We therefore conclude that our sliding hydrogel constitutes a versatile material for supporting differentiating stem cells to regenerate a broad range of tissue types. These sliding hydrogels can also serve as a useful tool for elucidating the effects of the molecular mobility of niche cues on stem cell-fate regulation in 3D.
Spatiotemporal Control of Human Cardiac Tissue Using an Optogenetic Platform
The connection between the functional geometry of the heart and its dynamic behavior has informed a number of recent therapeutic advances, such as targeted catheter ablation for refractory arrhythmias. Progress in arrhythmia management requires better characterization of cardiac behavior as a function of geometry, and development of novel spatiotemporal control schemes in biological systems.
We approach these questions in human cardiac constructs through the use of optogenetics, which allows for fine, spatiotemporal control of membrane polarization through light-based actuation. Channelrhodopsin-2 (ChR2) is a cation channel that depolarizes the cell membrane in response to blue light (Boyden et al., 2005) and can be used to excite cardiomyocytes. Halorhodopsin (eNpHR) is a chloride pump that hyperpolarizes the membrane in response to green light (Gradinaru et al., 2008) and can be used to block conduction.
ChR2- and eNpHR-expressing hiPSCs were created through lentiviral transduction. They were differentiated into cardiomyocytes (Burridge et al., 2014) and subsequently formed into cardiac constructs. Simultaneous illumination and imaging of optogenetic cardiac constructs was performed by integrating a projector and camera with custom optical hardware.
Localized conduction blocks of arbitrary spatiotemporal parameters were created through patterned illumination of halorhodopsin-expressing constructs. Similarly, patterned illumination of constructs expressing channelrhodopsin was performed to overwrite existing conduction patterns.
The flexibility of this platform enables systematic study of tissue arrhythmogenicity as a function of tissue geometry, and high-throughput testing of novel spatiotemporal control algorithms for arrhythmia control. The use of hiPSCs allows for the exploration of these topics in disease- and patient-specific contexts.
Preclinical Study of an “Off-the Shelf” Completely Biological AV Graft
1. Lawson, J.H. et al. Lancet
2. McAllister, T.N. et al. Lancet
3. Syedain, Z.H. et al. Tissue Eng Part A
Impact of Peritoneal Pre-Conditioning on Intimal Hyperplasia and Patency of Tissue Engineered Vascular Grafts
Biomedical Engineering, Florida Institute of Technology, Melbourne, FL.
Viability of tissue engineered vascular grafts (TEVGs) is limited by the lack of endothelial function, intimal hyperplasia, and stenosis. In our approach, we used pre-implantation in the rat peritoneal cavity to recruit autologous cells and improve graft patency, and endothelialization. The goal of this study is to determine the impact of initial conduit collagen percentage and peritoneal pre-implantation on the post-grafting response, including structural stability. Conduits were electrospun from poly(ɛ-caprolactone)/collagen blends, characterized, pre-implanted for 4 weeks as an “in vivo bioreactor,” and then grafted autologously into abdominal aortae for 6 weeks. Graft patency, luminal diameter, percent expansion, lipid oxidation, matrix deposition, and cell phenotype were evaluated using ultrasound, HPLC/mass spectrometry, histology, immunofluorescence, and PCR. The preliminary results include that grafts reproducibly expanded with systole/diastole, and percent expansion increased with increasing collagen content (6.8 ± 4.3 to 1.1 ± 0.57%) and decreased with peritoneal pre-implantation. Importantly, H&E stained-sections (n = 2) suggested that peritoneal pre-implantation reduced intimal layer thickness for 100% and 90% PCL. The 75% PCL conduits resulted in a thicker intimal layer than the other blends. Cells in the lumen of the TEVGs expressed vWF. Cells in the graft wall exhibited lower α-SMA expression with pre-implantation. This study suggests that there is a benefit of peritoneal implantation, although all conditions remained patent and endothelialized. Ongoing cell characterization will improve our understanding of this response. Further, we showed that TEVG composition impacts several responses, including: inflammation, cell infiltration, percent expansion, and intimal thickness. The 10% collagen condition has demonstrated some of the best combination of responses.
Seoul National University, Seoul, KOREA, REPUBLIC OF.
Artery and vascular obstructions are known as atherosclerosis. Atherosclerosis causes plaque to form within the vasculature can eventually leads to stroke or heart failure. Currently cardiac stenting is the most effective and a less invasive approach to treat the disease. However in-stent restenosis has been the most complex and chronic side-effect of the stenting treatment. In this study, to reduce the restenosis of stent, we used growth factor secreting stem cell coated stent. To evaluate the re-stenosis and potential therapeutic use of stem cell coated stent, we generated myocardial infarction (MI) model of swine. After 4 weeks of cardiac stenting treatment, we analyzed re-stenosis via Optical coherence tomography (OCT), micro-computed tomography (mCT) and immunostaining. We found controlled release of Hepatocyte growth factor (HGF) by mesenchymal stem cell successfully helped natural re-endothelializatin within the stent, thus reduced the stenosis markedly. Interestingly we also found Vascular endothelial growth factor (VEGF) releasing mesenchymal stem cell did not prevent re-stenosis in swine model. Our finding have significant implications for the future clinical stenting therapy. It appears that HGF secreting mesenchymal stem cell coating on stent would reduce side effects of cardiac stenting with superior re-endothelialization.
Bioengineering, University of California, Merced, Merced, CA.
Embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells and are attractive in vitro models of vascular development, therapeutic angiogenesis, and tissue engineering. However, distinct ESC and iPS cell lines respond differentially to the same microenvironmental factors. Developing improved/optimized differentiation methodologies tailored for, or applicable in a number of distinct iPS and ESC lines, remains a challenge in the field. Utilizing our stage-specific chemically-defined derivation methodology, we examined multiple combinatorial factors for directing endothelial cell (EC) fate in 4 ESC lines including: kinetics, cell seeding density, matrix signaling, as well as medium treatment with vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). By modeling the potentially interacting variables directing Flk-1+/KDR+ vascular progenitor cells (VPC) and VE-cad+ EC, we were able to identify key signals directing VPC in early and EC in late stage of commitments. The results indicate that temporal development in each of these stages is the most significant factor generating the desired cells. The generation of Flk-1+/KDR+ VPC from pluripotent ESC is directed predominantly by high cell seeding density and matrix signaling from fibronectin, while
Critical limb ischemia (CLI) is associated with a 5 year mortality rate in excess of 70% with limited effective therapies. The goal of this study was to determine if pulsed focused ultrasound (pFUS) would enhance homing of mesenchymal stromal cells (MSC) to CLI in an aged mouse model and reestablish perfusion compared to pFUS or MSC alone. CLI model was created by cauterizing the external iliac artery (EIA) on C3H mice (age = 10–12 months). Laser Doppler perfusion imaging (LDPI) was perform of lower extremities to confirm surgery and subsequently performed for weekly for 7 weeks post surgery. Mice were divided into 4 groups: saline (n = 8), pFUS (n = 8), MSC (n = 8), and MSC+pFUS (n = 17). Mice received either 3 consecutive days of saline, pFUS, MSC, or MSC + pFUS starting on day 14 post surgery. pFUS exposures was performed with a 1MHz transducer at 4MPa with DC 5%; in hamstring of ischemic limb. Histology of muscles for vascular cell density was performed at 7 weeks. LDPI demonstrated significant (p < 0.01) differences between (MSC + pFUS) versus saline, pFUS, and MSC groups when treatment was delayed 2 weeks after CLI. Perfusion significantly increased with the MSC + pFUS treatment out to 7 weeks compared to other cohorts. Histological examination of muscle revealed significant increase (p < 0.05) in CD31 + cells and vascular density treated with MSC + pFUS compared to other groups. This study demonstrates that pFUS enhanced homing of IV MSC to targeted muscle resulting in reperfusion and neovascularization in CLI model compared to MSC alone.
Enhanced Cutaneous Delivery of Polymer Complexed Dicer-Substrate Short Interfering RNAs using Carboxymethyl Cellulose-Based Dissolvable Microneedle Arrays for Effective Knockdown of Targeted Gene Expression
Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA.
Short interfering RNA (siRNA) therapeutics have significant potential for treatment of numerous cutaneous conditions by gene silencing. Further, dicer-substrate siRNAs (DsiRNAs) have recently increased potency in RNA interference. However, to date the clinical deployment of DsiRNA therapeutics has been limited by several considerations including lack of patient-friendly delivery technology to enable safe and reproducible administration to patients. Here, we exploited the use of carboxymethyl cellulose (CMC)-based dissolvable microneedle arrays (dMNAs) for effective cutaneous delivery of polymer complexed DsiRNAs. First, polyethylenimine complexed DsiRNA (PEI-DsiRNA) was prepared for effective intracellular delivery. To estimate the cellular uptake and skin penetration, PEI-DsiRNA was labelled with a fluorescence dye TYE563. Subsequently, CMC-MNAs with obelisk-shaped microneedles that incorporate PEI-DsiRNA were created using a micromilling/spin-casting method. The cellular uptake from PEI-DsiRNA and CMC-MNA embedded PEI-DsiRNA were compared after fabrication and subsequent 2 month storage of MNAs at 4 0C using in vitro cultured HEK-293T cells through flow cytometry. The activity of CMC-MNA embedded PEI-DsiRNA was determined through in vitro knock-down in rabbit fibroblasts CRL1414 using western blot. Finally, intradermal delivery of PEI-DsiRNA from CMC-MNAs into living human skin demonstrated cellular uptake capacity of PEI-DsiRNA and DsiRNA target knock-down activity. Specifically, MNA embedded DsiRNA retained knock-down activity for up to two months. It was also shown that CMC-MNAs effectively deliver greater than 85% of the MNA integrated PEI-DsiRNA into human skin in 20 min. Taken together, our results demonstrate the capacity of CMC-MNAs to preserve and deliver DsiRNA therapeutics to skin for effective knockdown of targeted gene expression.
School of Biomedical Engineering Science and Health Systems, Drexel University, Philadelphia, PA.
Alternatively activated “M2” macrophages are commonly believed to function at late stages of wound healing, behaving in an anti-inflammatory manner to mediate the resolution of the pro-inflammatory response caused by “M1” macrophages1. However, the differences between two main subtypes of M2 macrophages, namely interleukin-4 (IL4)-stimulated “M2a” and IL10-stimulated “M2c” macrophages, are not well understood. M2a macrophages are characterized by the secretion of anti-inflammatory cytokines like transforming growth factor-b (TGF-b) and the stabilization of angiogenesis1. However, the role and temporal response of M2c macrophages in wound healing are not well understood. Therefore, we performed whole transciptome sequencing (RNAseq) to identify biological functions and gene expression signatures of M2c macrophages compared to M1 and M2a macrophages plus an unactivated control (M0). M2c macrophages upregulated 28 genes compared to the other phenotypes (using an adjusted p-value cutoff of 0.05), many of which were related to vascular remodeling, including matrix metalloproteinase (MMP)-7 and -8, which were confirmed on the protein level with enzyme-linked immunosorbent assay (ELISA). Surprisingly, temporal gene expression analysis of three publicly available microarray data sets of human wound healing showed that M2c-related genes were upregulated at early times post-injury, similar to M1-related genes, while M2a-related genes appeared later. Taken together with previous work that M2c macrophages promote angiogenic sprouting and clearance of apoptotic cells, these results suggest M2c macrophages function at early stages of wound healing, while M2a macrophages act later. These results will aid in the design of immunomodulatory strategies in regenerative medicine.
1. Spiller K.L., et al. Biomaterials, 2014, 4477–4488.
Plastic and Reconstructive Surgery, Nagoya City Univesity, Nagoya, JAPAN.
Although a variety of synthetic materials have been used to reconstruct tissue defects, these materials are associated with complications such as seromas, fistulas, chronic patient discomfort, and surgical site infection. While alternative, degradable materials that facilitate tissue growth have been examined, these materials can still trigger a foreign body inflammatory response that can lead to complications and discomfort. In this report our objective was to determine the effect of placing a pedicled omental flap under a biodegradable, microfibrous polyurethane scaffold serving as a full wall thickness replacement of the rat abdominal wall. It was hypothesized that the presence of the omental tissue would stimulate greater vascularization of the scaffold and act to reduce markers of elevated inflammation in the patch vicinity. For control purposes a polydimethyl siloxane sheet was placed as a barrier between the omental tissue and the overlying micro-fibrous scaffold. Both groups were sacrificed 8 wk after the implantation and immunohistological and rtPCR assessments were performed. The data showed omental tissue placement to be associated with increased vascularization, a greater local M2/M1 macrophage phenotype response, and mRNA levels reduced for inflammatory markers, but increased for angiogenic and anti-inflammatory factors. From a clinical perspective, the familiarity with utilizing omental flaps for an improved healing response and infection resistance should naturally be considered as new tissue engineering approaches are translated to tissue beds where omental flap application is practical. This report provides data in support of this concept in a small animal model.
1. Thesis Dermatological Surgery Specialization: Gisela Reyes, 0676429-A1, UNAM. 2012.
Electrospun Curcumin/gelatin Blended Nanofibrous Mats Accelerate Wound Healing By Dkk-1 Mediated Fibroblast Mobilization And Mcp-1 Mediated Anti-inflammation
Automated Bioprinting of Customized Tissue Engineered Grafts for Tympanic Membrane Perforation Repair
Fischell Department of Bioengineering, University of Maryland College Park, College Park, MD.
Tympanoplasty, the surgical technique to repair a defect in the tympanic membrane with the placement of a graft, is performed over 55,000 times each year in the United States. The success of a tympanoplasty, however, is critically dependent on the surgeon's ability to measure the shape of the defect, hand-cut an appropriately sized graft, and accurately place it. The goal of this research is to create a 3D bioprinted graft to precisely fit an ear drum defect based on endoscopic imaging. To this end, we designed and bioprinted prototype gelatin-based implants. Puncture testing indicates that the implant can withstand up to 4.67 ± 2.22 kPa of pressure without displacement, which is sufficiently strong for use as a graft to snap into the anatomical defect. Next, we demonstrated that epidermal growth factor (0, 6, 13 μM) can support dose-dependent fibroblast migration rates (40.7 ± 100, 51.8 ± 112, 120 ± 102 μm/day) within the bioprinted gelatin hydrogel, a critical process in wound closure. We then tested our prototype implants in a chinchilla model. Varying tympanic membrane perforations were created (diameter: 2–5 mm) across in both ears in two chinchillas. We captured endoscopic images and used these images to size the customized 3D bioprinted grafts. All four grafts were successfully placed in the tympanic membrane perforations in this pilot study. Next, we will evaluate the effect of EGF and mesenchymal stem cell delivery on tympanic membrane regeneration. Completion of this work will demonstrate the feasibility of utilizing digital imaging directed bioprinting to create custom grafts for ear drum regeneration.
Biomedical Engineering, University of Michigan, Ann Arbor, MI.
Current clinical therapies cannot predictably regenerate bone-ligament interfaces in damaged tooth-supporting tissues. We previously developed a scaffold with 3D-printed, micropatterned features for guidance of osseous and soft tissue formation in vivo [1]. However, bone formation within the scaffold resulted from in vitro cell transduction with adenoviral (Ad) vectors expressing bone morphogenetic protein (BMP-7) prior to implantation. This study investigated a more clinically-relevant gene delivery approach by localized immobilization of AdBMP7 and platelet-derived growth factor (AdPDGF) onto scaffold ‘bone’ and ‘PDL’ regions, respectively, for evaluation in subcutaneous murine extraorthotopic model. The scaffold consisted of 3D-printed polycaprolactone (PCL) compartment for bone formation and poly(lactic-co-glycolic acid) (PLGA) film micro-patterned for guidance of PDL-like tissue formation. Covalent attachment of AdBMP7 and AdPDGF onto PCL and PLGA polymer surfaces was achieved using chemical vapor deposition (CVD)-based polymerization [2]. Control group contained empty Ad-vectors. Prior to implantation, PCL and PLGA scaffold regions were pre-seeded with human gingival fibroblasts and PDL cells, respectively. At 6 weeks, analysis of bone volume (BV) and tissue mineral density (TMD) was performed using micro-computed tomography. Results indicate significant (p < 0.05) bone formation in scaffolds with AdBMP7 (n = 6) relative to empty vector (n = 6), with total BV of 1.67mm3 (0.77mm3 localized to PCL region) and TMD of 215.9mg HA/cm3 (229.5mg HA/cm3, localized bone). These findings support potential of polymer-immobilized delivery of gene therapy vectors for in vivo tissue regeneration.
[1] Pilipchuk SP et al. (2016) Adv Healthc Mater. 5(6):676–87.
[2] Hao J et al. (2016) Adv Mater. 28(16):3145–51.
Institute of Cell & Tissue Engineering, College of Medicine, The Catholic University of Korea, Seoul, KOREA, REPUBLIC OF.
Currently available wound dressings have commonly functions of protecting wound from outside and preventing evaporation of exudate. Therefore, the recovery of wounds may be slow occasionally in severe wounds. Composites with growth factors involved in wound healing can be used as wound tissue engineering scaffolds for accelerating the healing. In the present study, A visible-light-cured glycol chitosan (GC) hydrogel system1 with a controlled release of growth factors (PDGF-BB, VEGF and PDGF-BB/VEGF)2 for accelerating wound healing. The wound healing efficacy was compared with those of Duoderm® using a wound mouse model. For the photo-curing, Computed tomography was performed preoperatively and at months 6 and 12 postoperatively. Defect volume was calculated through Osirix Dicom Viewer (Pixmeo, Apple Inc.). Bone filling percentage was the primary outcome of interest. Secondary outcomes included overall morbidity, surgical time and length of hospital stay.
Injectable Biomaterial Increases the Myogenic Capacity of Transplanted Human Skeletal Muscle Stem Cells in Vivo
Center for Genetic Muscle Disorders, Kennedy Krieger Institute, Baltimore, MD
Cellular therapies for muscular dystrophies have not attained clinical success due to low levels of donor cell survival and engraftment. We describe an injectable biomaterial hydrogel that promotes the viability and myogenic capacity of transplanted human skeletal muscle stem cells. The hydrogel, a 6% w/v 1:3 mixture of hyaluronic acid conjugated to N‐hydroxysuccinimide and decellularized skeletal muscle extracellular matrix, demonstrates optimal gelation properties and cytocompatibility in vitro. Incorporation of a murine monoclonal antibody against myostatin, a negative regulator of myogenesis, yields controlled release of the compound in vitro and increased bioactivity in vivo. Subfascial injection of the hydrogel into murine tibialis anterior muscles demonstrates spreading across the potential space between tendons, covering the inner surface and spreading into deeper tissue prior to degradation at three weeks. The hydrogel also exhibits several immunomodulatory properties positively correlated with myogenesis, including significant increases in CD206+ macrophage infiltration and expression of anti‐inflammatory cytokines. Notably, immortalized human myoblasts transplanted with the hydrogel demonstrated significantly increased levels of survival when compared to saline at 1 week post‐injection. At longer‐term timepoints, human myoblasts transplanted with the hydrogel reoccupy the endogenous progenitor cell niche and form human myofibers that restore expression of the protein dystrophin in immunodeficient dystrophic mice. Overall, this study represents a significant advance in the development of cell therapies for muscular dystrophy.
Ionically crosslinked alginate hydrogels can be permantly deformed and break under mechanical stimulation due to their low elasticity and brittleness. In this study, we engineered highly elastic, biocompatible, biodegradable, and tough hybrid hydrogels resulting from an interpenetrating polymer network (IPN)‐structure of ionically crosslinked alginate and photocrosslinked methacrylated gelatin (GelMA) and examined their utility as bone tissue engineering scaffolds.
IPN structured hydrogels were prepared by mixing two different polymers at an equal volume ratio and crosslinking them: ionically crosslinked alginate and photocrosslinked GelMA. The swelling ratio and mass loss of the hydrogels were evaluated in Dulbecco's Modified Eagle Medium (DMEM). The elasticity of the hydrogels was measured with unconfined cyclic compression testing up to 50% strain. Human mesenchymal stem cells (hMSC) were encapsulated and their viability assessed by Live/Dead staining. To evaluate the capacity of these gels to support osteogenesis, the hMSC/hydrogel constructs were subjected to strain controlled, unconfined, dynamic compression using a bioreactor.
Hydrogel degradation rate was controllable by varying the oxidation degree of alginate while swelling ratio remained relatively constant. While alginate‐only hydrogels exhibited significant permanent deformation after unloading, the IPN‐structured hydrogels fully recovered their original thickness after each unloading. The physical properties of IPN‐structured hydrogels were controllable, and their high elasticity was preserved during degradation. Encapsulated hMSCs maintained high viability in the IPN‐structured hydrogels, and mechanical stimulation enhanced their proliferation and osteogenic differentiation. This hydrogel system may be valuable for biomedical applications that require a biomaterial to fully recover from large strains and long‐term cyclic compression.
Bioactive Peptide Amphiphilic Nanofiber Gels Accelerate Burn Wound Healing: In Vitro and In Vivo Studies
Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA
Teaching and Research Institute, Sirio Libanes Hospital, Sao Paulo, BRAZIL
Materials Science and Engineering, University of Delaware, Newark, DE.
Biomedical Engineering, University of California, Davis, Davis, CA.
Macroporous composite scaffolds composed of bioactive glass (BG) and poly-lactide-co-glycolide (PLG) offer advantages for osteogenic potential and biodegradability compared to homogenous substrates. However, these scaffolds require novel strategies to promote cell adhesion and present instructive cues to associated cells to maximize their potential. We reported that cell-secreted extracellular matrix (ECM) produced by mesenchymal stem cells (MSCs) enhances survival of transplanted cells in vivo. The objective of this study was to examine the synergistic capacity of ECM-coated composite scaffolds to promote cell survival and instruct the osteogenic response of MSCs.
Macroporous composite BG-PLG scaffolds were coated with MSC-secreted ECM at increasing masses (0, 50, or 100 μg). Constructs were seeded with MSCs and cultured for up to 3 weeks in vitro to assess metabolic activity, osteogenic differentiation, and pro-angiogenic potential. MSCs were then seeded onto scaffolds and subcutaneously implanted into immunocompromised mice. Viability and persistence of implanted MSCs were observed via bioluminescent imaging, and scaffolds were explanted after 2 and 6 weeks for histological analysis of tissue formation.
Scaffolds coated with ECM possessed increased Young's moduli compared to uncoated scaffolds, yet treatment did not reduce porosity. The presence of ECM increased metabolic activity and decreased caspase activity while simultaneously stimulating secretion of pro-angiogenic factors by associated MSCs. MSCs on ECM-coated scaffolds exhibited improved persistence over 3 weeks in vivo, and histological analysis revealed more extensive tissue infiltration and osteogenic differentiation within ECM-coated scaffolds This work demonstrates the promise of ECM-coated composite scaffolds on MSC survival and their potential in tissue regeneration.
Biomedical Engineering, Cornell University, Ithaca, NY.
Human-shaped auricles containing bovine auricular chondrocytes (AuCs) successfully formed auricular cartilage following implantation. However, full ears require large (>200×106) numbers of cells, which cannot be achieved through standard monolayer expansion as chondrocytes undergo phenotypic dedifferentiation. Mesenchymal stem cells (MSCs) can differentiate into chondrogenic cells and enhance cartilage generation when co-cultured with chondrocytes. We have shown that human AuCs and MSCs encapsulated in collagen discs in a 1:1 ratio can generate auricular cartilage in vivo that is similar to AuCs implanted alone. Here, human AuCs and MSCs were combined in a 1:1 ratio, encapsulated in 10 mg/mL type I collagen hydrogel at a density of 25×106 cells/mL, and injected into a 6 year-old female human ear mold. Ear constructs were implanted subcutaneously in nude rats for 3 months. Gross-inspection showed that ear constructs developed cartilage-like color and appearance after 3 months, while maintaining major ear structure including the helical rim and lobule. Constructs featured full thickness cartilage generation with no necrotic core. Constructs contracted from initial dimensions of 5 × 3 cm length and width to a length of 3.0 ± 0.4 cm and width of 1.7 ± 0.3 cm. Ears also demonstrated elastic mechanical response to bending and featured positive staining for proteoglycan content and elastin fibers by Safranin-O and Verhoeff's stains. The successful maintenance of ear morphology, development of auricular cartilage tissue, and elastic mechanical properties demonstrates the potential for human AuCs and MSCs to form tissue engineered auricles using 50% fewer chondrocytes, a critical step towards clinical translation for ear reconstruction.
Biomedical Engineering, Illinois Institute of Technology, Chicago, IL.
Mesenchymal stem cells (MSCs) and endothelial cells (ECs) promote angiogenesis and osteogenesis in tissue engineering applications. Appropriate co-culture of MSCs and ECs can result in large volumes of vascularized bone tissue. We have previously shown that pre-vascularized bone tissue engineering scaffolds anastomose with host tissue and improve in vivo vascularization [1]. This study aimed to assess the stability of pre-formed vascular networks within fibrin gels in an in vivo bone model and their effect on bone regeneration. Five spheroids each containing 5000 cells (50% hMSC/50% HUVEC) were encapsulated in fibrin hydrogels and cultured for 3 weeks. Immunofluorescent staining for CD31 showed extensive formation of vessel-like networks in vitro. Gels were implanted in a critical sized cranial defect model in nude rats for 1 week and 4 weeks to assess the stability and perfusion of the implanted vascular networks as well as their effect on initial bone formation. Regenerated bone area was quantified with X-ray imaging. Samples were decalcified and processed for histological evaluation. Tissue structure was evaluated with H&E and Masson's Trichrome stains, and vascularization was assessed by CD31 immunostains and perfused isolectin. Preliminary results indicate that pre-formed vascular networks are stable in vivo and accelerate bone regeneration.
1. Mishra R, et al. Effect of prevascularization on in vivo vascularization of poly(propylene fumarate)/fibrin scaffolds. (2016) Biomaterials 77: 255–66.
Graduate School of Dentistry, Tohoku University, Sendai, JAPAN.
In the preclinical studies, octacalcium phosphate (OCP) and collagen composite (OCP/Col) achieved effective bone regeneration without cell transplantation and exogenous osteogenic cytokines. And it was associated with active structural bone reconstitution. After completion of an investigator initiated clinical study, the sponsor-initiated multicenter clinical trial for the bone defects of oral and maxillofacial region was begun to commercialize OCP/Col as a bone regenerative material from last year. This study investigated the effects of OCP/Col for osseointegration of dental implants in canine infrabony defects. Four male beagle dogs were used. After extraction of bilateral mandibular second and third premolars, four standardized infrabony defects of 5 mm depth around each dental implant site were prepared. After placement of dental implants, 12 of total 16 defects were filled with OCP/Col (4), autologous bone (4), or β-tricalcium phosphate (β-TCP) (4). And the remaining 4 defects were nothing implanted (Untreated). After 3 months, the specimens were fixed and bone-to-implant contact (BIC) was evaluated by histological and histomorphometric analysis. No significant differences between the BIC of OCP/Col and that of autologous bone, whereas the BIC of β-TCP was significantly lower than that of autologous bone. And the BIC of Untreated was significantly lower than those of three groups (OCP/Col, autologous bone, and β-TCP). These results suggest that OCP/Col may improve osseointegration in bone defects around dental implants as well as autologous bone.
Department of Biomedical Engineering, National Cerebral and Cardiovascular Center Research Institute, Suita, JAPAN.
Biomedical Engineering, Wake Forest Institute for Regenerative Medicine, WINSTON SALEM, NC.
Biomedical Engineering, University of Saskatchewan, Saskatoon, SK, CANADA.
Cardiac patch-based myocardium tissue engineering aims to repair myocardial infarction using implantable cardiac patches. Due to low density and weak X-ray attenuation of hydrogels, conventional radiography is unable to visualize hydrogel-based cardiac patches. Thus, it is crucial to develop non-invasive and quantitative imaging techniques for monitoring cardiac patch biodegradation during myocardium regeneration. This study aims to assess imaging performance of state-of-the-art synchrotron-based in-line phase contrast tomography (SIPCT) for visualization and quantitative assessment of an implanted hydrogel-based cardiac patch. A hybrid alginate-fibrin patch was fabricated using a 3D-bioplotter. Left anterior descending (LAD) coronary artery of rats was surgically ligated and the 3D-printed alginate-fibrin cardiac patch was implanted proximally below the ligation. Animals were sacrificed 7 days after implantation; the hearts were excised and embedded in soft tissue-mimicking gel and imaged using SIPCT at the Canadian Light Source synchrotron facility. SIPCT parameters were optimized; phase-retrieved images were reconstructed and then compared to the sample MRI images obtained from a clinical 3T MRI at Royal University Hospital. Contrary to MRI images, SIPCT clearly visualized the implanted cardiac patch microstructure with detailed information including the thickness of alginate strands (∼250 μm), fibrin-filled pore dimensions (∼700 μm), 3D cardiac patch volume and attachment to the myocardium, and the proximal LAD ligation. SIPCT-based quantitative assessments of the patch were consistent with measurements from 3D-bioprinter. SIPCT provided quantitative/qualitative assessments of the implanted cardiac patch, which was poorly detectable by MRI. SIPCT offers a great potential for monitoring implanted biodegradable hydrogel-based cardiac patches for myocardium tissue engineering.
Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC.
Therapeutic effects of stem cells are generally accepted, but their contribution to tissue regeneration remains poorly understood. Current evaluation methods for tissue regeneration require numerous samples and animals to adequately assess the outcomes, however this approach often yields inaccurate results due to the single-time-point information. In this study, we hypothesize that stem cell-based bone regeneration can be tracked and monitored noninvasively by multi-channel fluorescence imaging using highly functional near infrared (NIR) fluorophores. We developed amine-functionalized PCL-ran-PLLA ran-PGA (PCLG) copolymers with three different degradation rates. The PCLG copolymers were conjugated with ZW800-1 fluorescent dye, and placenta-derived mesenchymal stem cells (PDMSCs) were labelled with SCNF fluorescent day. The in vitro data showed that the scaffold degradation and cellularity were quantified in real-time. After fabrication of bone constructs composted of PCLG copolymer and PDMSCs using 3D bioprinting, the bone constructs were implanted in calvarial bone defect (7-mm) in rats. Using NIR fluorescence imaging, the in vivo scaffold degradation were measured in real-time. We are continually tracking the transplanted cell behaviors and scaffold degradation simultaneously by multi-channel fluorescence imaging with highly functional NIR fluorophores. It has been investigated the applicability of noninvasive fluorescent imaging technology for the stem cell-based tissue engineering applications.
1. Choi HS, et al. Nature Biotechnology, 31, 148, 2013.
2. Kim SH, et al. Scientific Report 3, 1198, 2013.
JPKInstruments USA Inc., Carpinteria, CA.
Besides structural and physico-chemical composition, the topography, roughness, adhesiveness and mechanical properties of biomaterials are relevant parameters which strongly affect cell differentiation and tissue formation, and are thus crucial for assessing biocompatibility in the human body. We have developed a multipurpose AFM device which allows comprehensive characterization of these properties and interactions on the nanoscale under physiological conditions and in combination with advanced optical microscopy. Our unique “Quantitative Imaging” (QI™) mode provides several sample properties like topography and the Young's modulus with one measurement. With the CellHesion® technique, the adhesion of a single cell to any substrate can be measured and validated. The NanoWizard® ULTRA Speed technique allows fast AFM imaging of dynamic processes with approx. 1 frame per second. Using QI™, we have characterized the topography and mechanical properties of challenging samples like living cells and tissue sections. The adhesion of single fibroblast cells to different surface modifications could be quantified and their suitability as cochlear implant coatings assessed [1]. Fast AFM imaging revealed collagen type I fibrillogenesis and the formation of the 67 nm D-banding in situ with high spatio-temporal resolution [2]. Here we are presenting an enhanced AFM, making this technique a valuable tool for biomedical research.
[1] Aliuos P. et al., Biomed. Tech. 55,66–68, 2010.
[2] Stamov D. et al., Ultramicroscopy 149, 86–94, 2015.
Microsphere - encapsulated Astrocytes Expressing Ngf Promote Axonal Growth
Astrocytes, the most abundant glial cells in the central nervous system, play a critical role in supporting the normal physiological function of neurons. Recent studies have revealed that astrocyte transplantation can promote axonal regeneration and functional recovery after spinal cord injury. Biomaterial can be designed as a growth-permissive substrate and serve as a carrier for astrocyte transplantation into injured spinal cord. In this study, we developed a method to generate collagen microspheres encapsulating astrocytes by injecting the mixture of collagen and astrocytes into the cell culture medium with a syringe controlled by a syringe pump. The collagen microspheres were crosslinked with 4S-StarPEG. The degradation rate of the microspheres crosslinked with 4S-StarPEG in the cell culture medium containing collagenase reduced compared with non-crosslinked microspheres. The viability of the cells in the crosslinked microspheres was higher than 90%. Astrocytes were transfected with plasmids encoding NGF-ires-EGFP genes by electroporation and were encapsulated in crosslinked microspheres. Then the amount of NGF secreted by the astrocytes was determined by ELISA assay. The level of NGF released into the cell culture medium was higher than that remains in microsphere or in astrocytes. When microspheres encapsulating astrocytes transfected with plasmids encoding NGF-ires-EGFP genes were added into the cultured rat dorsal root ganglion, the axonal growth was significantly enhanced by the NGF secreted by transfected astrocytes. In this study we show that the microspheres can be potentially used as a carrier of astrocytes to promote nerve regeneration in injured neural tissue.
Nerve Regeneration Using Lysophosphatidylcholine and Nerve Growth Factor
Peripheral nerve damage affects hundreds of thousands of people every year. This study tested the effectiveness of using lysophosphatidylcholine (LPC) in combination with nerve growth factor (NGF) to increase the healing rate of damaged left sciatic nerves in female rats. The rats were randomly divided into eight groups: Sham, Right Sciatic, Crush, LPC, LPC-NGF, Crush- LPC, Crush-NGF, and Crush-LPC-NGF (n > 6 per group). The healing of the nerves was measured by monitoring gait, electrophysiological parameters (compound muscle action potential amplitudes and nerve conductance velocities) and morphological parameters (total fascicular area, total myelinated fiber counts, fiber densities, fiber diameters, and g-ratio). Gait and electrophysiological parameters were measured three times a week. Morphological parameters were measured at three weeks and at six weeks. The LPC and LPC-NGF groups were not statistically different from the controls (Sham and Right Sciatic) at either of the morphological time points but were statistically different from the controls for the first three weeks for the electrophysiological parameters and gait. The LPC-NGF group did not differ from the LPC group at any time point for any of the parameters. Crush, Crush-LPC, Crush-NGF, and Crush-LPC-NGF groups statistically differed from the controls at week 3 for all parameters and only in the electrophysiological parameters at week 6. Crush-LPC, Crush-NGF, and Crush-LPC-NGF did not differ from each other or from the Crush group. One dose of LPC and NGF did not prove to be an effective treatment for peripheral nerve damage. Future work will test multiple injections of LPC and NGF.
Polymer Thin Film Device for Immuno-protective Encapsulation of Human Stem Cell Derived Insulin Producing Cells for the Treatment of Type 1 Diabetes
Type 1 diabetes is an autoimmune disease characterized by loss of functional beta-cell mass and subsequent insulin insufficiency that is currently being managed by routine insulin injections which have long-term side effects. Replacement of insulin-producing cells through islet transplantation has been successful in achieving insulin independence for type 1 diabetics but adoption is limited by the need for immunosuppression, limited cell source, and poor viability and performance of transplanted cells. We combine stem cell engineering technologies with immuno-protective biocompatible device engineering to overcome these challenges. Immuno-protective thin films were fabricated by spincoating polycaprolactone onto nanorod templated silicon wafers. Human stem cell derived insulin producing cells (hSCIPC) were encapsulated between the thin films. Glucose stimulated insulin secretion (GSI) was performed by incubating encapsulated cells in buffer containing 2 mM and 20 mM glucose. hSCIPC loaded devices were transplanted between the liver lobes in male NSG mice. hSCIPC viability was measured by bioluminescence imaging and serum was collected from animals for C-peptide measurement. We have demonstrated that hSCIPC encapsulated in immunoprotective devices are functional and exhibit mean GSI of 1.47. Cell viability in devices in vivo for one month was confirmed by bioluminescence imaging. Finally, human C-peptide was detected at 31.46 in mouse serum following one week of transplantation of hSCIPC loaded devices. We have demonstrated the fabrication and use of a biocompatible immuno-protective devices to transplant hSCIPC into pre-clinical animal models. This novel hSCIPC-IP-PCL cell-device system is a compelling translational solution to achieving insulin independence for type 1 diabetics.
Transplantation of Encapsulated Islets in the Presence of OxySite leads to Enhanced Islet Function in a Diabetes Model
Encapsulation of islets in semipermeable immunoisolating devices has been an attractive approach to avert host immune destruction, and potentially mitigate the need for immunosuppression. However, the success of this approach has been marred by the negative oxygen gradients that form within this completely avascular devices. We have developed an in situ oxygen generator termed OxySite. Herein, we sought to evaluate the ability of this biomaterial to sustain islet and beta cell viability and function in an animal model of diabetes. For this purpose beta cells or pancreatic islets were encapsulated within agarose hydrogels containing a blank PDMS disk (control) or with an OxySite disk (treated). Constructs were transplanted into the intraperitoneal (IP) space or the omental pouch (OP) of chemically induced diabetic rodents. Following the implantation of constructs containing beta cells embedded in an agarose hydrogel with an OxySite disk into diabetic mice, diabetes reversal and sustain normoglycemia was observed. This result was counter to the control group (blank PDMS), which failed to restore normal blood glucose levels. Furthermore, encapsulation of rat islets with an OxySite disk placed in the OP of diabetic rats resulted in 60% of the animals reverting to stable normoglycemia compared to none (0%) of the control grafts. In conclusion, incorporation of OxySite within encapsulating devices containing pancreatic islets leads to enhance cell viability and function in an in vivo model of diabetes. Thus, OxySite has the potential to heighten the performance of encapsulated cell based therapies.
The Central Role Of Talin-1 In Cancer Cell Extravasation In Metastases Dissected Through A Human Vascularized 3D Microfluidic Assay
IRCCS Istituto Ortopedico Galeazzi, Milano, ITALY.
• Significantly affected morphology and proliferation (P < 0.001) • Significantly reduced adhesion efficiency (P < 0.001) • Statistically decreased TEM (MDA-MB231 P < 0.05, HT1080 P < 0.01) • Statistically reduced migration in 3D matrix (P < 0.001)
[1] Cell1 47:275–92 (2011).
[2] Proc Natl Acad Sci USA 112:214–9 (2015).
Bioengineering, University of Pittsburgh, Pittsburgh, PA.
Cancer biology tools to recapitulate the microenvironment have largely remained unchanged: collagen gels and Matrigel have remained the gold standard for the past 50 and 33 years respectively. The stimulus for the present study was to develop a new cancer biology technology: disease-specific extracellular matrix (ECM) hydrogels.
A novel approach was used to develop ECM hydrogels from decellularized normal, inflammatory, and neoplastic adenocarcinoma (EAC) esophageal tissue and the distinctive effect of these hydrogels upon macrophage activation state was investigated. Matrix-bound nanovesicles (MBVs) were isolated from the ECM materials, which have been shown to rapidly and markedly affect cell phenotype. Important unanswered questions are: 1) What is the profile of extracellular miRNA contained within the ECM via MBVs to drive EAC progression? 2) How does diseased ECM activate an important cell type in an inflammatory driven cancer, the macrophages, via dynamic reciprocity?
The three types of ECM showed distinctive fiber networks by SEM and protein profiles by Silver Stain/SDS-PAGE. Inflammatory and neoplastic ECM activate macrophages (human THP1) to a dual “pro-inflammatory” (TNFalpha+) and “immunomodulatory” (IL1RN+) state, with increasing expression as ECM tumorigenicity increased by qPCR/ELISA. Top differentially regulated MBV miRNAs were notably related to epithelial-mesenchymal transition, a known mechanism of EAC progression; cancer; and macrophage activation by small RNA sequencing/pathway analysis, suggesting the ECM's direct role to dynamically instruct cell behavior.
In conclusion, a novel ECM hydrogel “progression series” was developed. A better understanding of diseased ECM MBV miRNA cargo and disease-specific macrophage activation will guide EAC regenerative medicine strategies.
Institute for Regenerative Medicine, Wake Forest Baptist Medical Center, Winston-Salem, NC.
Precision medicine - identifying treatments for patients based on their tumor genetic profiles - has gained significant traction. However, in practice, even after identification of key mutations, oncologists are often left with several drug options, suggesting that systems are necessary for prediction of effective treatments. Colorectal cancer (CRC) organoids were created by encapsulating CRC cells (Caco2 and SW480 - WT; HCT116 - KRAS MT; and HT29 - BRAF MT) in hyaluronic acid and gelatin hydrogels in microfluidic devices. Each type of CRC organoid was then subjected to a panel of clinical CRC drugs: 5-FU or oxaliplatin (1st line drugs effective in WT tumors), Tramatinib (an EGFR pathway drug effective in KRAS MT tumors), and sorafenib or regorafenib (EGFR pathway drugs effective in BRAF MT tumors). Following 48-hour treatments, organoids were assessed by MTS assays. There were clear differences in drug responsiveness that correlated with EGFR genetic states. Both Caco2 and SW480 organoids (WT) were particularly sensitive to 5-FU, and less so to the other drugs. HCT116 organoids were particularly sensitive both to 5-FU and to sorafenib. HT29 organoids were in general more resistant across the board, but displayed a trend of slight sensitivity to regorafenib. The results described here demonstrate that 3D tumor organoids can be successfully employed for screening drugs based on tumor genetic profiles. We are now transitioning to patient biopsy-derived tumor cells, and envision that we can screen potentially effective drugs using tumor-on-a-chip systems customized to individual patients, determining the treatments that are the most effective and safest to administer.
Mechanical Engineering, McGill University, Montreal, QC, CANADA.
3D cultures have been widely applied to cancer cell research and tissue regeneration. Using 3D bioprinting technologies we have developed a method to embed tumor cells and fibroblast cells into alginate/gelatin hydrogels to print heterogeneous model mimicking the in vivo tumor microenvironment. Cancer and fibroblasts cells (MDA-231 and IMR-90, respectively) were independently mixed into an alginate/gelatin hydrogel and subsequently printed using an extrusion-based bioprinter and crosslinked before culturing for 30 days. The samples were analyzed mechanically, physically, and biologically. The mechanical properties of the hydrogel were tuned in an effort to match them to the tumor microenvironment creating an internal viscosity ranging from 5 Pa·s to 1 Pa·s and an exterior shell with stiffness of 10 kPa to provide essential integrity. The high porosity of the hydrogel allowed the cell proliferation as well as cancer spheroid-like formation after 7 days, increasing their sizes during time. After 21 days of cultures, IMR-cells can be seen migrating to the cancer cells region and surrounding the spheroids mimicking stromal symbiotic tumorigenesis. The matrix exhibits high mechanical stability and the cancer cells formed spheroid morphology during culture yielding insights into tumorogenesis in 3D microenvironments. We have successfully used 3D bioprinting to create heterogeneous tumorigenesis models with high-throughput, low cost, and high reproducibility as a more realistic alternative to traditional cell culture and small animal tumor models.
Multiple Organ-on-a-Chip Platform for Metastasis Dynamic Studies
Wake Forest Institute for Regenerative Medicine, Winston Salem, NC.
Cellular phenomena involved in cancer metastasis have been studied under the “seed and soil” hypothesis or defined by anatomical and geometric routing of the lymphatic or blood vasculature [1]. Advances in modular and microfluidic layouts of organ-on-a-chip (OC) platforms, alongside integrated extracellular matrix (ECM) based scaffolds, help to recapitulate in vivo environmental composition and customized circulatory system organizations [2–3]. In the case of colorectal cancer (CRC), tumor cells predominantly metastasize to the liver, likely due to proximity lymphatic drainage [3]. However, are there additional characteristics of liver tissue (cells, ECM, signaling molecules, etc) that prime the liver as a site of CRC cell metastasis? In situ patterned hyaluronic acid hydrogel organs; HepG2/C3A (liver), A549 (lung), HUVEC (endothelium) and HCT 116 (colon) cells, were set up in a multi-organoid equidistant and “equi-geometrical” microfluidic perfusion chambers. We achieved the fabrication of a highly precise multiple OC device with >500 μm in situ patterned organs embedded in a 3D ECM based scaffold. The system provides in vitro mimicry for a week of in vivo environmental composition and physical flow. Future studies will assess the effects of the fluidic network design versus tissue environmental factors on colorectal cancer metastasis.
1. Skardal, A. Biomaterial. 33: 4565–4575. 2012.
2. Oleaga, C. Scientific Reports. 6:20030. 2016.
3. Skardal, A. Biotechnol Bioeng. 9999: 1–13. 2016.
The use of lab animals has substantial translational deficits for predicting human outcomes in a variety of medical disciplines, one reason for a growing interest in using dogs with naturally occurring diseases to better predict therapeutic outcomes prior to entering human clinical trials. The use of biomaterials and regenerative cellular techniques have been used for decades in veterinary medicine, often giving critical insight into clinical performance and outcomes.
Optimizing Soft Tissue Management And Spacer Design In Caprine Segmental Bone Defects
Biomedical Engineering, Cleveland Clinic, Cleveland, OH.
Amniox Medical, Atlanta, GA.
Osteoarthritis (OA) is a progressive degenerative joint disease and to date, no disease modifying osteoarthritis drug exist. Amniotic membrane and umbilical cord products have been used clinically in several diseases due to their anti-inflammatory and anti-scarring properties. In the present study, we sought to evaluate whether a particulate amniotic membrane and umbilical cord (AM/UC) matrix could aid in attenuating disease progression. Lewis rats underwent medial meniscus transection (MMT) to induce OA. Two weeks after surgery, animals received intra-articular injections of either 50 μg/μL or 100 μg/μL particulate AM/UC or saline control and were subsequently euthanized 1 or 4 weeks later. Cartilage degeneration was assessed using both histological scoring methods and equilibrium partitioning of an ionic contrast agent-microcomputed tomography (EPIC-μCT). EPIC-μCT analysis demonstrated that overall cartilage destruction was attenuated, with a significant increase in both cartilage thickness and volume as well as a significant decrease in total lesion area in animals injected with either dose of particulate AM/UC at one week, but only a high dose at four weeks post-injection. OARSI histology scores of tibial sections corroborated EPIC-μCT results, with overall joint destruction in animals injected with either dose of AM/UC tissue less than saline injected control animals at one-week post-injection, but only high dose AM/UC injected animals maintaining less overall joint destruction by 4-weeks post-injection. Intra-articular injection of particulate AM/UC tissue attenuates cartilage degradation in a rat MMT model of OA, suggesting that it may be able to slow joint destruction in patients with OA.
Local vs. Intravenous Injections of Skeletal Muscle Precursor Cells in Nonhuman Primates with Acute or Chronic Intrinsic Urinary Sphincter Deficiency
Multi-Scale Evaluation and Comparison of Common Muscle Injury Models
Bioengineering, University of California San Diego, La Jolla, CA.
Sigilon Inc., Cambridge, MA.
Bioengineering, Hanyang University, Seoul, KOREA, REPUBLIC OF.
A type 1 diabetes mellitus, an autoimmune disease, results from the destruction of the beta cell in the pancreas. Ideally pancreatic islet transplantation is a promising remedy because of its dynamic regulation of blood glucose level of patients. However, transplanted islets are rapidly eliminated due to host's immune responses. Therefore, patient should administer immunosuppressive agents such as tacrolimus to protect transplanted islets, which could induce several side effects to patients. It was reported that a high-mobility-group-box 1 (HMGB1) protein could play a crucial role in transplanted islet rejection. Therefore, it is possible that attenuation of HMGB1 activity can strongly contribute to successful regulation of immune reactions. Here we modified the islet surface with glycyrrhizin-chitosan bioconjugate to modulate the release of HMGB1 protein. We confirmed that the glycyrrhizin-chitosan bioconjugate could be stably immobilized to islet surface without any damage. Then xenogenically implanted the glycyrrhizin-conjugated islets could cure the blood glucose levels of the mice within normal range more three weeks without any immunosuppressants. For control islet implantation, they were rapidly rejected within a week. Currently, we are doing synergistic effect of glycyrrhizin-chitosan bioconjugate when accompanied with low-dose tacrolimus, a well-know immunosuppressant in islet transplantation. Collectively, we demonstrated the feasibility of glycyrrhizin-chitosan bioconjugate for successful outcome of islet transplantation.
Department of Biomedical Engineering, Drexel University, Philadelphia, PA.
A major challenge in engineering tissues for regenerative medicine is forming functional, perfusable vascular networks required for tissue survival in vivo. Macrophages, the primary cells of the inflammatory response, are known to be highly angiogenic, but exist on a spectrum of functionally diverse phenotypes; to date, their distinct roles in blood vessel development are unclear. The objective of this work was to elucidate the impact of macrophage phenotype on tissue vascularization, with the hypothesis that M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages act sequentially to promote blood vessel sprouting and anastomosis. Porous collagen scaffolds (Gelfoam, Pfizer) were pre-seeded with human adipose microvascular endothelial cells expressing tdTomato together with human adipose-derived mesenchymal stem cells as a model of blood vessel network formation. Unactivated THP-1 monocyte-derived macrophages (M0) were added at varying times during vessel growth, and the effects on network development in vitro were monitored over time using confocal microscopy. Interestingly, delayed addition (day 6) of M0 macrophages supported construct vascular development to a greater extent than early seeding times (days 0, 3). To delineate the role of macrophage phenotype, we characterized the effects of activated M1 and M2a macrophages on our 3D model of vascularization. Confocal microscopy revealed that both phenotypes induced changes in the formation of tissue vasculature. Most notably, while the individual phenotypes appeared to promote vessel sprouting, sequential M1 and M2a activation caused vast construct vascularization relative to control constructs without macrophages. Ultimately, this work will aid in the design of immunomodulatory tissues that promote vascularization and integration.
1) Krampera, et al. Cytotherapy. 2013.
2) Marklein, et al. Stem Cells. 2016.
Lipid Metabolism Modulates Immunomodulatory Properties Of Mesenchymal Stem Cells
Italian Institute of Technology, Genoa, ITALY.
Biomedical Engineering, Johns Hopkins University, Baltimore, MD.
Biologically-inspired materials have the potential to engineer target cells in vivo. We are particularity interested in designing polymeric particles that can mimic attributes of immune cells, such as antigen-presenting cells. These artificial antigen presenting cells (aAPCs) can be composed of poly(lactic-co-glycolic acid) (PLGA) with surface conjugated biomimetic proteins and designed to be biodegradable and safe while also capable of driving a cytotoxic T cell response as an immunotherapy. We have found that the interaction of an artificial polymeric particle's surface with the surface of a biological cell can be highly dependent on the physical properties of the particle including its size and shape. Through the use of an automated multidimensional film-stretching device, we can synthesize anisotropic particles on both the microscale and the nanoscale. We find that oblate ellipsoids, with a larger radius of curvature, lead to more potent aAPC activity than prolate ellipsoids, which in turn are more effective than spherical particles. Microspheres, which have a larger radius of curvature than nanospheres, are similarly more effective. Anisotropic particle shapes were determined to be especially critical for nanosized aAPCs, where they boost cytotoxic T Cell proliferation, reduce non-specific cellular uptake, and lead to longer blood circulation half-life. When evaluated in mouse models of melanoma, anisotropic aAPCs are found to enhance survival. Further, when PLGA aAPCs are combined with the checkpoint inhibitor anti-programmed death 1, we find a synergistic effect and improved efficacy at treating melanoma. This immunoengineering approach could be an enabling technology for cancer therapy and regenerative medicine.
Histogen Inc., San Diego, CA.
We have developed a bioengineered human extracellular matrix (hECM) to promote bone and cartilage regeneration for orthopedic indications. The insoluble collagen and sGAG-containing hECM is produced in vitro by human fibroblast cells grown under hypoxic and serum-free culture conditions using a controlled bioreactor system. Following initial experiments demonstrating regeneration of osteochondral defects in rats and rabbits, the hECM was evaluated in a large animal goat model of articular cartilage repair. Critical sized, full-thickness ostoechondral defects were surgically created in the medial femoral condyle of skeletally mature female goats. Defects were treated with hECM, and at 4 and 12 months post-treatment, animals were euthanized and the defect sites were photographed and evaluated by biomechanical indentation testing, micro-computed tomography imaging and histology. Macroscopic observation showed the hECM-treated defects exhibited substantial filling with a tissue resembling articular cartilage. The untreated, control defects displayed deep defects with minimal closure and evidence of erosion and collapse at the wound margins. Histology and imaging revealed that the hECM supported new, integrated subchondral bone growth and overlying hyaline cartilage composed of chondrocyte-filled lacunae surrounded by a proteoglycan-rich extracellular matrix. The untreated defects were characterized by voids or filled with undifferentiated, fibrotic tissue. The results of this study demonstrate the potential of hECM as a human-source material for the treatment of bone and cartilage defects in clinical orthopedic applications.
A Wireless Sensor for Real-Time Measurements of Mechanical Strain in a Rodent Model of Segmental Bone Regeneration
Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA.
Biomedical Engineering, University of North Carolina Chapel Hill and North Carolina State University, Raleigh, NC.
(1) Bodle et al. Stem Cells. 2016 Jun;34(6):1445–54.
Department of Orthopedic Surgery, Columbia University Medical Center, New York, NY.
Damages in the pelvic floor muscles often cause dysfunction of the entire pelvic urogenital system. Current treatments for the injury include physical therapy, autologous muscle flap transfer, and surgical interventions using synthetic and biological materials. However, none of them entirely address the problems associated with long-term restoration of normal anatomy and function in the injured pelvic floor muscle system. Engineering of functional muscle tissue constructs may provide a solution to this unmet medical need. However, the current muscle engineering techniques are limited by the ability to build sizable constructs with timely innervation for successful graft survival. To this end, this study aims to fabricate and optimize volumetric 3-D bioprinted skeletal muscle constructs with innervation capability for repairing pelvic floor muscle injuries. Bioprinted skeletal muscle constructs that mimic native skeletal muscle organization were fabricated by using a ‘bioink’ formulation consisting of fibrin-based hydrogel containing human muscle progenitor cells, and muscle tissue formation capacity was investigated in pelvic floor muscle injuries in rats. Our results demonstrate that the bioprinted cells within the engineered skeletal muscle constructs are able to maintain their viability and formed muscle fibers in vivo, suggesting that the engineered muscle constructs may contribute to the restoration of pelvic floor muscle function anatomically and functionally.
Characterization and Modification of 3D Silk Scaffolds for Kidney Tissue Engineering
Biomedical Engineering, Tufts University, Medford, MA.
Silk fibroin is a desirable biomaterial for tissue regeneration applications due to its expansive range of aqueous fabrication techniques, biocompatibility, long term stability and ease of chemical modification. For the purposes of kidney regeneration, we have evaluated the physical and chemical properties of bulk silk scaffolds. The physical and chemical structure of the silk scaffolds were characterized using scanning electron microscopy and Fourier transform infrared spectroscopy, respectively. To add tunable chemical functionality to silk scaffolds, a two-step process was performed to modified silk surfaces. First, avidin (pI 10.5) was adsorbed to the silk surface (pI 4.2) via electrostatic interaction followed by avidin/biotin interaction with target molecules. The feasibility of this concept was first demonstrated using biotinylated horseradish peroxidase. We also demonstrated binding and cleavage of a biotinylated MMP-cleavage peptide using type I collagenase and fluorescence. Future work will utilize an MMP-cleavage peptide as a linker for tissue-state dependent release of molecules to induce cell differentiation. Finally, though silk can be degraded by numerous physiological enzymes (i.e. MMPs, chymotrypsin), we compared the enzymatic stability of silk scaffolds to commonly used biologically derived scaffolds (i.e. collagen and fibrin) used for tissue regeneration. The silk scaffolds were significantly more resistant to proteolysis than commonly used biologically-derived materials. This is a key feature for relatively stable systems are needed, yet the biomaterial is fully degradable, for rebuilding a kidney. The biomaterial persistence with retention of slow degradability and ease of modification will be utilized for long-term in vitro tissue development and translation in vivo.
Tissue Engineering of Tracheas in a Rabbit Model
Baylor College of Medicine, Houston, TX.
Patients who have long-segment tracheal stenosis or atresia have a mortality rate of 77% and a 100% respectively [1]. Using a scaffold-free methodology to produce de novo tracheal replacement units, termed “neotracheas” [2], 31 White New Zealand rabbits were implanted with neotrachea constructs fabricated from autologous skin and cartilage tissue. Chondrocytes were harvested from rabbit ears, expanded and formatted into sheets as described [2]. To fabricate a neotrachea, a silicone tube (6.0–7.5 mm dia) was wrapped with platysma muscle, a cartilage sheet and a skin graft and implanted proximal to the trachea. At 8–12 weeks post-implantation, segmental reconstruction was performed. Of the 31 rabbits implanted, 2 were used to assess t-tube tolerance, 1 was implanted with buccal mucosa, and 6 were used to assess skin viability. Eight implants had displaced cartilage rings due to handling errors, and 3 were too weak for segmental reconstruction. The remaining 11 were used for segmental reconstruction. Histologic examination showed that the lumen was patent and viable epithelium lined the implants. The results demonstrated that autologous cells can be used to construct a neotrachea with a viable epithelial lining and a ring of cartilage providing structural support. Early difficulties with the formation of structural sound cartilage rings were attributed to disruption of the implant due to handling. Future studies will focus on the fabrication of a mucosal epithelial layer to provide a natural barrier to infection and particle clearance.
1Fuchs JR et al. (2002) J Pediatr Surg.
2Weidenbecher et al. (2009) Laryngoscope.
The development of a new medication/drug is a cost and time intensive process, attributed to process inefficiencies and poor predictability in preclinical phase. Recent trends have swayed to a “learn and confirm” process rather than the traditional multi-phased development process. However, there is currently no systematic validation approach that bridges the gap between experimental data, computational simulation, and patient results by examining metabolic profiles. There is a lack of hepatic 3D models that could help evaluate hepatotoxicity in vitro. In this regard, first we evaluated the metabolism of the popular drug, acetaminophen, using kinetic data from literature in a computational simulation. Metabolic profiles of acetaminophen and metabolites from simulation were compared with the metabolic profiles of patients to assess the validity of those data. Secondly, we used 3D chitosan-gelatin porous structures colonized with either hepatocytes alone (HepG2, HepaRG) or in presence of various ratios of endothelial cells. Experiments were performed using acetaminophen at conditions used in simulation for comparison purposes. Acetaminophen conversion and yield of metabolites were analyzed and compared with simulation/clinical data to determine which combination demonstrated greater organotypic behavior. Evaluation of multiple kinetic and transport constants via simulation led to metabolic profiles that matched well with those of patients found in literature. Experimental results showed improvements in cocultures on 3D scaffolds, yielding acetaminophen metabolic profiles that more closely matched patient profiles. In summary, our method aides in development of 3D liver models and data validation.
Modeling of VTL C3A Cell-Secreted Protein Dosage During ELAD Treatment for Acute Alcoholic Hepatitis
Research and Development, Vital Therapies, Inc., San Diego, CA.
The ELAD System, an investigational cell-based liver treatment for severe acute alcoholic hepatitis (sAAH), recirculates ultrafiltrated plasma (UF) through four hollow-fiber cartridges containing metabolically-active human-derived VTL C3A cells. Estimating treatment delivery of human proteins with a human cell-based product presents unique challenges. The study objective was to evaluate methods for estimating dosage of proteins related to our understanding of mechanism of action (MOA) during ELAD treatment. ELAD C3A cell cartridge conditioned media samples from single-pass perfusion production systems were collected and immunoassayed for concentrations of interleukin-1 receptor antagonist (L-1Ra), alpha-1-antitrypsin (AAT), gelsolin, amphiregulin (AR), transforming growth factor alpha (TGFα), and vascular endothelial growth factor (VEGF)–analytes associated with anti-inflammatory, hepatoprotective, and/or anti-fibrotic responses. Mean ELAD treatment time (94.3 h), UF recirculated volume (286 L) and VTL C3A cell glucose consumption rate (2.6 g/h) from our recent phase 3 clinical study (VTI-208) of the intent-to-treat (ITT) sAAH population were modeled against manufacturing steady-state production rates of the selected proteins to yield estimated delivered doses of 76–222 μg IL-1Ra, 864–2,517 mg AAT, 196–572 mg gelsolin, 29–83 μg AR, 45–131 μg TGFα, and 11–33 mg VEGF. The estimated protein dose was 2.7-fold higher when using UF volume in the modeling compared to treatment time and glucose rate. These data assume that values measured in manufacturing conditioned medium would be similar to those delivered to a treated subject and do not account for variations in secretion levels due to factors within individual subject's plasma; however, this will be addressed in the on-going clinical study.
Rotator cuff tears (RCTs) are a common orthopaedic disorder in the US with 70,000 repairs performed annually. Repair of RCTs with suture techniques fails 20–57% due to lack of enthesis regeneration. Our lab has demonstrated enthesis regeneration in a sheep ACL repair model. We hypothesized that engineered tendon grafts (ETG) will also be effective for RCTs repair in a full-thickness RCT model. Bone marrow stromal cells from adult male sheep were used to fabricate ETGs. The ETGs were used as an underlay in combination with a double row suture repair (N = 12) and compared to a double row suture-only (SO) repair (N = 11) in female sheep. After 6-mo, both the repaired and contralateral shoulders were explanted and radiographed. The infraspinatus tendon and muscle was isolated for biomechanical testing, histology, and PCR. The mean tangent modulus of the contralateral, SO, and ETG repair was 106 ± 24 MPa, 21 ± 7 MPa, and 33 ± 16 MPa, respectively. The modulus of ETG repaired tendon was 11% higher compared to SO repair, but not significantly different (p = 0.33). Histology of ETG repaired enthesis showed graded zones resembling native enthesis. The enthesis of SO repair did not have graded zones, indicating poor regeneration. In summary, utilization of ETG for repair is capable of enthesis regeneration and could potentially be used for clinical repair of RCTs.
Evaluation of In-Vitro Generation of Submandibular Salivary Gland
Biomaterilas Department, OKAYAMA UNIVERSITY, OKAYAMA, JAPAN.
A significant challenge to tissue regeneration is the requirement of the regenerating tissue to be adequately vascularized and innervated. These considerations are essential for volumetric muscle loss injuries, as the current standard of care for these injuries is limited by an incomplete restoration of muscle strength or function as a result of incomplete re-innervation of the implanted tissue. In this study, we hypothesize that endothelial cells present in nascent vascular networks secrete factors that will enhance axonal growth. Specifically, we investigated how the axonal growth of chicken or rat dorsal root ganglia (DRG) explants or human induced neural stem cells (hiNSCs) were affected by co-culture with human umbilical vein endothelial cells (HUVECs). Axons grew toward cell clusters of HUVECs, and axon length was significantly longer in HUVEC co-culture conditions with respect to DRGs or hiNSCs grown in monoculture on PDL. Axons were significantly longer when grown on HUVECs that were seeded at lower densities. A cytokine array was performed to identify factors secreted by HUVECs that may support axonal growth, and based on these findings brain-derived neurotrophic factor was neutralized. There was a significant reduction in axonal growth when incubated in HUVEC-conditioned medium and in direct co-culture with HUVECs with neutralizing antibodies. These data suggest that HUVECs secrete neurotrophic factors in physiologically relevant concentrations that significantly enhance axonal growth, and that directing the angiogenic response in regenerating tissues may encourage re-innervation. These findings will ultimately be used in future studies to generate complex 3D model systems to study these processes.
Disease Modelling with Mesenchymal Stromal Cells and Induced Pluripotent Cells Reveals an ER Stress Related Cellular Pathology in Patients with Familial Osteochondritis Dissecans
REMEDI, National University of Ireland, Galway, Galway, IRELAND.
Familial osteochondritis dissecans (FOCD) is a rare orthopedic condition with autosomal dominant pattern of inheritance. Patients with FOCD can develop short statue, polyarticular, and early onset osteoarthritis (OA). FOCD has been reported in over 50 families. Despite much speculation, the definitive causation associated with FOCD development still remains unclear.
Our present study focuses on a FOCD family from northern Sweden (NS) with a known mutation on aggrecan gene, which results in a Valine-Methionine replacement in the G3 aggrecan C-type lectin domain (CLD). The objective of this project was to investigate the cellular pathogenesis of FOCD-NS using disease specific stem cell derived FOCD-NS models.
To this purpose, we isolated patient bone marrow-mesenchymal stromal cells (BM-MSCs) and generated induced pluripotent stem cells (iPSCs) from patients' skin fibroblast. Disease phenotype was examined during chondrogenesis of BM-MSCs and iPSCs.
Our findings revealed that the animo acid replacement in the aggrecan CLD triggered unfolded protein response (UPR) in rough endoplasmic reticulum (rER) of FOCD-NS chondrocytes. This further leaded to aggrecanase in patient samples. Disturbed cell function gave rise to defective extracellular matrix (ECM) production and assembly. Studying the ECM composition of patient samples showed that biological markers associated OA were highly unregulated in later developed chondrogenic nodules.
Our study discovered a cellular pathogenesis of FOCD-NS by studying the chondrogenesis of FOCD-NS stem cell models. Further application of this novel approach will reveal more mechanisms of FOCD-NS and sheds further light on the critical role of UPR and aggrecanase in pathology of early onset OA.
Institute for Regenerative Medicine, Wake Forest Baptist Medical Center, Winston-Salem, NC.
Glioblastoma (GBM) is a cancer of the brain that despite maximal therapy, infiltrates normal brain, are surgically incurable, and universally recur in most patients. Unfortunately, few 3D in vitro models of GBM exist that faithfully recapitulate the tumor architecture and microenvironment seen in vivo in humans. To address this limitation, the cell lines U138 and U373 were formed into spheroids were encapsulated in a hyaluronic acid/gelatin hydrogel that better mimics the makeup of brain extracellular matrix than most biomaterials. We studied tumor growth, migration distances and velocities, proliferation, and viability as effects of conditions including Wnt signaling manipulation, different glucose concentrations, and different matrix stiffness values using MTS assays, microscopy, and confocal imaging. WNT modulated environments lead to marked decreased migration in U138 spheroids with change in phenotypical appearance of the cells. In different glucose concentrations, U138 spheroids had reduced proliferation but increased migration in no glucose environment compared with high glucose and low glucose. In different stiffness environments the notable observation was that U373 cells had increased proliferation in stiffer environments, while U138 cells did not. Our results demonstrate that this platform is an effective tool to study environmental manipulations of the GBM tumor microenvironment, drug testing, and subsequent changes in disease progression kinetics. We are currently working to adapt this system for use in biomarker identification and personalized precision medicine by employing patient-derived cells.
Bioengineering, Rice University, Houston, TX.
Biohybrids, a combination of naturally derived tissue and polymers, can both mimic the complexity of the natural ECM, as well as incorporate tools for manufacturing and maintenance of shape and strength. A material engineered to replace complex, compliant tissue as is found in the cardiovascular environment should strive to both mimic the native mechanical properties as well as encourage regrowth and remodeling as fast as possible. We have developed a 3D printable and degradable material by combining polyethylene glycol diacrylate (PEGDA) and homogenized pericardium tissue. This study aimed to 1) characterize the homogenized pericardium and confirm its inclusion in the hydrogel network, 2) design a specific resin to 3D print scaffolds using digital light processing (DLP) and 3) evaluate the biohybrid gel for cell viability, differentiation and remodeling of the scaffold. Successful homogenization of the tissue component resulted in a powder with confirmed collagen, elastin, and glycosaminoglycan content using Western blotting and component specific assays. Crosslinking with visible light reduced the primary amine concentration in the pericardium by 96%. This result together with compression testing and FTIR analysis confirms successful inclusion of the homogenized tissue into the biohybrid gel. Specific shape formation of the biohybrid using DLP 3D printing can be accomplished with 1mm resolution. Human mesenchymal stem cell (hMSC) viability and preferential growth when compared to PEGDA hydrogels has been evaluated using live/dead imaging. This work supports the further investigation to 3D print specific shapes that can be used to study hMSC differentiation and cell-initiated remodeling of the hydrogel.
3D Printing of Polylactic Acid Microspheres in Polycaprolactone Scaffolds for Tissue Regeneration
Biomedical Engineering, University of Cincinnati, Cincinnati, OH.
To date 3D printing has made custom polycaprolactone (PCL) or polylactic acid (PLA) implants that give mechanical strength to healing tissues. PCL and PLA are of particular interest in this study because of their melting temperatures of 60oC and 155oC, respectively. In addition, PLA can also be used to create drug or other biologic loaded microspheres. Because of the relatively high melting temperature and low heat transfer coefficient of PLA, it was hypothesized that biologic-loaded PLA microspheres could be extruded with PCL powder to create 3D printed, bioactive constructs without melting the PLA barrier which serves to protect the encapsulated biologic materials. PLA microspheres with diameter ranging from 20 to 200 microns were created by single emulsion, mixed with 100 micron PCL powder by sonification, and made into 20 wt% PLA:PCL filament by extrusion at 57oC. The filament was fed to a Flashforge Dreamer which printed experimental discs with a nozzle temperature of 75oC, bed temperature of 37oC, and nozzle diameter of 400 microns. The scaffolds were analyzed using microscopy to verify microsphere embedment and are also being tested in vitro for cell viability, although the results are not yet available. Because it has been shown that PLA can encapsulate many different types of drugs and decellularized extracellular matrices, the hybrid scaffolds proposed herein have the potential to inspire an entirely new generation of patient specific, bioactive scaffolds for many applications including osteochondral augmentation, craniofacial reconstruction, and otolaryngology.
A 3D Printed Retinal Culture System for Photoreceptor Maturation
The functional maturation of retinal photoreceptors from retinal stem cells (RSCs) are essential for personalized in vitro drug screening and potential in vivo retinal neuron repair. The expansion of ganglion cells is tightly related to the spatial arrangement of surrounding cell types such as the retinal pigment epithelium (RPE) and distribution of growth factors during embryonic eye development. While many studies have established systems to facilitate the maturation process, no study has demonstrated in vitro differentiation of ganglion cells from RSCs. The application of 3D printing technology to tissue engineering has enabled patterning of multiple cell types in a hierarchical biomimetic manner that can promote cell differentiation. Here we present a 3D hydrogel-based co-culture model that facilitates differentiation of photoreceptors from RSCs by positioning RSCs and RPE cells in a physiologically relevant 3D environment to mimic their original positioning during development stages. In comparison to 2D co-culturing system and single cell type model, our 3D construct show both phenotypic and developmental enhancements in the RSCs over weeks of culture. We used real-time, non-invasive quantitative phase imaging and immunochemistry staining to analyze the cell morphology changes. Our electrophysiological results also identified cell function by their distinctive discharge and membrane properties. By incorporating multi-electrode arrays (MEAs) to process the electrical pulses generated by activated photoreceptors, this system could be further developed into a hybrid device for cell functional assessment and drug screening.
3D Bioprinted BioMask for Facial Skin Reconstruction
Burn injury to the face remains one of the greatest challenges in wound care due to the varied contours and complex movement of the face. In addition, repairing of the facial wound limits the use of a traditional wound vacuum-assisted closure (VAC) and the effectiveness of cut-to-fit skin substitutes. Unfortunately, current treatment strategies following facial injuries often lead to scarring, infection, graft failure and poor cosmetic outcome. Development of an effective treatment modality will greatly improve the quality of life and social integration of the affected individuals. In this study, we developed a customized engineered skin substitute that snuggly fits onto the complex contour, shape and architecture of facial wounds. This is achieved by 3D bioprinting a BioMask, consisting of customized structure combined with skin cells, including keratinocytes and fibroblasts. The BioMask, including porous polyurethane layer and skin cells, were applied to mouse full-thickness skin wounds. H&E-stained histological sections of skin samples, harvested at 14 days, showed clear differences in the quality of the epidermal layers near the center of the wound areas between control (non-treated) and BioMask groups. The results showed that the Effective and rapid restoration of aesthetic and functional facial skin would have a significant impact on the affected individuals as well as huge cost benefit.
1. Cheng XG, Yoo JJ, Hale RG, Davis MR, H-W Kang, and Lee SJ, 3D printed biomaterials for maxillofacial tissue engineering and reconstruction - A review, OJBMR 1(3), 34, 2014.
Biodegradable Unsaturated Polyesters Synthesized from Antifungal Monomers
Bioengineering, Rice University, Houston, TX.
The combination of patient-specific cells with scaffolds could result in improved regeneration of human tissues. Decellularization of the native tissue is the first step in this technology. Effective decellularization uses agents that lyse cells and remove all cellular materials, leaving intact collagenous extracellular matrices (ECMs). Preserving the underlying structure and basement membrane components are of crucial importance to retain regenerative potential of a decellularized scaffold. In this study, the impact of five decellularization agents (0.1 N NaOH, 1% peracetic acid, 3% Triton X-100, 1% sodium dodecyl sulfate (SDS), and 0.05% trypsin/EDTA) on renal tissue was examined to comprehensively compare a broad range of agents including acidic/basic solution, ionic/non-ionic detergents, and protease enzyme. The NaOH solution induced the most efficient cell removal (>90% DNA removal, p-value <0.05), and resulted in the highest amount of human renal cell viability and proliferation after recellularization, although it also produced the most significant damage to collagenous fiber networks, glycosaminoglycans (GAGs), and fibroblast growth factor (FGF). The SDS solution led to less severe damage to the ECM structure, but it resulted in less proliferation of reseeded cells. Peracetic acid and Triton X-100 preserved ECM components intact. However, these agents alone were not efficient in removing cellular materials. We suggest NaOH as the most efficient agent to remove cellular materials combined with non-ionic Triton X-100 as an optimum procedure for decellularization of renal tissue.
Denervation Properties of Botullinum Neurotoxin Type A in Complex with Chitozan
Academician E.N. Meshalkin State Research Institute of Circulation Pathology, Novosibirsk, RUSSIAN FEDERATION.
Botulinum neutotoxin type A (BoNT-A) is used in many medical areas. Disrupting the process of membrane fusion within the cell, BoNT-A prevents the release of acetylcholine at the synaptic cleft and in fact denervates muscles. Chitosan, the partially deacetylated derivative of chitin, is a linear mucopolysaccharide, composed of glucosamine and N-acetyl glucosamine units linked by covalent bonds. Chemical modification enables to obtain nanoglobular form of chitosan, when dissolved in water, transformed into a sol (chitosol), but maintains biocompatibility and functional amine groups. We compared denervation properties of BoNT-A in mixtures with chitosol and other biocompatible mucopolysaccharides (heparine, nadroparin, hyaluronic acid) after intramuscular injection in rats using electromyography and histological methods. And discovered that chitosol 2-fold increased the threshold of electrical stimulation and prolongated denervation effect of the BoNT-A in comparison with other mucopolysaccharides or pure toxin. Heparin also increased denervating ability of the toxin, but did not have long-lasting effect. Immunohistochemical analysis with antibodies specified to light- and heavy-weight BoNT-A subunits confirmed that the toxin-chitosol complex remained at the injection site. Thus desired effect was already achieved by a single dose, while was a reduction of botulinum toxin side effects. We believe this method can be used in cardiac surgery for the treatment of arrhythmias, or in aesthetic medicine.
Current methods for obtaining tissue coverage and soft tissue augmentation to repair volumetric muscle loss involve the use of existing host tissue to construct muscular flaps or grafts. In many instances, this approach is challenged by the host muscle tissue availability and donor site morbidity such as functional loss and volume deficiency. The present study aimed to overcome these limitations by utilizing endogenous stem/progenitor cells via a biofunctionalized extracellular matrix (ECM)-derived scaffold. To this end, we developed a novel insulin-like growth factor binding protein 3 (IGFBP-3)-conjugated decellularized muscle ECM scaffold for the controlled delivery of IGF-1. The release kinetics and in vitro biological properties, including cellular proliferation and migration, were examined. The result showed that the IGFBP-3 conjugated ECM scaffolds provided the sustained release and increased biological activity of IGF-1 via the IGF-1 binding domain. Therefore, we found significant higher cellular proliferation and migration in the ECM scaffolds compared with controls. Our current data suggest that IGF-1 delivery from biofunctionalized ECM scaffolds may promote host cell recruitment, which can result in accelerating muscle regeneration in situ. In vivo assessment using a rabbit TA muscle excision model is currently being performed.
Antibacterial Nanofibrous Matrices of Nonmulberry Silk Sericin for Wound Repair
An established cosmetic constituent, silk protein sericin is a promising biomaterial with potential of healing wounds [1, 2]. Environmental microflora hinders wound repair and thus incorporation of antibacterial properties is the requisite for absolute therapy. Nanosystems provide superior means for sustained delivery of bioactive molecules. In this study, we develop cost effective, nonimmunogenic, antibacterial nanofibrous matrices using tropical nonmulberry Antheraea mylitta sericin as a wound dressing. Water based polymer (polyvinyl alcohol) is homogenized with sericin to fabricate nanomatrices at optimized parameters and subsequently crosslinked in glutaraldehyde vapors. Their morphology, bonding interaction, tensile strength, structural integrity, blood compatibility and immunogenicity are evaluated. The in vitro potential of wound healing is determined using human dermal fibroblasts in terms of their adhesion, morphology, viability and metabolic activity. These matrices are further functionalized by incorporating cephalexin hydrate via electrospinning and the anti-bacterial properties are investigated. The nanofibrous matrices are porous with the fiber diameter of 200–380 nm and 80% stable in over two weeks. The results indicate that the presence of sericin improves the tensile strength (upto 6 MPa), cell adhesion (99%) and morphology (elongated). The fabricated matrices show minimal immunogenicity, enhanced antibiotic entrapment with gradual release and good antibacterial activity indicating their application as a carrier vehicle with the prospect of wound dressing.
1. Aramwit et al., 2012. Waste Manag Res
2. Kundu et al., 2008. Prog Polym Sci
Recellularization of Porcine Liver Scaffolds Using Primary Porcine Endothelial Cells and Hepatocytes
Liver transplantation is the only definitive treatment for chronic liver failure, however it is severely limited by the availability of donor organs. Whole organ engineering based on decellularization/recellularization techniques has been proposed to alleviate organ shortage. This study aimed to test the feasibility of recellularization of porcine liver scaffolds with primary porcine endothelial cells and hepatocytes, and examine whether the recellularized liver construct possesses functional capabilities. Porcine hepatocytes were isolated by a liver perfusion using collagenase, and the isolated hepatocytes were seeded into the re-endothelialized liver scaffold via direct parenchymal injection, followed by bioreactor culture. Histological and functional analyses were performed to evaluate the recellularization efficiency. Cell characterization analysis revealed that the isolated liver cells showed high yield and cell viability (70–80%) of the primary hepatocytes and maintained the phenotypes during cell culture. Histological and immunohistochemical data showed that the repopulation of primary hepatocytes within the re-endothelialized porcine liver scaffolds resulted in cellular organization with maintenance of hepatocyte phenotypes during the perfusion bioreactor culture period. Importantly, the recellularized liver constructs demonstrated urea and albumin secretion. These outcomes indicate that the isolation of primary porcine hepatocytes and subsequent recellularization within the re-endothelialized liver constructs are feasible, and the use of this approach may contribute as a potential treatment option for chronic liver failure.
Struggling to Prepare an Injectable Self-Assembling Human Cardiac Matrix and Facing Unexpected Failure
Restoring myocardium integrity is the most ambitious and demanding undertaking of cardiovascular regenerative medicine. Several attempts to recover myocardium integrity and contractile function by either direct injection of stem cells in the heart wall or functional three-dimensional tissue constructs of scaffolds and cells, were made in the past decade. Obviously, an injectable preparation of decellularized extracellular matrix (d-ECM), avoiding open chest cardiac surgery would be a tremendous innovation in cardiac regenerative medicine. Aiming at preparing human cardiac d-ECM solution to be used as stem cell delivery medium and embedding scaffold, we decellularized ECM incubating cardiac samples first with 1% SDS for 12 h, then with 1% Triton X-100 for 30 min.1 To obtain an injectable form, we lyophilized and dissolved d-ECM in pepsin solution as previously reported.2 Although we scrupulously followed the described methodology, d-ECM did not dissolve. Adjustments were made until, with a treatment of 24 h at 37°C on tube rotator, d-ECM dissolved. It is reported that d-ECM-pepsin solution spontaneously gels at 37°C in about 1 h2. Again, strictly following previously reported procedures, our results differed significantly. We struggled with several adjustments of temperature, time, d-ECM batch and concentration in pepsin solution, but we never had the d-ECM gelling. Since in previous reports d-ECM was obtained from animal models or human pericardium, we infer that gelling depends on collagen content and, hence, they do not make a good substitute for ECM of human cardiac myocardium.
1. Ott H et al. Nat Med 14(2), 213, 2008.
2. Singelyn JM et al. Biomaterials 30(29), 5409, 2009.
Long-Term Transplantation of Bioengineered Porcine Kidney Constructs Seeded with Autologous Cells
Kidney transplantation is the definitive treatment for end stage renal disease (ESRD). The availability of transplantable kidneys is limited. Recent advances in the field of bioengineering whole kidney constructs have provided a promising solution to address the shortage. Previously, we have developed decellularization and recellularization methods that allowed for efficient recellularizaiton including reendothelialization of vasculatures and repopulation with renal cells using acellular porcine kidney scaffolds. To make this technology amenable for clinical translation, this study aimed to evaluate vascular patency of bioengineered porcine kidney seeded with autologous cell sources using a heterotopic implantation pig model. Decellularized kidney scaffolds processed from native porcine kidneys were recellularized with porcine primary endothelial and renal cells, followed by implantation at the iliac site of pigs. During implantation, blood perfusion through the kidney implant was examined by CT imaging. At 1 week after implantation, the implant was harvested and processed for histological analysis. Results of CT scan demonstrated evidences of partial blood perfusion within the implants. Histological and immunochemical analyses confirmed the vascular patency and viability of the seeded renal cells with maintenance of renal phenotype during the implantation. These results demonstrate that long-term implantation of engineered porcine kidney constructs is possible and this approach may lead to the development of an alternative treatment method for patients with ESRD.
Optimization Of Porcine Kidney Decellularization Methods For Successful Implantation
Kidney transplantation is currently the only definitive solution for the treatment of end-stage renal disease (ESRD), however transplantation is severely limited by the shortage of available donor kidneys. Recent progress in the development of bioengineered whole organs based on decellularization/recellularization of native organs has enabled pre-clinical in vivo studies using small animal models; however, these in vivo studies have been limited to short-term assessments. Previously, we developed a decellularization protocol that effectively removes cellular components from normal pig kidneys and the resultant decellularized kidney scaffold maintained vascular patency short-term after implantation (less than 2 hr). To address the limitation of the short-term assessment, this study aims to optimize decellularization methods that are able to preserve functional vascular architecture for long-term implantation. We compared various decellularization protocols and assessed the effects of the decellularization on the maintenance of porcine renal vasculatures using angiography, vascular corrosion casts, and scanning electron microscopy (SEM) analysis of the casts. Particularly, the vascular casting technique was efficiently used to analyze normal morphology and functional architecture of the vascular luminal structures. Our results demonstrate that the decellularization protocol using a combination of Triton X-100 and SDS was most effective in preserving microvasculatures of the renal scaffold and that the optimized method may contribute to vascular patency for long-term implantation.
Fabrication of Electrospun TPU/Fibroin Vascular Grafts with Biomimetic Structure
Mechanical Engineering, University of Wisconsin-Madison, Madison, WI.
The demand for small-diameter blood vessel substitutes has been increasing due to a shortage of autograft vessels and problems with thrombosis and intimal hyperplasia with synthetic grafts. In this study, hybrid small-diameter vascular grafts made of thermoplastic polyurethane (TPU) and natural silk fibroin were fabricated by the electrospinning technique using two different customized collectors, the striated collector and the assembled rotating collector, to mimic the layered and micro-wavy structure of natural blood vessel. The former method produced a continuous aligned-random fibrous sheet which could form tubes with alternative aligned- and random-oriented layers, followed with methanol post-treatment to induce the transition of fibroin protein conformation and the formation of crimped structure. Ultrafine nanofibers and nanowebs were found on these electrospun samples, which effectively increased the surface area for cell adhesion and migration. The other method, the assembled rotating collector, was able to generate tubes with circumferential-aligned wavy fibers due to the dynamic “jumping rope” collecting process. The wave size was influenced by the material compositions, rotating speed, and length of the collector. Cyclic circumferential tensile test results showed compatible mechanical properties for grafts made of a soft TPU/fibroin blend compared to human coronary arteries. In addition, cell culture tests with endothelial cells culture exhibited high cell viability and good biocompatibility of these samples, suggesting the potential of applying electrospun TPU/fibroin grafts in vascular tissue engineering.
Biotechnology, Universidad Autónoma Metropolitana, Mexico City, MEXICO.
The neem leaf extract (NE) is a natural product obtained from the Azadirachta indica tree. Its composition is very complex including proteins, polysaccharides, sulphurous compounds, polyphenolics, aliphatic compounds and terpenoids, such as azadirachtin (AZA). It has been attributed antimicrobial properties of AZA and used for the treatment of leprosy, epistaxis, gastrointestinal infections, biliary disorders and for topical medication in the treatment of ulcers. AZA concentration (1762 ppm) was determined in a methanolic NE by UltraPerformance Convergence Chromatography. Later on, methanol was changed to ethanolic NE and used for further studies. Antimicrobial capacity of NE was determined in an AZA concentration range of 0.44–3.52 ppm on Pseudomonas aeruginosa, Escherichia coli and Staphylococcus epidermidis. The minimum inhibitory concentration (MIC) was determined at 0.44 ppm for all tested microorganisms. Scanning electron micrographs showed absence of cell division and high number of cells in elongation and septum formation of bacteria cultured with NE at MIC. Human peripheral lymphocytes cultures were also exposed to NE. The mitochondrial and lysosomal activities were 24% and 20% higher than control as determined by MTT and neutral red assays. The mitotic index decrease 86% with regard to the control confirming the cytotoxicity of NE. Therefore, NE has a high antimicrobial capacity but displays cytotoxic activity to human lymphocytes.
Bioengineering, University of Pittsburgh, Pittsburgh, PA.
Hydrogels have become an intriguing option for the 3D culture of secondary follicles and maturation of oocytes; however, several hurdles continue to hinder human translation of this technology for female patients looking to restore fertility. Our group has developed a novel biomaterial derived from decellularized porcine ovarian tissues to closely mimic the ovarian microenvironment and study how a dynamic extracellular matrix (ECM) affects follicle development. Porcine ovarian tissues were decellularized using mild detergents, lyophilized and digested through a natural enzymatic process. Ovarian hydrogels were formed spontaneously at 37°C after neutralizing the pH and balancing the salt concentration of solubilized ovarian ECM. Sohlh1-mCherry newborn mouse ovaries were cultured on top of ovarian hydrogels at varying ECM concentrations to examine the effect of ECM stiffness on follicle development. After 7 days in culture, ovaries were imaged using confocal microscopy to identify populations of Sohlh1+ cells, indicating the presence of early oocytes. A volumetric analysis showed no significant difference in the number of primordial follicles between the 2 mg/mL ovaries and the wild-type control group. Immunohistochemistry (IHC) was performed using a NOBOX-specific marker that localizes in oocytes at each stage of folliculogenesis. The effect of stiffer hydrogels was clearly illustrated by IHC showing larger oocyte size and a definitively developed matrix. Our results suggest an increase in ECM stiffness could drive follicle activation and assist with oocyte maturation. We have also verified that ovarian hydrogels can support the culture of ovarian tissues and may be used as an alternative biomaterial for fertility preservation.
University of Southern California/Children's Hospital Los Angeles, Los Angeles, CA.
Tissue engineered organ transplants success depends on the biomaterials capacity to promote a pro-healing response. Multiple studies, including ours, have demonstrated the use of extracellular matrix (ECM) from animal organs as a platform for tissue engineering. Most recently, discarded human organs have also been proposed as a scaffold source. However, little is known of how the immune properties vary between diseased and healthy ECM and artificial scaffolds. We characterized the morphology and composition of decellularized renal ECM derived from WT and diseased (Alport Syndrome) mice and compared their in vitro effects on macrophage activation with that of synthetic scaffolds commonly used in the clinic (collagen type I and poly-L-(lactic) acid). We show that difference in fibrous protein deposition and cytokine content exists between WT and Alport kidneys. Yet, both WT and Alport renal ECM induce macrophage differentiation mainly towards a reparative (M2) phenotype, while artificial biomaterials towards an inflammatory (M1) phenotype. Together, these data support the notion that natural ECM, even if derived from diseased kidneys promote a M2 protolerogenic macrophage polarization, thus providing novel insights on the applicability of ECM obtained from discarded organs as an ideal scaffold for tissue engineering.
Department of Biomedical Engineering, Vel Tech Multi Tech Engineering College, Chennai, INDIA
1. M. Nakamura, K. Arimoto, Takeuchi, T. Fujii, A rapid extraction procedure of human hair proteins and identification of phosphorylated species, Biol. Pharm. Bull. 25 (2002) 569–572.
2. T. Fujii, D. Ogiwara, M. Arimoto, Convenient procedures for human hair protein films and properties of alkaline phosphatase incorporated in the film, Biol. Pharm. Bull. 27 (2004) 89–93.
Cell Transplantation Centre, Pauls Stradins Clinical University Hospital, Riga, LATVIA.
Different cell populations from bone marrow are used in various clinical trials for cardiac diseases during last decade. Four clinical studies are on going in our institution and enrol patients with cardiac diseases, coronary disease and type 2 diabetes, patients with osteoarthritis. Density gradient is used to separate bone marrow mononuclear cells. Cell processing looses are significant. To find out critical control points we analysed processing process and differences in cell yields between operators performing cell extraction. Bone marrow mononuclear cells were isolated using Ficoll density gradient centrifugation. Cells were counted using flow cytometry for mononuclear cell total counts, CD34+ population count and viability analysis. The patients who underwent bone marrow aspiration followed by cell isolation received cell suspension for transplantation. Two cells processing for separate patients were performed at once. Same standard operation procedures were applied. Processing looses between operators performing cell extraction were analysed. Bone marrow samples from eight patients were processed. Mononuclear cells were extracted. Operator performances were compared. Operator A average bone marrow mononuclear cell yield in starting material was 9.97 ± 9.98%, CD34+ population yield 75.46 ± 79.67%. Operator B average bone marrow mononuclear cell yield in starting material was 24.68 ± 14.8%, CD34+ population yield 70.42 ± 44.84%. Operator A average cell viability in starting material was 45.24 ± 9.55%, after cell processing 42.96 ± 23.66%. Operator B average cell viability in starting material was 49.85 ± 5.48%, after cell processing 69.52 ± 6.65%.
Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, JAPAN.
Clinical translation of engineered tissues for regenerative medicine has been limited by the inability to provide three-dimensional (3D) tissue engineered constructs with efficient vascular networks. Endothelial cells are a critical component of blood vessels, and therefore the generation of patient-specific endothelial cells is vital for creating vasculature within tissue engineered constructs. Since embryonic vascular development occurs in the setting of a 3D extracellular matrix environment, we hypothesized that 3D polymeric scaffolds will be more efficient than two-dimensional (2D) membranes in inducing endothelial differentiation of human pluripotent stem cells and their subsequent morphogenesis into vascular network-like structures. 3D fibrous polymer scaffolds composed of polycaprolactone (PCL) and polyethylene oxide were fabricated by electrospinning and were characterized by scanning electron microscopy to be approximately 800 μm in thickness with 14 μm average fiber diameter and an effective pore diameter of 53 μm. Human pluripotent stem cells were differentiated within 3D scaffolds or control 2D PCL membranes in the presence of endothelial growth factors for 5 days. The efficiency of endothelial differentiation was assessed by gene and protein expression quantifications using immunocytochemistry, polymerase chain reaction and flow cytometry for endothelial markers, which revealed that the 3D scaffolds consistently exhibited greater endothelial differentiation, compared to 2D substrates. Furthermore, when compared to 2D films, the 3D scaffolds promoted increased network formation by increasing the levels of endothelial branch points by 75%. Collectively, these results demonstrate that compared to 2D, 3D scaffolds were more effective in inducing endothelial differentiation and in generating vascular network-like structures in vitro.
Development of Silk Fibroin/Polycarbonate Composite Scaffold for Tissue Engineering Material
Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, JAPAN.
Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo, JAPAN.
Biomedical Engineering, Boston University, Boston, MA.
Viable engineered tissues should replicate native tissue structural organization. Moreover, environmental cues such as substrate stiffness have significant effects on cell phenotype; thus physicochemical properties of the substrate should also mimic that of native tissue. Here we introduce a novel cell sheet system with tunable substrate stiffness, surface chemistry, and micropatterning. In this system, maintenance of native substrate stiffness was an important driver of optimal cellular differentiation. Confluent cell sheets could easily be harvested and stacked to mimic 3D structure of native tissue while maintaining cellular orientation and high viability. In vivo studies showed that C2C12 sheets grown on native muscle stiffness (10–13kPa) substrates actively recruited blood vessels, and differentiated and fused with the host tissue to form myotubes. Our system has the potential to be used to rapidly fabricate functionalized 3D thick tissues from multiple stacks of cell sheets, and thus may open up new opportunities in tissue engineering.
Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, JAPAN.
Beating Human Heart Tissue From hiPSC-derived Cardiomyocytes And Porcine ECM
Chemical Engineering, Brigham Young University, Provo, UT.
Beating human heart tissue was created in vitro using non-immunogenic scaffolds generated from decellularization of porcine hearts combined with cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs). Whole porcine hearts were decellularized to create 3D scaffolds capable of supporting the human cells mechanically and biochemically. From the left ventricle of the decellularized hearts, 300 μm thick, 10 mm round slices were prepared and mounted on glass coverslips. Human iPSCs were differentiated into cardiac progenitors and 4 days after differentiation, these cells were seeded onto the cardiac slices. Ten days after recellularization, clusters of differentiated CMs started to beat spontaneously. Immunofluorescence images showed confluent coverage of CMs on the decellularized slices and the effect of the scaffold was evident in the alignment of the CMs in the direction of the collagen fibers. A resazurin-based viability assay showed attachment and survival of 75% of the seeded cells up to 14 days after recellularization. The clusters continued to beat for 60 days. This study demonstrated the biocompatibility of decellularized porcine hearts with human CMs and the potential of these scaffolds for use in cardiac tissue engineering. Future studies will be directed toward 3D perfusion recellularization of whole decellularized hearts and repopulation of the scaffolds with fibroblasts and endothelial cells along with the CMs, as well as adding mechanical and electrical stimulation to obtain more mature beating tissue.
Fixed pericardial tissue is used for commercially available xenograft valve implants, has proven tissue durability, but lacks the capability to remodel and grow. Decellularized pericardial tissue has the promise to out perform fixed tissue and remodel, but the decellularization process has been shown to damage the collagen structure and reduce mechanical integrity of the tissue. Therefore, a comparison of uniaxial tensile mechanical properties was performed on native, fixed, decellularized, and sterilized pericardial tissue, where twelve specimens were tested in both the circumferential and longitudinal direction for a total of twenty four specimens in each group. The results showed that the average ultimate tensile strength was found to be 43.4, 26.8, 8.07, and 23.5 MPa respectively. As expected, the results showed a statistically significant (P < 0.5) difference between the ultimate tensile strength of 1) decellularized vs. sterilized and 2) decellularized vs. fixed tissue. Scanning electron micrograph of the tissues showed larger pores sizes for the decellularized tissue (>5um) compared to the fixed tissue, possibly allowing for tissue ingrowth from the host cells. Although comparatively decellularized pericardial had lower mechanical properties in the groups studied, the decellularized pericardial tensile strength was still three times higher than the average native porcine aortic cusp (2.69 MPa) tensile strength, therefore making it a viable candidate for tissue engineered valvular applications.
Guiding Vascular Network Formation by Scaffold Inner Architecture
Department of Engineering, Cambridge University, Cambridge, UNITED KINGDOM.
Vasculature is essential for effective transport of oxygen and nutrients, as well as removal of waste products in the living tissues. Therefore in vitro pre-vascularization approaches enabling the survival of engineered tissues upon implantation need to be established. We propose endothelial cells in co-culture with osteoblasts within collagen scaffolds with tailored pore architecture to create vascular networks. We aim to understand the spatial aspects of micro-vascular assembly, and to identify the key parameters that need to be controlled to promote and regulate these complex processes. Scaffolds with tailored pore geometries were produced by manipulation of the freeze-drying conditions, and characterized by scanning electron microscopy and X-ray computed tomography. Vascular organization and inter-cellular interactions were assessed using histology, immunohistochemistry, confocal microscopy and ELISA. We produced and characterized scaffolds with isotropic (randomly aligned) and anisotropic (aligned in one axis) pore geometries and various pore diameters. Our results show that scaffold pore architecture significantly affects endothelial cell morphology, infiltration and proliferation in both mono- and co-cultures. Additionally, we demonstrate that the presence of osteoblasts promotes endothelial cell organisation into elongated multinucleated structures, penetrating deep within the anisotropic scaffolds. These multinucleated structures create 3-dimensional vascular-like networks with high expression of endothelial markers. This work concerns a novel approach to identify the architectural parameters and to investigate the mechanisms that regulate vascularization in vitro. Little is known about the role of 3-dimensional pore geometry on endothelial cells behavior, since anisotropic scaffolds have not been investigated previously for growth of vessel structures.
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Tecnology, Tokyo, JAPAN.
Novel Techniques for Electrospinning Transcatheter and Surgical Cardiac Valve Prostheses with Potential for Endothelialization and Durability
Valve replacement is the most effective treatment for aortic valve disease, however mechanical prostheses are thrombogenic and bioprosthetic prostheses have limited durability. To address these limitations, we developed novel techniques for electrospinning valve prostheses from polyurethane nanofibers. Electrospun nanofibers provide excellent cell adhesion and mechanical properties, which can facilitate endothelialization and durability. The objective of this study is to fabricate three novel valve prostheses and asses their functionality. The first design is a tube-on-stent valve which can be implanted minimally invasively and does not require sewing for assembly, which can cause asymmetries and stress concentrations. The second design is a tube-on-crown valve which can be implanted surgically and also does not require sewing for assembly. The third design is proof-of-concept for a tube-in-tube valve which can be made from biodegradable polymers to allow for remodeling and growth within a pediatric patient. By using a multilayered fabrication approach with carefully designed masks to define the regions of adhesion between layers, valve prostheses without structural defects and with mobile cusps were fabricated. Functionality was assessed using a bioreactor and an accelerated wear tester based on ISO 5840 standards. All three valve prostheses demonstrated functional cusps with appropriate opening and closing actions. Effective orifice area was >1.00 cm2 (indicating proper opening) and maximum diastolic pressure gradient was >95 mmHg (indicating proper closing) for all three valve prostheses. In conclusion, three novel valve prostheses were successfully fabricated by electrospinning and functionality was verified. Future studies will assess endothelialization and durability.
Regeneration Capacity of Valvular Interstitial Cells from Diseased Heart Valve
Regeneration of diseased valve tissues depends on the regeneration capability of the residing valvular interstitial cells (VICs). In this study, we investigated the regeneration potential of VICs from diseased heart valves in vitro on nanofibrous substrates that morphologically mimic the valve-leaflet structure. VICs from the aortic heart valve-leaflets of surgical patients (n = 4, calcified valve) and human cadavers (n = 4, healthy valve) were cultured for 3 weeks on randomly oriented electrospun polycaprolactone nanofibrous substrates, which provided a completely disease-free environment. Proliferation, cell morphologies, collagen depositions, and gene and protein expressions of the VICs were investigated to assess their potential in heart valve regeneration. VICs from healthy valves showed higher proliferation and cell-spreading on nanofibrous substrates compared to VICs from diseased valves. VICs from calcified valves showed higher collagen deposition compared to their non-calcified counterparts irrespective of gender and age of the patients. Vimentin expression was higher from the VICs of healthy valves of male patients than of female patients. In the male patients, vimentin expression was higher in cells from calcified valves of older patients and from non-calcified valve of younger patients. Expressions of α-smooth muscle actin and type I collagen were quite similar to that of vimentin. However, proliferation, growth and gene expressions among the VICs from patients of various ages and genders, and with/without heart valve diseases, were not significantly different. Thus, it can be concluded that VICs from a diseased heart valve have the potential to regenerate in a disease-free environment.
High Content Screening Platform For Human Engineered Cardiac Tissues Maturation And Drug Discovery
University of Toronto, Toronto, ON, CANADA.
Poly(glycerol sebacate) (PGS) has been successfully used to produce tissue engineering scaffolds for various applications. The elasticity and rapid degradation rate of this polymer has been associated with improved extracellular matrix (ECM) deposition rates and quality, particularly relating to elastin fibres, in the in vitro production of tissue engineered vascular grafts. However, the utility of PGS is limited by the requirement for high temperatures and extended reaction times to crosslink and cure the prepolymer during scaffold manufacturing. Here, we have developed a novel photocurable form of PGS with improved processing capabilities: PGS-M. By methacrylating the secondary hydroxy group of the glycerol units in the PGS polymer chain, the material is rendered photocurable, rapidly crosslinking on exposure to UV light at ambient temperatures. Our results have shown that the mechanical properties of PGS-M can be tuned by varying the molecular weight of the prepolymer and the degree of methacrylation, both of which can be independently controlled. PGS-M has also demonstrated excellent biocompatibility and rapid degradation under physiological conditions. Using a novel additive manufacturing method, porous tubular scaffolds have been produced from PGS-M with suitable structure, porosity and mechanical properties for use in vascular graft tissue engineering. These scaffolds were then seeded with vascular smooth muscle cells and cultured under physiological flow in a bioreactor, producing tissue engineered blood vessels. Future work is set to examine these vessels in a small animal model, as vascular grafts.
Transcatheter Tissue-Engineered Venous Valve
1. Sathe RD, Ku DN. Flexible Prosthetic Vein Valve. J Med Device. 2007;1:105.
“Off-the-Shelf” Tissue-Engineered Transcatheter Aortic Valve
Dept of Biomedical Engineering, University of Minnesota, Minneapolis, MN.
1. Webb and Dvir, 2015, JACC 8(8), p1092–1092.
2. Syedain et al, 2015 Biomaterials 73, p175–84.
The New York Stem Cell Foundation Research Institute, New York, NY.
(1) Pallotta I et al., 2015.
(2) Lian X et al., 2013.
(3) Spiller KL et al., 2015.
(4) Hansen A et al., 2010.
Hitachi, Ltd., Saitama, JAPAN.
For cost reduction by mass production of high-quality cells, we have developed the automatic cell culture equipment. Aiming the manufacture of the induced pluripotent stem cell (iPSC) derived dopaminergic progenitors for Parkinson's disease treatment [1] with this equipment, we have studied the proliferation of iPSCs and the differentiation to dopaminergic progenitor cells. The cell culture process with this equipment differs from the conventional manual cell culture by introduction of a closed system and large culture vessels with 500 cm2 of culture surface. We have evaluated the modified culture process by analyzing the cultured cells with microarray analyses. As a result of the first half of differentiation in a large culture vessel with a closed system, microarray analyses showed that gene expression profiles were almost equal to that of the manual culture, and correlation coefficient of the gene expressions was as high as 0.99. In addition, after the latter half of the differentiation of these cells, the gene expressions (NANOG, FOXA2, etc.) were almost the same. Therefore, we consider that the developed culture process for automatic cell culture equipment is applicable to manufacturing the iPSC derived dopaminergic progenitors.
[1] Doi D. et al., Stem Cell Reports 2, 337, 2014.
In cardiac tissue engineering approaches to treat myocardial infarction, cardiac cells are seeded within three-dimensionalporous scaolds to create functional cardiac patches. However, current cardiac patches do not allow for online monitoringand reporting of engineered-tissue performance, and do not interfere to deliver signals for patch activation or to enable itsintegration with the host. Here, we report an engineered cardiac patch that integrates cardiac cells with flexible, freestandingelectronics and a 3D nanocomposite scaold. The patch exhibited robust electronic properties, enabling the recording ofcellular electrical activities and the on-demand provision of electrical stimulation for synchronizing cell contraction. We alsoshow that electroactive polymers containing biological factors can be deposited on designated electrodes to release drugs inthe patch microenvironment on demand. We expect that the integration of complex electronics within cardiac patches willeventually provide therapeutic control and regulation of cardiac function.
Design And Fabrication Of A Well-set, Cost-effective Device For Mechanical And Biochemical Stimulation In Cardiac Tissue Engineering Applications
It is confirmed that mechanical stretch and gradient of different components have vital control over cell morphology, proliferation, lineage commitment, and differentiation. However, most systems have low throughput, restricted small size, are costly and not user-friendly.
In this work, we have designed and fabricated a stretching device for studying different stretching regimes on 3D structures. A user-friendly interface has been developed to control different parameters like the frequencies. Also, to investigate the gradient of different components, like growth factors and drugs, a mold has been designed for fabrication of a flexible chamber of 4 cm2 square as a platform for 3D cell culturing. The bottom of the chamber with a porous membrane has been utilized to simulate interface of the pericardial cavity in native heart. So, pericardial-like fluid flows in below chamber and 3D cell structure places in upper chamber. The whole set-up has been first tested using human fibroblasts in a combination of decellularized extracellular matrix and fibrin hydrogel which is responsible for the 3D support of the structure. Stretching the cells seeded in the hydrogel for 48 h with elongation of 20% applied at 0.5 Hz frequency provided alignment parallel to the direction of stretch. We are currently repeating experiments with H9C2 cells in 5 mm-thick 3D structure.
Regarding the data, cell stretch within 3D constructs is one of the most effective and physiologically relevant stimulations for tissues. We fabricated a cost-effective, well-set stretching device and 3D tissue culture platform with appropriate function to use in in-vitro cardiovascular tissue engineering and drug screening.
Development of a Tissue Engineered Diabetic Cardiomyopathy Model
Effect of Separation Distance on the Properties of HepG2 Spheroids in Micropatterned Cultures
Spheroid (three-dimensional cell aggregate) culture is a promising technique for tissue engineering and drug screening studies. We made interaction between neighboring spheroids the focus of this study, and we evaluated the effect of spheroid separation distance on their growth and function by using a micropatterned culture. We fabricated a culture chip which had 37 collagen spots (300 μm in diameter) in a hexagonal arrangement on a glass substrate, as the micropatterning of spheroids. Three similar chips with different gaps (distance between the collagen spots) of 500 (gap 500), 1000 (gap 1000), and 1500 μm (gap 1500) were designed to investigate the effect of separation distance on spheroids. HepG2 cells formed spheroids on each collagen spot via cell proliferation. Although the spheroid sizes increased throughout the culture period, the growth was the highest in the gap 1500. Additionally, in the gaps 500 and 1000, the spheroid growth in the outside region was higher than that in the inside region, but there were no significant differences in spheroid size across the regions of gap 1500. Furthermore, albumin production function was highest in the gap 1500 and decreased in the gaps 1000 and 500 in that order. These results indicate that the interference between spheroid-spheroid occurs when the separation distance on spheroids is less than or equal to 1000 μm.
Characteristics of Mouse iPS Embryoid Bodies Cultured on a Microwell Chip
Embryoid bodies (EBs), which are generated by aggregation of iPS cells, can promote the initial differentiation of iPS cells. A microwell chip, in which the microwells of several hundred micrometers were regularly fabricated on a culture substratum, is a promising platform for generating EBs. In this study, the EB properties of mouse iPS cells were compared between microwell chip and traditional hanging drop (HD) cultures. We fabricated a chip that contained 139 microwells (500 μm in diameter) on a poly-methylmethacrylate plate, and evaluated the EB growth and the expressions of differentiation gene markers. Mouse iPS cells formed a single EB in each microwell within 1 d of culture, and the EB size increased by cell proliferation. However, the growth of EB in the chip culture was lower than that in the HD culture. The differentiation patterns of EBs were compared at 7 d of culture. The expression of hepatic markers (TTR and AFP) in the chip culture was higher than that in the HD culture, while the expression of vascular markers (Flk1 and PDGFRβ) in the chip culture was lower than that in the HD culture. These results indicate that the difference of EB generating method affects to the decision of differentiation fate of iPS cells, and that the microwell chip culture prefers hepatic differentiation.
Patient Customizable Hybrid Biofabrication of Seamless Multilayer Tubular Tissue
Biofabrication of the tubular organs such as the blood vessels, gastrointestinal tracts, and trachea is attracting substantial attention to date. Numerous surgeries are performed to bridge surgical gap, aiming to restore the functions of organs. Biofabrication represents a promising approach in the regeneration of tubular organs. Tubular organs are typically comprised of a tubular aligned outer extracellular matrix (ECM) together with smooth muscle cells (SMC) and a luminal layer of endothelial or epithelial cells. Here we present a hybrid biofabrication of a model tubular organ by melt-drawing of a tubular scaffold that mimics the outer ECM layer, followed by bioprinting of the lumen to recreate a barrier to the luminal content. Both the layers supported cell growth. The outer tubular scaffold was fabricated by elastic poly(L-lactide-co-ɛ-caprolactone) (PLC) microfibers with customizable dimensions and elasticity formed during melt-drawing process, which can cater to the biomechanical properties of various tubular organs. These highly aligned microfibers allow SMC to grow into the tubular shaped tissue following the 3D fiber alignment. Cell-laden poly(D-lactide-co-glycolide) (PLGA) microspheres were then printed in the lumen of the scaffold. The bioprinted internal tissue was found to fit well to the lumen of the outer melt-drawn scaffold. The multi-layered construct can be easily handled and could possess sufficient strength to support fluid and bolus flow. This work demonstrates a novel hybrid biofabrication method for the seamless multi-layered tubular tissues, which provides insights into the practical production of functional tubular organs.
Calcium Alginate Honeycomb Mesh for Handling Delicate Materials
University of Tsukuba, Tsukuba, Ibaraki, JAPAN.
School of Chemical, Biological, and Materials Engineering, University of Oklahoma, Norman, OK.
Bone tissue engineering is a promising alternative to traditional approaches for the intervention of bone tissue defects; however it presents many challenges including the ability to monitor cell growth and differentiation, and deposition of in vitro generated extracellular matrix in real time. Meeting this challenge will be required in order to meeting FDA regulatory demands and realize the clinical significance sought by the field. This study aims to provide the framework for meeting such demands and securing FDA clearance. Constructs comprised of 85% porous spunbonded poly(L-lactic acid) scaffolds seeded with 1 million rat mesenchymal stem cells were cultured in a flow perfusion bioreactor with osteoinductive media. Throughout culture periods of up to 14 days, levels of key metabolites (oxygen, glucose, and lactate) and soluble proteins (osteoprotegerin and osteocalcin) were quantified and correlated to such properties as cellularity and mineral deposition (both requiring scaffold sacrifice to determine). The data demonstrate key correlations such as initial increases then leveling off of oxygen consumption and osteoprotegerin production rates. Moreover, trends in many ratios of these metabolites and secreted proteins seem promising for monitoring purposes, such as a spike in the ratio of OPG produced per glucose consumed that corresponds to the onset of mineral deposition, followed by a decrease and subsequent leveling off. These trends can be used to develop metrics for the real-time monitoring of constructs in vitro and determination of optimal implantation timing, both of which will be required for FDA clearance. This concept applies also to other TE constructs.
Characterization and Preparation of 3D Printed Scaffolds with Highly Porous Strands
3D printing technique of solid freeform fabrication (SFF) method is good tool for the scaffold fabrication of tissue engineering, because it is able to make specific structure as like damaged tissue. PCL is most widely used as the biomaterial because of good mechanical properties, biocompatibility and biodegradability. Additionally, it is easy to fabricate interconnected 3D structure using 3D bio-printer due to low melting point. However, PCL has some insufficient property for cell growth such as hydrophobicity and dense strand. Highly porous structure and hydrophilic surface of tissue engineered scaffold were eligible to more effective for cell attachment. In this study, we fabricated polycaprolactone/Pluronic F127 (PCL/F127) composite scaffold that improved the porosity and the hydrophilicity via 3D-printing system. The PCL and PCL/F127 scaffolds exhibited uniform interconnected strands in SEM observation. PCL scaffold had no pore in strands and the PCL/F127 scaffold had nano (∼200 nm)-micro pores in strands. Compared to the PCL scaffold, PCL/F127 scaffold had a hydrophilic surface (contact angle measurement ≈0°). Although the PCL/F127 scaffold (4.09 ± 0.11 MPa) had a lower compressive modulus than PCL scaffold (5.09 ± 0.10 MPa), the PCL/F127 scaffold surface was fully spread and covered by the cells because of porous and hydrophilic strands. These results indicated that our developed scaffolds may be useful for rapid tissue repair in biomedical engineering.
Exploring Tissue Engineering of Skin for New Automation Technologies
Product Development Group Zurich - Swiss Federal Institute of Technology Zurich, Zurich, SWITZERLAND.
Tissue engineering of skin is a prominent and proceeding field of research, but still not established as a clinical treatment, despite a high clinical demand. In the ongoing effort of the Tissue Biology Research Unit (TBRU) in Zurich to bring this treatment into clinical application, Phase I clinical studies were recently completed using a novel type of autologous bioengineered dermo-epidermal skin graft. For this study, the tissue engineering protocol includes primarily manual steps. Our new Advanced Therapy Medicinal Products indicate a high potential for better efficiency, scar-less healing, and production robustness, which are essential to meet the high demand for future applications. To address this need, the TBRU and the Product Development Group Zurich initiated the “SkinCreator” project with the goal to ease the manual production process with new biomedical devices. Thus far, the manual process was analyzed from an engineer's perspective to identify the most promising areas for new manufacturing technologies. Pressure mapping, fluid flow tracking and optical measurements were carried out for the process of skin-graft production. We were able to monitor the pressure distribution throughout the process of plastic compression of certain hydrogels. The corresponding results are used to understand the critical engineering parameters to develop devices tailored to the autologous tissue engineering of skin for an automated production, including a new type of bio-reactor and novel technical platforms.
Many three-dimensional bioprinters use hydrogels as ink. However, current hydrogel inks require deleterious crosslinking agents (ultraviolet, pH or chemical) to gel. Thermogelling hydrogels have been investigated as injectable scaffolds that solidify when their temperature is raised inside the body and do not require crosslinking agents. However, they have not been investigated as bioprinter inks. We began with an inexpensive, commercial printer kit ($699.00) with open source control software and designed a three dimensional (3D) printed extruder that accepts hypodermic needles. Due to the printer's small footprint, sterile printing was achieved by placing it inside a 4-foot sterile hood and disinfecting it with isopropanol, and 20 minutes of UV irradiation. The effect of particulate removal, solution pre-cooling, degassing, and extruder movement speed on fiber diameter and printability were examined. Neuroblastoma, pre-stained with carboxyfluorescein succinimidyl ester, were encapsulated in hydrogel, processed to remove particulates and entrapped air, and successfully printed through a 34 g needle. Printed structures were incubated in medium for five days without observable contamination. The structures were examined under florescent microscope. Cells were well distributed throughout the structure and were viable. In conclusion, solution cooling is necessary to prevent premature gelation and consistent production of 3D structures with uniform cell distribution.
Bio-pick, Place, And Perfuse Instrument For Building Perfusable Large Proto-organs
Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI.
Singapore Centre for 3D Printing (SC3DP), Nanyang Technological University (NTU), Singapore, SINGAPORE.
Bioprinting is an emerging research field that has recently attracted tremendous attention; it offers a highly automated, advanced manufacturing platform for fabrication of cell microarrays for RNA analysis, formation of controllable, uniform-sized tissue spheroids and analysis of cellular interactions in a highly reproducible manner. Particularly, the drop-on-demand (DOD) bioprinting enables higher degree of control over the cellular densities and positioning. There is currently an unmet need to mitigate cellular damage during the bioprinting process and this work aims to investigate the effect of polyvinylpyrrolidone (PVP)-based bio-inks for optimized DOD printing. It has been reported that PVP has an influential role in accelerating tissue maturation in a dose-dependent manner; hence this work pioneers the novel use of PVP-based bioinks in 3D bioprinting. We first evaluate a range of PVP-based bio-inks (0–3% w/v) for their printability in our microvalve-based (DOD) bioprinting system; following which the effects of PVP-based bio-inks on the cellular viabilities and cellular output consistency of human fibroblasts (ATCC® SCRC-1041™) were characterized. Our findings highlighted that significant improvements in both the cellular viabilities (>95%) and cellular output consistency (1.24 ± 0.91 in 0.5 mil cells/ml, 3.00 ± 1.06 in 1.0 mil cells/ml, 5.22 ± 1.18 in 1.5 mil cells/ml and 7.58 ± 1.63 in 2.0 mil cells/ml) were observed in the PVP-based bio-inks, indicating that a more viscous bio-ink (∼ 8 mPa.s for PVP bio-inks) is favourable over control group (∼ 0.8 mPa.s for culture medium) for bioprinting applications.
Institute of Medical Sciences, Canterbury Christ Church University, Chatham, UNITED KINGDOM.
This device allows entry into organ engineering for every cell culture laboratory.
Tissue Engineering and Textile Implants, Institute of Applied Medical Engineering, Helmholtz-Institute, RWTH Aachen University, Aachen, GERMANY.
Cell spraying is described as fast and effective method to coat surfaces with a thin layer of cells.1 Previously, we developed a novel method for cell spray application based on a clinical endoscope.2 This method allows endoscopic atomization of cells in a thin layer of two-component fibrinogen glue. Here, we built a set-up which allows automated and reproducible atomization of cells with defined parameters and systematically investigated the influence of varying air flow (80–200 ml/s) and cell suspension flow (0.4 and 0.8 ml/s) on droplet sizes and cell survival. Live-dead staining directly after atomization showed that increasing flow rates cause a decrease in cell survival. Similarly, a correlation between decreasing droplet diameter and cell survival was seen. However, survival rates of up to 90% were achieved. With the presented results, we are able to set thresholds for future nozzle designs and atomization parameters to maximize efficiency and cell distribution while minimizing cell damage.
1. Cohen M, Bahoric A, Clarke HM. Aerosolization of epidermal cells with fibrin glue for the epithelialization of porcine wounds with unfavorable topography. Plast Reconstr Surg 107, 1208, 2001.
2. Thiebes AL, Reddemann MA, Palmer J, Kneer R, Jockenhoevel S, Cornelissen CG. Flexible Endoscopic Spray Application of Respiratory Epithelial Cells as Platform Technology to Apply Cells in Tubular Organs. Tissue Eng Part C 22, 322, 2016.
School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore, SINGAPORE.
In recent years, bioprinting has been one of the fastest developing technology in the area of biomedical science. One method of printing of cells involves the use of hydrogel as an encapsulating agent to help provide the mechanical support and micro-environment for the cells in a 3-Dimensional space. However, printing of hydrogel is often challenging as the mechanical strength of the hydrogel predominantly weak. One approach to overcome these challenges is the use of a support material to retain the shape during printing. However, current printable support materials such as Pluronic are not suitable for printing small structures. The objective of this study is to manufacture a new blend of support material to improve the overall form of the printed scaffold without affecting cell viability and functionality. Herein, we have been successful in developing hydrogels with 100um diameters. This was accomplished by using a blend of Pluronic/Alginate as a support material and calcium supplemented gelatin methacrylate as the cell encapsulated hydrogel. The interaction between the alginate and calcium forms an inhibitory wall which partitions the two types of gel. Cytotoxicity of the gel was tested using HUVEC (Human Umbilical Vein Endothelial Cells) and have shown similar results to the gels fabricated using traditional casting techniques. Future study, will examine the effects of the process of printing on other naturally occurring hydrogels to mimic the exact microenvironment of the cells found in nature.
Human Mesenchymal Stem/Stromal Cells (hMSCs) are a critical raw material for Regenerative Medicine and Tissue Engineering. To move basic research to the clinic, high numbers of functional cells, isolated and grown in xeno-free (XF) conditions, will be needed to develop commercial RegenMed products and to mitigate safety issues related to xeno components. In this study, we developed and tested XF media for expansion of hBM-MSC. We hypothesized that hBM-MSC expanded in XF growth media will maintain comparable growth and quality parameters compared to hBM-MSC grown in bovine serum-containing medium (BSC), while maintaining scalability of design and favorable bioprocess economics of production. Over multiple cell lots, XF media expanded-hBM-MSC were highly proliferative (doubling time 24.4 +/− 0.5 hr), and had comparable functionality to BSC hMSC, including cell surface markers (CD73, CD90, CD105, and CD166), angiogenic cytokine secretion (VEGF, IL8, TIMP1, TIMP2, FGF, HGF), immunomodulation (IFNgamma-inducible IDO), and multi-lineage differentiation. An economic analysis of RoosterBio XF media showed that, like our BSC media system, the XF formulation demonstrated significant cost- and time-savings compared to other XF MSC systems (cost and time to 100M and 1B cells). Therefore, XF grown hMSC are comparable across XF and bovine-containing media and maintain favorable BioProcess economics; confirming our hypothesis. We believe that XF media and highly efficient hMSC systems will benefit and accelerate the clinical translation of therapeutics, as well as the industrialization and commercialization of Regenerative Medicine cellular based products.
Design, Fabrication, and Mechanical Properties of High Resolution 3D Printed Tissue Engineering Scaffolds
Department of Plastic Surgery, The Ohio State University, Columbus, OH.
A goal in tissue engineering (TE) is to regenerate tissue that has been lost to trauma or disease. Of primary importance is the design and fabrication of the scaffold, which serves as a temporary platform to hold biofactors and facilitate the growth of neotissue. Advances in the synthesis of poly(propylene fumarate) (PPF) allow for oligomers with highly controllable molecular weight and low PDI, while additive manufacturing (AM) allows for the production of complex scaffolds. Using a custom algorithm implemented in MATLAB, we show that gyroid-derived scaffolds can be created with porosity ranging from 2% to 98%, continuous curvature, and uniform mass distribution. Furthermore, the pore and strut dimensions can be easily tailored on a per case basis. Scaffolds were fabricated out of a PPF/DEF resin by photocrosslinking on an EnvisionTEC mask projection stereolithography unit with high resolution (X,Y: 42 μm; Z: 50 μm). We demonstrated fabrication with scaffold strut sizes as thin as 125 μm. Unconfined mechanical compression (ASTM D695) of specimens manufactured at vertical and 45 degree angles showed no significant difference in yield stress (P = 0.393) or Young's Moduli (P = 0.520). Testing of porous structures yielded the same results. Mechanical properties were significantly lower (P < 0.05) than cast specimens. Therefore, we find that orientation of layers does not have a significant effect on the mechanical properties of AM PPF constructs, though the AM process as a whole does. For example, the surface texture is dependent on build plane orientation.
One of the motivations in engineering cardiac tissue is inducing cell alignment for effective contractile force. Methods such as patterning growth factors, cell seeding on anisotropic scaffolds and electromechanical stimulation have been commonly used to direct cell alignment. These methods have shown only cell alignment in 2D planar manner which is unrepresentative of the 3D configuration of a native myocardium. In this work, we bioprinted a tissue construct with 3D variation in term of cell orientation and layer thickness as per design blueprint. Gelatin/Alginate/Gelatin Methacrylate containing 10E6/mL of C2C12 cells was printed using extrusion-based bioprinting technique. In order to recapture variations in the orientation of cell alignment across the native myocardium, a 0–90° grid pattern was designed with pores to facilitate nutrient transport. Cell-hydrogel struts of 224 ± 41 um were obtained. Cell alignment across struts of different plane was observed at day 7 of culture without any external stimulant. C2C12 cells displayed alignment parallel to the direction of bioprinting, with distinct difference in cell alignment orientation at different plane. This method is simple with high potential to scale up for organ printing.
Synthetic Hydrogel Microcarrier Coatings for Cell Expansion
Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI.
Lunenfeld-Tanenbaum Research Institute, Toronto, ON, CANADA.
Tissue Spheroids (TS) formed by aggregates of cells have been widely used in the field of tissue engineering. The cell viability and the increase of proliferation rate are function of the contact of cells with the culture medium. Nutrition, organization and growth in cells are largely determined by diffusion mechanisms. Due to the characteristics of a 3D environment, some zones within the TS are not equally exposed to the environment conditions impacting in the formation of microenvironments with decreased oxygen rates, nutrients and soluble factors produced by cellular metabolism which leads to the formation of low proliferation areas and therefore necrosis (cell death). Lockyballs can act as supports for cells to increase the diffusion of nutrients and oxygen in the TS. We also present a biological model that demonstrates the cell growth using CompuCell3D (CC3D) software. The first stage of the work consisted in the generation of different 3D models by the Computer Aided Design (CAD) software Rhinoceros 5.0. The CAD model was imported into finite element method software (ANSYS R16) and into volume element method software (Star-CCM) to perform computational simulation, which consisted of structural analyses and computational fluid simulation (CFD). CFD simulations were essential to predict the diffusion phenomenon in the lockyball. CC3D simulation allowed rapid and intuitive simulation of cellular and multi-cellular behaviors in the context of tissue formation. The development of new microscaffolds models can enhance the regenerative capacity of 3D tissues and computational simulations were essential to predict an optimal design and other critical aspects.
Expansion of Adipose-derived Stem Cells Using Defined 3D Scaffolds and Perfusion Bioreactor for Stem Cell Therapy
Stem cell therapy requires millions to billions of stem cells grown in GMP level facilities. The microenvironment of cells can affect the yield and quality of stem cells used for therapy. Conventional 2 dimensional (2D) cell culture does not mimic how cells grow in vivo and does not depict the 3 dimensional (3D) microenvironment of cells. Growing cells in 3D more closely mimics the physiological condition of cells. At 3D Biotek we have developed a polystyrene scaffold that allows the cells to be grown in 3D mimicking how cells are grown in the body. Our previous work has shown that adipose-derived stem cells (ASCs) can grow on these scaffolds and can be induced to differentiate into osteogenic, adipogenic, and chondrogenic lineage specific cell types. Here we show that ASCs grown on 3D scaffolds can be expanded using our novel perfusion bioreactor. After 2 weeks in the bioreactor, ASCs were expanded by almost 16 fold and flow cytometry analysis demonstrated that these cells maintained their cell identity and “stemness” after the 2 week period. Using our bioreactor not only we were able to expand stem cells, but compared to other bioreactors currently being used, our system has much better ease of use and minimizes space, reagents, labor and ultimately cost without affecting quality and ultimately result in the potential for large scale expansion of stem cells for stem cell therapy.
Extracellular Matrix Proteins Support A Native Phenotype Of Human Liver Endothelial Cells And Increase Their Proliferation In Vitro
Liver Sinusoidal Endothelial Cells (LSECs) have specialized functions, essential for liver function. However, we are unable to expand LSECs in vitro because they lose their normal phenotype when outside of their microenvironment. We hypothesize culturing LSECs on liver ECM would maintain a native phenotype and growth in vitro. LSECs use integrins to interact with their surrounding ECM. Collagen is a receptor for integrins α1β1 and α5β1. In a fibrotic or diseased state LSECs begin to express laminin-binding integrins α6β1 and α2β1. Thus, integrin expression in LSECs could determine if the cells maintain a healthy phenotype in vitro. In our studies, human LSECs (hLSECs) (purchased from ABM Inc.) were plated on different ECM proteins in different concentrations: Plastic, Collagen-I: 5μg/cm2, Fibronectin: 1μg/cm2, Laminin: 1μg/cm2 and were grown in culture for 1 week. In parallel, hLSECs were seeded on decellularized liver scaffold discs. Immunofluorescence and qRT-PCR were used to test for hLSEC phenotype. Cells were counted daily to determine cell proliferation. Our results show hLSECs plated on plastic did not express integrins. Integrins α5 and β1 are expressed by cells when plated on collagen and fibronectin coated plates. There was little to no expression of α5, but an increased expression of integrin α6 on laminin coated plates. Our results also show that hLSECs proliferate better in the presence of fibronectin and collagen as opposed to plastic and laminin. These results indicate that we are able to recapitulate the native LSEC phenotype in vitro by providing the cells with specific liver ECM proteins.
Crossing Kingdoms: Using Decellularized Plants as Pre-Vascularized Tissue Engineering Scaffolds
Worcester Polytechnic Institute, Worcester, MA.
A major element limiting the clinical translatability of tissue-engineered grafts is the lack of a perfusable vascular network. Most current biofabrication techniques are unable to provide patent microvasculature, effecting the required oxygen diffusion in engineered tissues. Current focus has shifted towards biomimetic approaches, such as whole organ decellularization. However, mammalian tissues are in short supply, variable, and extensive research is needed before they become a viable clinical option. A more readily available and tunable tissue type could yield improved graft availability at reduced costs.
There are surprising structural similarities between plant and animal vasculature, despite different mechanisms of fluid transport. We, thereby, decided to investigate if plants and their innate vasculature could serve as perfusable scaffolds for tissue engineering. Standard perfusion decellularization techniques were applied to different plant species, including spinach leaves. Leaf vasculature remained patent post-decellularization and were able to support transport of microparticles ranging from 1–100 μm in diameter. The smallest particles, which are similarly sized to a red blood cell, could be perfused completely through the scaffold. In order to investigate the decellularized leaf's use as a tissue-engineering scaffold, leaves were successfully recellularized with a variety of human cells. Endothelial cells, mesenchymal stem cells, fibroblasts, and human pluripotent stem cell-derived cardiomyocytes all adhered to the surface and remained viable for up to 21 days. By crossing kingdoms, we demonstrated the potential use of decellularized plants to act as pre-vascularized scaffolds for tissue engineering applications. Their use could provide a cost-efficient, “green” technology for creating vascularized mammalian tissues.
Biomedical Engineering, Rensselaer Polytechnic Institute (RPI), Troy, NY.
Reconstructing functional volumetric tissue in vivo following implantation remains a critical challenge facing cell-based approaches. Several pre-vascularization approaches have been developed to increase cell viability following implantation. Structural and functional restoration was achieved in a preclinical rodent tissue defect; however, the approach used in this model fails to repair larger (>mm) defects as observed in a clinical setting. We propose an effective cell delivery system utilizing appropriate vascularization at the site of cell implantation that results in volumetric and functional tissue reconstruction. Our method of multiple cell injections in a progressive manner yielded improved cell survival and formed volumetric muscle tissues in an ectopic muscle site. In addition, this strategy supported the reconstruction of functional skeletal muscle tissue in a rodent volumetric muscle loss injury model. Results from our study indicate that our method may be applicable to repair larger volume damaged tissues with the potential for clinical use.
In Vitro Microvasculature Model Using Genetically Engineered Cells
LiMMS/CNRS-IIS (UMI 2820), Institute of Industrial Science, The University of Tokyo, Tokyo, JAPAN.
The endothelial cells, which form the luminal tissue lining blood vessels, are involved in sprouting angiogenesis, a process by which new blood vessels form and which is important to the development and functions of most organs. Angiogenesis is also involved in diseases such as vasoproliferative retinopathies and cancers. We develop in vitro models of vasculatures in a healthy or diseased state in order to provide the vascular biology research field with alternative tools to existing assays. In this study, we generated a micrometer-scale tubular scaffold within a collagen matrix by using a needle-based tissue-engineering technique. The scaffold was prepared within a PDMS chip made by easy-to-use microfabrication techniques. We genetically altered human umbilical vein endothelial cells (HUVECs) by RNA interference, seeded them within the tubular scaffold and allowed them to grow. Finally, we exposed the cells to the angiogenic protein VEGF in order to study the angiogenic capacity of our model. The engineered cells formed a vascular tube, as shown by the formation of a monolayer with a central lumen and the establishment of adherens-junctions between the cells. Furthermore, they were able to from angiogenic sprouts under VEGF treatment. These results show that genetically engineered HUVECs can be used to form an in vitro vascular model. Therefore, it should be possible to study the effect of endothelial cells defects on endothelium properties and functions using such new devices.
Children's Mercy Hospital, Kansas City, MO.
Bone marrow mesenchymal stem cells (MSCs) have been investigated as a potential cell population for tissue engineering heart valve (TEHV) scaffold seeding; however, MSCs constitute only a small fraction of the larger mononuclear cell (MNC) population. Lengthy expansion periods are required prior to scaffold seeding. The objective of this study was to evaluate the efficacy of direct scaffold seeding with MNCs, followed by bioreactor conditioning, as a means of generating MSC populations on the decellularized aortic valve leaflet. Human aortic valves were decellularized, seeded with human MNCs, and conditioned in a pulsatile bioreactor for 3 weeks. Recellularization, cell phenotype, and leaflet biomechanics were evaluated. Following conditioning, the leaflet surface showed extensive cellular coverage, though regions of cell layering were observed. Regions of cellular penetration into the interstitium of the leaflet were observed; however, areas devoid of cells were also present and cell density remained below that of the native leaflet. Real-time PCR demonstrated a >10 fold (Log2RQ) up-regulation of the MSC markers CD72 and CD90, as well as down-regulation or absence of macrophage (CD68) and leukocyte markers (CD45), compared to the initial MNC population. Under biaxial loading, circumferential (1.18 ± 0.09) and radial (1.74 ± 0.10) stretch ratios were similar to cryopreserved and decellularized samples (p > 0.065). The results of this study indicate the MSC sub-fraction within the larger population of MNCs dominates leaflet recellularization following bioreactor conditioning. This may be a useful strategy, as it eliminates the need to isolate and expand the MSCs prior to scaffold seeding.
Wyss-Harvard and BU, Boston, MA.
Several mechanical pathways coordinate cell adhesion and migration during morphogenesis. In both angiogenesis and maintenance of vascular barrier function, the interplay between cytoskeletal forces and cell polarization is not completely understood for lack of 3D organotypic in vitro systems allowing for monitoring events in real time and at single cell level. Here, we engineered a vascular biomimetic platform conductive to multicellular seeding, fluid flow, and application of soluble gradients. We investigated the role of endothelial VE Cadherin and pericyte N Cadherin as important mediators of the supracellular cohesion. In detail, we found that VE Cadherin can sense cytoskeletal tension and rearranges its topographical distribution at cell-cell junctions, thus promoting collectiveness and compressive stretch of invading capillaries. On the perivascular side, N Cadherin is spatially rearranged at the intercellular junctions following to inflammation-mediated changes in the RhoA and Rac1 activation state. In our model, vascular integrity depends on VE Cadherin and N Cadherin-based adhesions strengthening. Overall, our in vitro system opens new avenues for developing proangiogenic and antivasculitis therapeutic strategies specifically directed against andothelial and perivascular molecular targets.
1. Loo C. Y., Sudesh K.:. MPJ, 2, 2, 31–57, 2007.
2. G. Q. Chen, Chem. Soc. Rev. 38:2434–2446, 2009.
The authors would like to thank technical staff from University College London. Partial funding was provided by the European Commission's “Seventh Framework Programme” under Grant Agreement No. 604251 (ReBioStent).
Controlled Presentation of Growth Factors to MSC/HUVEC Co-Culture Aggregates with Incorporated Gelatin Microparticles for Microvascularized Tissue Engineering
1. Rouwkema J, et al. Trends Biotechnol. 26, 434–441, 2008.
2. Dang PN, et al. Stem Cells Transl Med. 5(2), 206–17, 2016.
Engineered Organotypic Human Culture Model To Examine Morphogenetic Fusion In Vitro
Heterotypic cell-cell interactions regulate tissue fusion during embryonic development. For example, sonic hedgehog and fibroblast growth factor (FGF) signaling between palatal epithelial cells and palatal mesenchyme mediates palatal fusion. Morphogenetic fusion during heart development, neural tube closure, and palatal fusion are all dependent on epithelial-mesenchymal interactions (EMIs) and have largely been studied with mouse models and Ex Vivo organ cultures. We sought to engineer embryonic palate-like tissue in vitro using spheroid culture of human mesenchymal stem cells (hMSC). hMSC spheroids exhibited osteogenic phenotype (increased alkaline phosphatase activity and increased BGLAP and RUNX2 gene expression) upon culture in osteo-induction medium, which suggests that spheroid culture supports osteogenic differentiation. We developed a novel procedure to coat osteogenic hMSC spheroids with human primary epidermal keratinocyte progenitor cells (hPEKp) and demonstrated that heterotypic cell spheroids fused over the course of 4 days in non-adherent culture. FGF inhibition reduced fusion as quantified by the persistence of epithelial cells in the contact zone between spheres, which agrees with FGF-dependence of palatal fusion. Further, perturbation of epidermal growth factor signaling reduced spheroid fusion, which corroborates evidence of a crucial EGF/TGFβ3 switch that could be a target for cleft palate teratogens. This prediction also agrees with increased EGFR expression observed during spheroid fusion. We hypothesize that engineered hMSC/hPEKp spheroids will enable interrogation of signaling pathways crucial to morphogenetic fusion for predictive toxicology. This abstract does not necessarily reflect EPA policy.
3D Bioprinted In Vitro Cardiac Tissue Model
Bioengineering of functional cardiac tissue constructs composed of cardiomyocytes has many utilities, including surgical repair and enhancement of cardiac tissue as well as developing an in vitro tissue model for drug discovery, screening and toxicity studies. However, the complexity of myocardium, structurally and functionally, still presents many challenges for these applications. The cardiac tissue possesses highly organized structures with unique physiological and biomechanical properties. In this study, we applied 3D bioprinting strategy to fabricate functional and contractile cardiac tissue constructs. Rat neonatal heart tissues were obtained to isolate cardiomyocytes, and the cells were suspended in a fibrin-based hydrogel bioink. Cell-laden hydrogel was printed through a 300-micron nozzle by pneumatic pressure. The bioprinted cardiac tissue constructs showed spontaneous contraction after 3 days post-printing and demonstrated synchronized contraction after 14 days in culture, indicating of cardiac tissue development and maturation. Cardiac tissue formation was confirmed by immunostaining with antibodies specific to α-actinin and connexin 43, which showed aligned, dense matured cardiomyocytes. The bioprinted cardiac tissue constructs also showed physiological responses (beating frequency and contraction forces) to known cardiac drugs (epinephrine and carbachol). Moreover, tissue development of the printed cardiac tissue could accelerate by Notch signal blockad. Our results demonstrated the feasibility of printing functional cardiac tissues that could be used as a reliable and reproducible model pharmacological applications.
Spheroid Culture System Confers Differentiated Phenotype and Functional Advantage to Maturing 3T3-L1 Adipocytes
Biomedical Materials Science, University of Mississippi Medical Center, Jackson, MS.
An In Vitro Chondro-Osteo-Vascular Triphasic Model of the Osteochondral Complex
Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA.
1. Alexander PG et al. (2014) Exper Biol Med 239, 1080.
2. Lozito TP et al. (2013) Stem Cell Res&Ther 4(Suppl 1), S6.
Nanoscience and Microsystems, University of New Mexico, Albuquerque, NM.
Drugs tested on animal models and on humans do not always produce the same results in both species; the divide between the two can be bridged by a reliable in vitro lung model. Because alveoli is a target for several drugs, an alveolar model can be a platform for both designing drugs and studying lung diseases. It should allow for gas exchange, growth of alveolar epithelial cells, extracellular matrix production, and have similar mechanical properties to alveoli, creating an environment conducive to normal metabolic activity and cellular responses. Existing artificial lung models that use polymeric membranes to inhabit lung cells are limited by the lack of biocompatibility or the necessary strain profile. We report the fabrication of flexible polyurethane membranes of 15 μm thickness, which imitate the alveolar wall, using spin coating techniques, and an efficient technique to consistently handle and integrate these delicate membranes into microfluidic devices. The membranes were characterized for their elastic properties using a microfluidic-based bulging test system. 6 kPa fluidic pressure was applied to execute cyclic stress on the membranes for two weeks without failure. Finally, Human Small Airway Epithelial Cells (HSAECs) were cultured on the polyurethane membrane. Increased surfactant and aquaporin production were observe compared to standard culture plates and also investigated the effect of mechanical stretch on the airway epithelial cells. Our results suggest a use of this artificial alveolus in the development of an effective platform for rapid drug screening.
LA-UR-16-23747
Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA.
CIBiS, The University of Tokyo, Meguro-ku, Tokyo, JAPAN.
The vascular barrier is one of the important function to understand an effect of drugs and role of cancer microenvironment. Blood vessel increases its permeability in response to inflammatory factors, such as vascular endothelial growth factor (VEGF). To measure vascular permeability, Miles assay is used to evaluate permeability in vivo. But detailed biological functions are difficult to assess in animal experiment. On the other hand, a transwell assay which uses a monolayer of endothelial cell onto a semi-permeable membrane is used as an in vitro assay. However, this technique has limitations as it fails to see the vascular functions in a tubular structure and the cellular interactions with the extracellular matrix. Here we developed an in vitro microvasculature system to evaluate vascular permeability. The microvasculature was prepared by culturing human umbilical vein endothelial cells (HUVECs) within the micro-fabricated collagen gels. We investigated the barrier function of microvascultures with the marker of adherens junction VE-cadherin, and a fluorescent molecule. Following exposure to VEGF, we introduced fluorescent dextran (70 kDa) into the microvasculatures. The vascular permeability was evaluated by using confocal microscopy to observe the leakages of the fluorescent molecule into the surrounding collagen gel. We observed that VEGF led to a ruffling of cell membranes and an increase of vascular permeability. Moreover, VEGF-induced vascular permeability was prevented by pre-incubating the vasculatures with vascular permeability inhibitors/modulators. These results showed that an in vitro vascular model can be a reliable method to study drugs targeting vascular permeability.
Biomedical Engineering, Worcester Polytechnic Institute, Worcester, MA.
Tissue engineered blood vessels (TEBVs) have the potential to serve as 3D in vitro models to study disease and test prospective therapies. Certain vascular diseases, such as intimal hyperplasia, are characterized by focal changes in smooth muscle cell (SMC) phenotype within vessel walls. However, current methods for fabricating TEBVs create homogenous tubes not conducive to simulating focal heterogeneity. To address this challenge, we developed a modular approach for fabricating homogenous or heterogeneous TEBVs from fused ring segments. Human SMCs were seeded into agarose molds where they self-assembled into cell-only tissue rings. We also demonstrated that degradable gelatin microspheres can be incorporated within rings during self-assembly to mediate localized growth factor delivery. The goal of this study was to fabricate TEBVs with focal regions of microsphere incorporation and evaluate fusion between rings with and without microspheres. First we assessed ring fusion and spatial localization of SMCs within tubes by fabricating rings without microspheres from SMCs pre-loaded with alternating fluorescent red or green CellTracker™ dye and acquiring images daily over 7 days of fusion. Cells maintained spatial positioning within tubes, suggesting SMCs remain localized to their respective ring segments following fusion. We then fabricated tubes with focal regions of fluorescently-labelled rings with incorporated microspheres, and demonstrated retention of microsphere spatial positioning following ring fusion. Our modular approach to fabricating TEBVs may be used for spatially-controlled microsphere-mediated growth factor delivery, potentially creating localized SMC phenotypic changes characteristic of diseases such as intimal hyperplasia.
Wake Forest Institute for Regenerative Medicine, Winston Salem, NC.
Detailed architectural arrangement within in vitro organ-on-a-chip (OC) platforms is important to their successful application [1]. OC platforms are often fabricated with customized PDMS structures. A wide variety of human OC models have been created, but rarely have there been reports of fabrication of these devices using extracellular matrix (ECM)-like materials to generate the devices themselves [2]. We have fabricated an ECM-inspired hydrogel device with a high density of human derived cells and an integrated endothelial 3D microfluidic network. A suspension of HepG2 cells in 10% gelatin methacryloyl (GelMA) was partially UV cross-link, utilizing a 3D microfluidic mold. The hydrogel microfluidic layer was placed on top of a bottom layer, followed by full UV cross-linking and fusion of the system, creating a closed perfusion system within the GelMA device. Subsequent perfusion of HUVECs for seeding was performed inside the microfluidics. We have developed a viable cell-loaded hydrogel construct along with an integrated endothelialzed 3D microfluidic network that integrates two different cell types in one single system. We have also incorporated a plug-in system inspired by LEGO complementary female/male features. These discreet features will allow us to discard the use of tubing or metal connectors to bring together multiple modular organ system-based devices.
1. Huh, D. Nat. Protocols.
2. Oleaga, C. Scientific Reports.
Developing an Improved Three-dimensional Co-culture System of Hepatocytes on Silk Scaffolds
Creating long-life fully functional liver tissue in vitro is highly sought after for bioartificial liver (BAL) development, drug discovery and toxicity screening or even transplantation therapy. However, despite the exceptional regenerative potential of the liver tissue in vivo, the issue of replicating the viability and function of the native hepatocytes in vitro has yet to be fully addressed. This is mainly due to the “dedifferentiation” of hepatocytes after their isolation in vitro. In the present study, an improved 3D co-culture system of hepatocytes was established in which rat primary hepatocytes were co-cultured with hepatic stellate cells, on silk porous scaffolds. Silk scaffolds with incorporated extracellular matrix provided a suitable microenvironment for maintaining the viability, morphology and gene expression of the primary hepatocyte in vitro. The presence of stromal cells promoted primary hepatocyte to generate cellular aggregates with well-organized 3D architecture. These aggregates exhibited proper morphology similar to liver tissue in vivo. A well-maintained functionality of hepatocytes was also observed in the co-cultures, evidenced by their significantly increased albumin secretion and urea synthesis when compared to the monolayer cultures. Additionally, this 3D multicellular culture model displayed a better-maintained metabolic activity of CYPs during the cultivation. Thus, the co-culture model constructed offers a robust tool to better understand cell-cell/cell-ECM interactions and other elements involved by the tissue microenvironment modulate hepatic tissue phenotype and functions, allowing us to gain fundamental biological insight as well as develop functional tissue constructs for therapeutic and drug-screening applications. (This work was supported by NSFC81173125 and 31470952).
Development of A Novel Renal Organoid Model Using Whole Kidney Cells for Drug Screening
Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC.
Motorneuron (MN) diseases are progressive, debilitating disorders resulting from the loss of muscle innervation. In vitro models of the neuromuscular junction (NMJ), the connection between MNs and muscle fibers, have been developed to examine mechanism(s) causing dysfunction associated with specific MN diseases; however these co-culture models with myotubes and MNs have randomly oriented myotubes with immature synapses that contract asynchronously. Mechanically patterned (MP) extracellular matrix with alternating soft and stiff stripes improve current in vitro NMJ models by inducing myoblast durotaxis to stripes where they align and differentiate to form patterned myotubes. MP substrates support increased differentiation, fusion, significantly larger acetylcholine receptor (AchR) clusters, and increased expression and localization of MuSK and Lrp4, two cell surface receptors required for NMJ formation. Primary MNs cultured with patterned myotubes formed functional synapses that more faithfully recapitulate mature in vivo NMJs by supporting extensive neurite outgrowth with processes that co-localized with myotube AchRs. Most importantly, robust spontaneous contractions were observed, indicating that functional synapses were developing. Co-cultures containing myotubes and embryonic stem cell-derived motorneurons expressing channelrhodopsin were optically stimulated to demonstrate “on-demand” NMJ functionality and connectivity via individual myotube contraction. The mechanically patterned NMJ platform described here represents a significant improvement over current in vitro NMJ systems by creating a NMJ-in-a-dish model that more accurately recapitulates in vivo physiology, enables the exploration of patient-derived cells to elucide disease pathology, and creates a better platform for NMJ drug discovery.
Model of Microvascular Pulmonary Inflammation Modulated by Extracellular Matrix Mechanical Properties
Bioengineering, Temple University, Philadelphia, PA.
The overall goal of this research is to elucidate the effects of matrix mechanics and composition on the activation of pulmonary endothelial cells by inflammatory cytokines. The hypothesis to be tested is that increasing matrix stiffness in the (patho) physiological range will exacerbate the response of cultured endothelial cells to inflammatory stimuli. To test this hypothesis, we are culturing control and TNF-alpha stimulated rat pulmonary microvascular endothelial cells on hydrogels with tunable stiffness between ∼2 kPa to ∼20 kPa (controlled using rheometry and compression testing), modeling the stiffness of healthy vs. fibrotic lung tissue respectively. As cellular readout we are assessing, through RT-qPCR, immunocytochemistry, and monocyte adhesion, basal expression and up regulation of vascular cell adhesion molecule-1 (VCAM-1) in quiescent and TNFa stimulated cultured endothelial cells. Our preliminary data indicates that basal expression of VCAM-1 increases in response to stiffer substrates, suggesting that pathological tissue stiffening (e.g. in pulmonary fibrosis) could exacerbate endothelial cell responses to inflammatory insult. Currently, studies are underway to evaluate the cells' response to stimulation with TNFa. Our model of microvascular pulmonary inflammation, mimicking normal and fibrotic lungs, is aimed at establishing a correlation between substrate stiffness and inflammation. Our results will contribute to a mechanistic understanding of disease pathologies such as idiopathic pulmonary fibrosis, in which treatment is just about limited to a full lung transplant and facilitate testing of new drug therapies.
Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA.
Juvenile Osteochondritis Dissecans (JOCD) is an increasingly common condition that predominantly affects adolescent and young adults, and progresses to early onset osteoarthritis. JOCD involves the formation of an avascular lesion in the subchondral bone with secondary effects in the overlaying articular cartilage. Previous research has been limited to retrospective clinical studies, and the pathophysiological mechanisms associated with the onset and progression of JOCD have yet to be fully characterized. There is a need for improved understanding of the pathological mechanisms involved in JOCD in order to develop more effective treatment options and prevent joint degeneration in this pediatric population.
The purpose of this study is to establish pluripotent stem cell-based in vitro models of JOCD chondrogenesis and osteogenesis. We generated and validated iPSCs from 2 healthy and 2 JOCD pediatric patients. Characterization of iPSCs (IHC, PCR, and embryonic body formation) confirmed the pluripotent nature of the cells. Subsequent differentiation to mesenchymal stem cells was confirmed using FACS analysis, PCR and trilineage differentiation analysis. We hypothesize that our iPSC in vitro models of JOCD will show protein dysfunction and accumulation in the rough endoplasmic reticulum as a hallmark of the disease, as seen in familial and equine OCD. Using these patient-specific pluripotent stem cell in vitro models, we aim to elucidate the cellular pathophysiology of JOCD as well as provide a test bed for therapeutic interventions.
Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA.
2D osteogenic culture is widely used but known to be poorly reflective of the in vivo cell niche. With the application of induced pluripotent stem cells (iPSCs) to disease modeling, there is a critical need for a robust 3D osteogenic culture method. Despite positive characterization of iPSC-derived mesenchymal-like stem cells (iPSC-MSCs) by surface markers and multipotency, current studies have shown that iPSC-MSCs display gene expression profiles of the paraxial, lateral and intermediate mesoderm lineages over time, which are different from those seen in primary preparations of MSCs and which may influence their osteogenic differentiation potential. The goal of this study was to develop a robust method for 3D differentiation of patient-specific iPSC-MSCs. The hypothesis underlying this study is that pre-conditioning of iPSC-MSCs with osteogenic media yields a more homogeneous population of pre-osteoblastic cells, which in turn exhibit a higher osteogenic potential in a scaffold-free 3D culture system. We used previously characterized iPSC-MSC lines from healthy donors with no known skeletal diseases. Pre-conditioned cells were cultured in monolayer in osteogenic differentiation media for 7 days. Micromass cultures were harvested from preconditioned and non-preconditioned groups after 7, 21 and 35 days in osteogenic media. Both groups demonstrated osteogenesis by Von Kossa and ALP staining; however, the pre-conditioned groups showed greater calcium deposition. Data presented will include characterization of the extracellular matrix, including osteocalcin, osteopontin and collagen immunostaining.
Biomedical Engineering, Korea University, Seoul, KOREA, REPUBLIC OF.
This study describes that a simple microfluidics 3D compartmentalized culture systems which have employed mixed culture of Cornea fibroblast Keratocyte cells and epithelial cells in 3D gels. And the effective of flow rate and viscosity on the microchannel dimensions are investigated through the use of the commercial CFX 16.1, ANSYS CFX Inc. This microfluidics system enables information-rich in vitro assays. CFD results that the effects of fluidic density and pressure at each channels was simulated in figure 2. Figure 3 shows that image of co-culture of Keratocyte cells and EP cells in 3D microfluidic system. The Keratocyte cells and EP cells in compartmentalized culture is observed at interface. In conclusion, our PDMS-based 3D microfluidics cell culture system may help overcome barriers to a limitation in a scientific method to investigate pathogenesis and treatments of TGFBI-linked corneal dystrophy.
ICVS/3B's Associate Laboratory, University of Minho, Guimaraes, PORTUGAL.
Osteoarthritis (OA), a prevalent chronic condition with a striking impact on life quality, represents an enormous societal burden that increases greatly as populations' age. Yet no approved pharmacological intervention, biologic therapy or procedure prevents the progressive destruction of the OA joint. Based on bilayered structures that have been previously suggested for osteochondral (OC) applications (Oliveira et al. 2006) and on the potential of methacrylated gelatin (GelMA) and methacrylated gellan gum (MAGG) for different tissue engineering applications (Silva-Correia et al. 2013, Tasoglu et al. 2014), we set a dynamic platform for the in vitro recreation of an OA 3D in vitro model. Since OA is an inflammatory and degenerative disorder affecting cartilage and subchondral bone, we created 6 hybrid formulations recreating a 3D controlled subchondral bone and cartilage integrated microenvironment. Fat pad adipose derived stem cells (ASCs) were isolated from Hoffa's body obtained from healthy Patients, characterized by flow cytometry and their performance in the developed 3D structures assessed. GelMA formulation showed the best cell adhesion and proliferation, but the life-time of this one in culture is shorter due to the faster degradation in vitro comparing to MAGG based structures. According to this we proceeded with the best hybrid formulation, GelMA-MAGG 2:1, for OC co-differentiation using a dual-chamber bioreactor designed for the establishment of co-cultures in a single 3D structure (Canadas et al. 2014). This approach solved challenges of 3D cell culture in interfaced tissues as OC and will ultimately be used for OA in vitro modeling.
Intra-articular administration of corticosteroids and viscosupplements is widely used to treat osteoarthritis (OA) with inconsistent results. Numerous drugs exist with potential to modify OA when administered via intra-articular injection, but are limited by rapid clearance from the joint through synovium. Synovium is comprised of multiple cell populations and a high density of draining lymphatic vessels that contribute to short drug residence times (e.g., 10 minutes for 370 Da). Our goal is to engineer a 3D model synovium and fluidic chamber to reproduce the intra-articular, synovium, and lymphatic compartments of the diarthrodial joint as a drug-testing platform. Morselized human synovial tissue was conjugated to an amine-reactive polymer to control model porosity. The model construct was incubated within a fluidic test chamber with both water and a bolus of drugs to estimate permeability and t1/2 of drug clearance, respectively. In a second approach, the model was crosslinked with synoviocytes type A (macrophages) and type B (fibroblasts), and overlaid with a human fibroblast-like synoviocyte cell line (FLS) to form a “leaky” barrier representative of native synovium. Cellular constructs were challenged with TNFα or IL1β to simulate OA pathology followed by bolus delivery of therapeutic drug candidates. In vitro values for drug clearance and drug activity in model synovium can be used to benchmark test drugs and screen drug doses and formulations of relevance to intra-articular drug delivery.
A Microphysiological Approach to Elucidate Factors Disrupting Epithelial:Mesenchymal Interactions in Orofacial Clefting
Biomedical Engineering, UW-Madison, Madison, WI.
Proper cell–cell contact and communication are essential for the correct development and survival of higher order organisms. In order to study complex cell interactions that occur in vivo, model systems that are able to recapitulate high density 3D cell–cell interactions in vitro are key to advancing new biotechnologies, therapeutics, and tissue engineering applications. Herein, we show a new strategy to rapidly and efficiently generate complex multiple cell line containing spheroids and tissues in microfluidic flow without the use of scaffolds, molecular biology, or metabolic biosynthesis. We demonstrate this strategy by assembling various combinations of cell types with an interfacial cell to cell click chemistry in microfluidic flow to generate a range of spheroid types and oriented tissue multilayers composed of stem cells. The method relies on the integration of microfluidics, liposome fusion, bio-orthogonal chemistry, and cell surface engineering to rapidly click co-culture cell assemblies in flow. The scaffold-less constructs were imaged with brightfield microscopy, fluorescence microscopy, and confocal microscopy alongside cell staining for stem cell differentiation studies to visualize and study both spheroid assembly and tissue formation. These results are significant as a first step towards recapitulating high cell dense tissues such as liver, heart and neural tissue with microscale architecture, using a simple, mild and rapid methodology which forms profuse cell-cell contacts influencing stem cell differentiation.
O'Brien PJ., Luo W., Rogozhnikov D., Chen J., Yousaf MN. Spheroid and tissue assembly via click chemistry in microfluidic flow. Bioconjug Chem. 26. 1939–1949 (2015).
Electrophysiology and Drug Responses of Engineered Heart Slices
Geometric Microwells with Synthetic Substrates for Neural Organoid Generation
Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI.
Reference
1 Lancaster et al. Nature 501, 373, 2013.
Biomedical Engineering, Columbia University, New York, NY.
Cardiac tissues formed from human induced pluripotent stem (hiPS) cells have limited utility due to their immature, fetal-like phenotype. We report the development of adult-like human heart muscle from co-cultures of human induced pluripotent stem cell derived cardiomyocytes and supporting fibroblasts in native hydrogel subjected to three weeks of electromechanical conditioning at an increasing intensity. The resulting engineered tissues recapitulated many of the molecular, structural, and functional properties of adult human heart muscle, including well-developed registers of sarcomeres, networks of T-tubules, mature calcium homeostasis, comprehensive responses to beta-adrenergic stimulation, and a positive force-frequency relationship. The level of maturation was confirmed by comparison to human fetal heart tissue, and probed by studying physiological responses to drugs and by modeling heart disease. The measured drug EC50 values corresponded to plasma levels in patients, while the patient-specific model of Timothy Syndrome recapitulated the disease phenotype and the effects of clinically used drugs. We show here that adult-like human heart muscle can be engineered in vitro and used for predictive studies of drugs and modeling of cardiac disease.
Institute for biomaterials and biomedical engineering, University of Toronto, Toronto, ON, CANADA.
An in vitro co-culture model of nerve and skeletal muscle cells can serve as a useful platform for studying neuromuscular junction (NMJ) formation, maturation and function as well as neuromuscular disorders. Here we report a 3D culture model of innervated skeletal muscle tissue generated from human myogenic cells co-cultured with motor neurons derived from human embryonic stem cells (ESCs). 3D aligned muscle fibers produced from human cells demonstrated clustering of acetylcholine receptor (AChR) as shown by α-bungarotoxin staining. ESC derived motor neurons were able to innervate the muscle fibers and form NMJ within 14 days of co-culture as confirmed by co-localization of AChR and synaptic vesicle glycoprotein synaptophysin on striated muscle fibers. Analysis of AChR cluster size and number revealed that the presence of motor neurons increases muscle fiber maturation. Indeed expression of adult subunit of the AChR (epsilon) revealed a progressive maturation of the NMJ with time in 3D co-cultures. To confirm the functionality of NMJs, calcium handling and contraction forces of muscle tissues were evaluated following biochemical activation of motor neurons using glutamate. In addition our electrophysiological measurements revealed the functional connectivity between our muscle fibers and motorneurons. Action potentials were recorded using sharp microelectrode recoding of myofibers post biochemical stimulation of neurons. Our results indicate that our 3D culture platform supports the maturation of neuromuscular junction better than current 2D culture models. We envision that our platform will be used as a model to study neuromuscular disorders and as a drug-screening platform for preclinical studies.
The generation of biomimetic in vitro tissue models that can recapitulate the physiological functions of native tissue has been suboptimal due to the challenge of patterning cells in a 3D and precise manner. Depending on the type of tissue, different types of cells are organized in specific three-dimensional spatial arrangements across the extracellular matrix. For instance, hepatocytes and fibroblasts in the liver lobule are not in direct physical contact, but they must be spatially close enough to exchange biochemical cues that help maintain the differentiated functions of the hepatocytes. Microfluidic technology can produce monodisperse cell-laden microgels with programmable microenvironment, but accurately and reproducibly patterning cells in desired spatial configurations is still a big challenge, which requires judicious device designs and careful selection of biomaterials. Here, we used a poly(dimethylsiloxane) microfluidic device to generate double-layered microgels by flowing a sodium alginate precursor solution with cells, a calcium chloride crosslinking solution, and a methacrylated gelatin (Gel-MA) solution with cells through an optimized tripod-shaped microchannel, which formed an alginate core upon contact between Ca2+ ions and alginate molecules and a Gel-MA shell after photopolymerization. The thickness of the gel layers, which determines the spatial separation between the cells, can be tuned by varying the flow rate of the input solutions. The physically separated cells demonstrated high viability in culture. These preliminary results suggest that these microfluidic core-shell microgels can contribute to the generation of biomimetic tissues with functions that more closely mimic those of native tissue.
Engineering a Reconstituted Lung with Conditionally Reprogrammed Human Bronchial Epithelial Cells
We have created an Ex Vivo reconstituted lung system by seeding conditionally reprogrammed primary human bronchial epithelial cells (HBECs) into a decellularized murine lung matrix in a bioreactor with simulated breathing. By inducing differentiation over the course of 12 days of perfusion in the bioreactor, we have produced reconstituted lungs that display a pseudo-stratified upper airway epithelium and alveolar structures. We decellularized murine lungs by perfusing them with a CHAPs solution for 3 hours and a DNAse solution for 1 hour at 37 degrees C, which removed >95% of the DNA while preserving the ultrastructure. We conditionally reprogrammed human basal progenitor HBECs to maintain their stemness during in vitro expansion through co-culture with irradiated J2 3T3 fibroblasts in medium containing a Rho-associated protein kinase inhibitor. We confirmed the formation of a bronchial pseudo-stratified epithelium and alveolar formation in the reconstituted lungs by histology, Western blotting, and immunofluorescence staining. Conditionally reprogrammed HBECs cultured in standard air liquid interface conditions for 12 days do not form a pseudostratified epithelium or alveolar cells, but after 4–5 weeks they form mucous and ciliated epithelial cells. Tissue-engineered lungs have the potential to be a platform for simulating complex lung diseases in a controlled ex-vivo environment. The life extension protocol we've developed permits rapid scale-up of primary patient derived HBECs and clonal selection without the need for genetic manipulation; facilitating the study of rare lung diseases such as cystic fibrosis in tissue engineered models.
3D Bioprinting Technology and its Potential for Orthopedics
Department of Orthopaedics, Faculty of Medicine, Comenius University in Bratislava, Bratislava, SLOVAKIA.
3D printing recently shows a great impact in orthopaedics worldwide, mostly in manufacturing dimensionally accurate human anatomy models from high resolution image data, exported in common medical file format DICOM (digital imaging and communication in medicine). It is valuable tool in virtual diagnostic and presurgical planning of bone deformities, malunion, bone tumor or complex intraarticular fractures. This method is used mostely I preoperative surgical planning and explaining the complex surgical operations to the resident doctors and patients. In the orthopaedic oncology it may also be a valuable tool in virtual diagnostic and presurgical planning. Aditive manufacturing of custom orthopedic implants and surgical guides or instruments is one of our aims to offer these customized implants at reasonable unit costs without loss of quality. In terms of bioprinting technologies, soon we will be able to print the bone, but achieving such goal we need to consider a structure that is to some extent both flexibile and strong, contains colagenous matrix components, and is favorable to mineralization and vascularization. It is also necessary to maintain the porosity an the ability to match the graft to the defect site. We have lounched a recent study with materials capable of mediating reconstruction of large bone defects, using the medical-grade PCL-TCP. These scaffolds with combination with BMP-7 and stem cells (bone marrow and adipose), could be a promising equivalent of bone healing compared to autologous bone graft. Supported by Grant KEGA no. 071UK-4/2016.
Electrical and Computer Engineering, Old Dominion University, Norfolk, VA.
Culturing epithelial cells in 3D conditions has proven to be a superior biomimetic method for modeling epithelial cell structure and function as compared to traditional 2D culture techniques. However, because the cellular and physical constituents of a functional mammary niche are complex, methodologies that allow for precise placement of cells within 3D matrices would provide a more robust exploration of mammary development. Using a low-cost microextrusion-based, 3D bioprinting device developed in our laboratory (1), we generate high throughput arrays of breast epithelial cell clusters to optimize organoid generation in a 3D hydrogel. Our investigation provides information on key properties of epithelial cell branching and morphogenesis in a 3D hydrogel. By varying the amount of printed cells inside a 3D hydrogel, the number of cells required for the reliable formation of organoids at 7 and 14 days post-printing was found to be 30 and 15, respectively. Using this information, we found a minimum spacing of 350 μm and 750 μm is required to reliably generate fusion among these organoid structures at 7 and 14 days post-printing. This investigation furthers our understanding of mammary gland development by providing a method to generate repeatable, large branching structures in 3D.
Reference
1. Reid, John A., et al. “Accessible bioprinting: adaptation of a low-cost 3D-printer for precise cell placement and stem cell differentiation.” Biofabrication 8.2 (2015): 025017–025017.
Encapsulation Of 3d-cylindrical Disc Scaffolds In Hydrogel Beads With Mesenchymal Stem Cells For Rabbit Calvarial Defect Repair
Oral and Maxillofacial Surgery, National Dental Centre Singapore, Singapore, SINGAPORE.
Reference
Schantz JT, et al. Repair of calvarial defects with customized tissue-engineered bone grafts II. Evaluation of cellular efficiency and efficacy in vivo. Tissue Eng. 2003;9 Suppl 1:S127–39.
Center for Craniofacial Regeneration, Columbia University Medical Center, New York, NEW YORK, NY.
Columbia University, New York, NY.
Center for Craniofacial Regeneration, Columbia University, New York, NY.
Seoul National University, Seoul, KOREA, REPUBLIC OF.
Orthodontics, Tufts University, Boston, MA.
Tooth and associated alveolar bone loss are a
Maintained Stemness of Human Periodontal Ligament Stem Cells Following Prolonged Storage of Extracted Teeth
Department of Periodontology, School of Dentistry, Chonbuk National University, Jeonju, KOREA, REPUBLIC OF.
Human periodontal ligament stem cells (hPDLSCs) are readily accessible and promising mesenchymal cell source for therapeutic strategies. For clinical applications of stem cell therapies, prolonged maintenance of the hPDLSC stemness is critical. The purpose of the present study is to evaluate the maintenance of hPDLSC stemness following storage of the periodontal ligament (PDL) in growth, proliferation, and differentiation capabilities. Following premolar extraction (N = 10), hPDLSCs were isolated immediately (n = 5) and after 1 week (n = 5) of tooth storage in a growth medium, followed by hPDLSC primary culture. The hPDLSCs were evaluated in colony-forming and proliferative abilities, immunophenotypes and differentiation capabilities (osteogenic, adipogenic, and chondrogenic) followed by real-time polymerase chain reaction (PCR) confirmation. The results showed that storing the PDL did not affect the stemness of hPDLSCs. The hPDLSC activities from the PDL immediately after harvest and following 1 week of PDL storage were comparable in colony-formation and proliferation capabilities, and immunophenotypes. The osteogenic, adipogenic, and chondrogenic differentiation capabilities of the hPDLSCs were unaltered after PDL storage. The real-time PCR analyses confirmed the maintenance of relative gene expression levels for osteogenic (RUNX2, ALP, and OCN), adipogenic (PPAR-γ and LPL), and chondrogenic (aggrecan and collagen types 2 and 10) differentiations. Within the limitation of the study, the hPDLSCs harvested immediately from the PDL and following 1-week PDL storage were shown to have maintained the important characteristics of mesenchymal stem cells.
It has been known since the discovery of the biological role of NO in the 1980s, that supplying NO donors such can have many beneficial effects in different conditions by stimulating stem cells and modulating the immune response, however, there also exists a substantial risk of side-effects with long-term use. Excess NO can inhibit mitochondrial metabolism by binding to cytochrome c oxidase (CCO) and can also produce reactive nitrogen species (Peroxynitrite) by interacting with reactive oxygen species (ROS). To avoid these potential damaging side-effects we propose to combine the use of NO donors with four additional components. Initially, we believe that the addition of antioxidants such as Thiols, Polyphenols and Vitamins can neutralize ROS and RNS. Secondly, we believe that application of appropriate wavelengths and dosages of light will dissociate NO from CCO (and other storage sites) thus restoring mitochondrial ATP production and stimulating healing in many situations. Thirdly, we think detoxification will remove the pathological free radical generators and increases the nitric oxide bioavailability Lastly, by delivering electrons to the body through electrotherapy, we might help to saturate the free radicals with electrons, thus eliminating underlying oxidative stress, stabilizing mitochondria, preventing further formation of pathological free radicals and increasing the nitric oxide bioavailability. This combination therapy may be applied to manage oxidative stressed related diseases such as degenerative diseases, immunological diseases, cancers and a broad range of unmet medical needs involving chronic inflammation with an emphasis on pain management.
Semi-quantitative Assessment of Protein and Cell Content in Engineered Vascular Tissue with Dispersive Raman Spectroscopy
Biomedical Engineering, Florida Institute of Technology, Melbourne, FL.
Small-diameter tissue engineered vascular grafts (TEVGs) must match the adjacent artery compliance to improve long-term viability. Raman spectroscopy has been used to non-destructively assess components of different TEVG strategies, including cardiovascular cells and 2D tissues. However, Raman has not been applied to semi-quantitatively assess 3D engineered-tissues with both high- and low-scattering components. Thus, this study will use dispersive Raman for analysis of initial composition and cellular remodeling of natural/synthetic blend materials previously used for TEVGs. Electrospun meshes were prepared with 100% poly(ɛ-caprolactone) (PCL), 90% PCL/collagen, and 90% PCL/fibrinogen. Meshes were characterized using SEM, XPS, and Raman. Raman spectra were normalized to the 1110 cm−1 PCL peak (n = 6 samples, 10 spectra/sample). Meshes had similar fiber diameters ranging from 0.63 ± 0.20—0.83 ± 0.23 μm. Smooth muscle cells were seeded (7,500 cells/cm2) and cultured for 7–21 days. Differences in cell proliferation, contractile markers (RT-PCR, immunofluorescence), and early-markers of mineralization were observed, especially with collagen incorporation. Raman analysis of pure materials demonstrated differences in fingerprint spectra. After electrospinning, the strong PCL signal prevented resolution of many of the distinctive peaks. However, peak integration showed increases in 740 cm−1 (p < 0.036) and 1050 cm−1 (p < 0.0003) peaks for protein-blended samples. Additional statistical differences at 740 cm−1 were observed after cell culture. For all groupings (dry, cultured, and incubated without cells), the first principle component (principle component analysis) was consistently attributed to variance from PCL scattering. Interestingly, fibrinogen-containing meshes exhibited the greatest variance after culture. We are further investigating these results. Overall, we have validated that Raman spectroscopy can be used to assess synthetic/natural blended TEVGs.
Department of Oral Biology, University of Leeds, Leeds, UNITED KINGDOM.
Finlay et al., in vitro Engineering of High Modulus Cartilage-Like Constructs, Tissue Engineering: Part C, 22:4, 2016.
Department of Oral Biology, Institute of Medical and Biological Engineering, Leeds, UNITED KINGDOM.
1. Finlay et al. Tissue Eng Part C. 22. 2016.
2. Lukinavičius et al. Nature Methods 11:7. 2014.
When employing imaging modalities to evaluate in vivo response to implanted biomaterials, it is crucial to make quantifiable diagnostic conclusions about the biomaterial-host interactions and degree of tissue repair. micro CT is a commonly used imaging technique which provides high resolution and non-invasive visualization for hard tissue, especially on the microarchitecture, volume, surface area, tissue thickness and spatial distribution. In our study, we aim to create micro CT visible hydrogels for longitudinal tracking of scaffold degradation and tissue formation. For this purpose, we decided to create micro CT visible hydrogels by combining our hydrogel constructs with gold nanoparticles (GNPs). The alterations in mechanical properties of the hydrogels will be evaluated via dynamic mechanical analysis (DMA). Cytocompatibility and bone tissue engineering potential of the hydrogel-GNP constructs will be analysed using rat derived mesenchymal stromal cells (MSCs). Different size and concentration of GNPs will be combined with hydrogels to optimize the micro CT visibility, cytocompatibility and bone tissue engineering potential of GNP-hydrogel constructs in wet and dry state.
Pulsed Focused Ultrasound Mechanically Activates Trpc1 Channels To Enhance Local Stem Cell Homing
A Novel Reporter System For Monitoring Of Skeletal Muscle Differentiation
Enhancing Regulatory Review of Modeling and Simulation for Regenerative Medicine Products
Online Monitoring Of Mechanical Properties Of 3D Tissue Engineered Constructs For Quality Assessment
ISTM, Keele University, Stoke on Trent, UNITED KINGDOM.
Instituto Israelita de Ensino e Pesquisa, Hospital Israelita Albert Einstein, São Paulo, SP, BRAZIL.
Porcine kidneys were cannulated through the renal artery/vein, and heparinized. Subsequently, the kidneys were harvested and frozen at −80°C. A novel bioreactor composed of fully autoclavable components in a closed circuit was produced, where a peristaltic pump perfuses sodium dodecyl sulfate (SDS) detergent from a 20 L carboy to an organ chamber, where the kidney receives this “affluent” through its renal artery, consummating the removal of the organ's cellular component. The same peristaltic pump aspirates detergent upon leaving the organ, partially by the renal vein and partially by extravasation through the interstitium, and this “effluent” is collected in another 20 L carboy.
Cryopreserved kidneys were thawed, and, in a sterile laminar flow cabinet, the kidney is placed in the autoclaved organ chamber, with its connectors coupled to urethral catheters used for the cannulation as above. The organ chamber is then linked up with the additional bioreactor components, such as the pump and carboys. Initial tests showed that, during 24 h of continuous 1% SDS perfusion at 15 mL/min, the kidney changes progressively and gradually from a deep red to a bright white color, initiating at the inferior pole and advancing towards the superior pole, becoming uniform after 24 h. Afterwards, the kidney is perfused with ultrapure water, to remove detergent remnants, and then perfused with PBS to osmotically equilibrate the scaffold.
No contamination or odors were noted upon using the bioreactor. The results indicate that the bioreactor is capable of producing renal scaffolds in a closed-circuit system that may be scaled up with ease.
1. Mansfield EG, Greene VK, Auguste DT. Acta Biomaterialia 33 176–182, 2016.
Toward Whole Liver Engineering: Human Induced Pluripotent Stem Cell-Derived Hepatocytes and Endothelial Cells for Recellularization of Liver Scaffolds
Improvement of Penile Histomorphological Structure and Function with Stem Cell in a Rat Model of Neurovascular Erectile Dysfunction
Development Of A Hollow Fiber-type Bioartificial Liver Using Genetically Engineered Hepatoma Cells In Three-dimensional Culture Configuration
A bioartificial liver (BAL) using cultured hepatocytes is one of the therapeutic alternatives to liver transplantation. The most critical issue for the development of BAL is obtaining sufficient mass of functional hepatocytes. In this study, we designed a hollow fiber type bioartificial liver immobilized genetically engineered hepatoma cell line, Hepa/8F5, in which enhanced liver functions could be induced by overexpression of liver-enriched transcription factors. Hepa/8F5 cells were inoculated into a device comprised of hollow fibers and cultured in a three-dimensional culture configuration. To activate exogenous genes, doxycycline (Dox) were added to the culture medium. We assessed cell number, some liver-specific functions such as ammonia removal and albumin secretion. Hepa/8F5 cells immobilized inside the hollow fibers showed high growth activity without Dox. The growth of Hepa/8F5 cells was inhibited after induction by Dox. The liver-specific functions of ammonia removal and albumin secretion were up-regulated after Dox addition. These result indicated that switching between proliferation and expression of differentiated functions could be controlled by the inducible exogenous genes in the genetically engineered hepatoma cells. Hepa/8F5 cells express adequate liver functions per cm3 of the HFs compared with primary mouse hepatocyte in a suitable culture condition. It is expected that the BAL with Hepa/8F5 cells is effective in the treatment of liver failure. In future work, we design BAL with Hepa/8F5 cells and assess the therapeutic effect for liver failure animal model.
Yamamoto H et al., Biochem. Eng. J. 60, 67–73, 2012.
Generation Of Functional Hepatocyte-like Cells From Human Induced Pluripotent Stem Cells In A Three-dimensional Culture Using Hollow Fibers
Proposing A New Pathology Discipline and New Banff Classification: Tissue Engineering Pathology
In the next decades, transplantation will include a variety of tissue engineering approaches thus enlarging the field at least ten fold. Transplantation pathologists of today will become tissue-engineering pathologists. Publications in regenerative medicine are increasing rapidly every year with the number in 2016 (6,752 estimated) likely to double that in 2013 (3,333). The literature on pathological reaction to scaffolds for implantable tissue growth began in 1967 but in an understated way. It is only in 2016 that a new consequential discipline of tissue engineering pathology can be said to begin and could benefit from a Banff Classification of tissue engineering pathology that can be an extension of the existing Banff classification of allograft pathology https://en.wikipedia.org/wiki/Banff_Classification. The original Banff classification of allograft pathology is 25 years old, but in 2016 will have more publications relating to it (estimated 100) than in any previous year. We propose starting the creation of such a new Banff classification of tissue engineering pathology now with interdisciplinary consensus discussions to further develop and finalize the classification at the 2017, 2019, and 2021 Banff Transplant Pathology meetings. The ultimate aim would be to assure the safety and efficacy of stem cell generated organs and to extend tissue engineering transplantation to everyone in need. Ways of categorizing abnormalities in tissue-engineered organs are suggested. Traditional light microscopy is just one of several modalities of examination likely to be employed in this new pathologic discipline. The use of soluble biomarkers is also likely to play an important role.
Heterotopic Implantation of Bioprinted Human Liver Tissue into a NOD/SCID Mouse
The critical shortage of donor organs creates an urgent need for novel treatments for patients with inborn errors of metabolism and end stage liver failure. Conventional cell therapy and tissue engineering approaches to treating liver disease and injury have been hampered by low cell retention, poor engraftment, poor graft durability and complications including portal hypertension. Next generation technologies such as 3D bioprinting are an essential step towards clinical success. Here, using the NovoGen bioprinting platform, we report successful fabrication, implantation and engraftment of a bioprinted human liver tissue (BHLT) containing endothelial cells, hepatic stellate cells (SC) and hepatocytes (Hep) in a NOD/SCID mouse. Human albumin, transferrin, alpha 1 anti-trypsin and fibrinogen were detected in the circulation of test animals as early as 7 days, and for at least 28 days post-implantation. Histopathologic evaluation of implanted BHLT at Days 7, 14 and 28 revealed retention of the fabricated tissue geometry with defined areas of non-parenchymal cells (NPC) containing human CD31-lined perfused vasculature and desmin-positive SC. Adjacent to the NPC regions were areas of dense polarized Heps, closely supported by cells phenotypically consistent with SC. Heps in the BHLT also stained positive for albumin, Factor VII, fibrinogen and ornithine transcarbamylase. HLA staining confirmed limited NPC migration into the surrounding mouse liver and no evidence of Hep migration outside the BHLT. The rapid vascularization, sufficient tissue engraftment and retention, evidence of synthetic function and graft durability demonstrate the clinical promise for bioprinted tissues to treat critical liver diseases.
Gelatin-based Biodegradable Ureteral Stents For The Treatment Of Urothelial Tumors Of The Upper Urinary Tract Cancer
3Bs Research Group, Guimarães, PORTUGAL.
Upper urinary tract urothelial carcinoma (UTUC) accounts for 5–10% of urothelial carcinomas and is a disease that has not been widely studied as carcinoma of the bladder. To avoid the problems of conventional therapies, such as the need for frequent drug instillation due to poor drug retention, we developed a biodegradable ureteral stent (BUS) impregnated by supercritical fluid CO2 (scCO2) with the most commonly used anti-cancer drugs, namely paclitaxel, epirubicin, doxorubicin, and gemcitabine. The release kinetics of anti-cancer therapeutics from drug-eluting stents was measured in artificial urine solution (AUS). The in vitro release showed a faster release in the first 72h for the four anti-cancer drugs, after this time a plateau was achieved and finally the stent degraded after 9 days. Regarding the amount of impregnated drugs by scCO2, gemcitabine showed the highest amount of loading (19.57 μg drug /mg polymer: 2% loaded), while the lowest amount was obtained for paclitaxel (0.067 μg drug /mg polymer: 0.01% loaded). A cancer cell line (T24) was exposed to graded concentrations (0.01 to 2000 ng/ml) of each drug for 4 and 72 hours to determine the sensitivities of the cells to each drug (IC50). The direct and indirect contact study of the anti-cancer biodegradable ureteral stents with the T24 and HUVEC cell lines confirmed the anti-tumoral effect of the BUS impregnated with the four anti-cancer drugs tested, reducing by 75% of the viability of the T24 cell line after 72h and demonstrating minimal cytotoxic effect on HUVECs.
Servicio de Urología, IIS Aragón. HU Miguel Servet, Zaragoza, SPAIN.
Using decellularized organs has important advantages in kidney tissue engineering due to biomechanical, composition and microstructure similarities (1,2). However, recovering the renal function after recellularization, and prior to implantation, poses significant challenges. A system capable of maintaining the renal function ex-vivo was developed in this work. Aiming to explore the capabilities of the system, porcine kidneys were harvested and subjected to normothermic perfusion (3) under different ischaemia conditions. The evolution of parameters as intrarenal resistance, urine production, fractional sodium excretion and creatinine clearance during the perfusion process were measured. Also cellular damage makers were analysed and histological studies where carried out in order to evaluate tissue damage. The obtained results show the capability of the system to maintain and recover the renal function. The application of the system to tissue engineered kidneys will help determining the functionality of the grafts before implanted in the recipient.
1. Hoshiba T, Lu H, Kawazoe N, Chen G. Expert Opin Biol Ther. 2010 Dec;10(12):1717–28.
2. Sullivan DC, Mirmalek-Sania SH, Deegana DB, Baptista PM, Aboushwareb T, Atala A, Yoo JJ. Biomaterials 2012; 33(31): 7756–64.
3. Hosgood SA, Barlow AD, Dormer J, Nicholson ML. J Transl Med. 2015;13:329.
Produced via electrospinning, poly(l-lactide-co-caprolactone) (PLCL) membrane, which has porous structure and biodegradable property, has been of interest in medical fields. The porous structured electrospun membrane exudates fluid from the wound, does not build up under the covering, and does not cause wound desiccation. Extracellular matrix (ECM) derived by tissue decellularization has applications as a tissue engineering scaffold and for support of cellular regeneration. Especially, heart decellularized ECM (hdECM) has various pro-angiogenic factors that can induce angiogenesis for wound healing. In this regard, the nanofibrous electrospun hdECM-based hybrid scaffold (NEhdHS) which is PLCL membrane including hdECM component, was tested as a wound dressing to provide fundamental biochemical cues as well as physical cue for wound healing. This approach is expected to furnish effective wound dressing with reduced scarring to provide porous structure and pro-angiogenic factors. We first demonstrated the effectiveness of the proposed decellularization protocol through analysis of dECM components and the mechanical properties of fabricated the NEhdHS. The in vitro angiogenesis analysis of the NEhdHS, using co-culture system with human fibroblasts and human umbilical vein endothelial cells, confirmed their biocompatibility and showed that the NEhdHS significantly enhanced angiogenesis compared with that of PLCL scaffold or PLCL including gelatin scaffold. We also studied the effectiveness of the NEhdHS in vivo. Using rat excisional wound splinting model, we showed that covering upside the wound of the NEhdHS significantly reduced scarring in wound healing process compared with others. We conclude that the NEhdHS has potential applications for wound dressing based upon its unique properties.
Fibroblast And Osteoblast Growth And Viability On Bio-nanocomposite Films And Scaffolds Produced From Biological Chitin Nanowhiskers And Enzyme-mediated Poly(ɛ-caprolactone) Assieted By Organic Compressed Fluid Technology
Chemistry, National University of Mexico, Mexico, MEXICO.
Nanoparticles from biologically obtained chitin were used as nanofiller embedded in a poly-ɛ-caprolactone matrix, which was produced by an enzymatic route using a non-toxic compressed 1,1,1,2-tetrafluoroethane medium. The biologically-mediated chitin was transformed into nanoparticle size by acidic hydrolysis in a novel process at low temperature (4°C) that enhances the nanoparticle size distribution. The bio-nanowhiskers were analyzed by transmission electron microscopy, light diffraction and powder X-ray diffraction. Homogeneous distribution of the biopolymer nanowhiskers in the polyester matrix was conducted by a non-aqueous methodology. The confocal and atomic force microscopies reveled the nanofiller distribution. Films of the nanocomposite were also characterized by infra-red and wide-angle X-ray diffraction spectroscopies, mechanical, thermal, static contact angle, water absorption, erosion and atomic force microscopy to assess the adequate properties of the produced materials for tissue engineering applications. The latter analysis suggest that the nanoparticles might play a significant role in the polyester crystallization giving a more ordered structure. Finally, the growth of human fibroblasts and osteoblasts on the nanocomposite films showed good biocompatibility with adhesion and proliferation for both type of cells. Further, 1,1,1,2-tetrafluoroethane was used to produce macroporous scaffolds of the nanocomposite, which were employed for osteoblast growth studies.
Application of Antimicrobial Peptide-functionalized Silver CNTs Against Staphylococcus Infection in a Full Thickness Human 3D Skin Model
Infections of skin wounds are commonly caused by cutaneous opportunistic pathogens such as Staphylococcus species. Due to newly arising antibiotic-resistant strains of Staphylococcus, treatment of such wounds becomes difficult with prolonged healing times. Present study investigated the antibacterial application of antimicrobial peptide (AP)-functionalized silver coated carbon nanotubes (fAgCNTs) against Staphylococcus aureus infection in a full thickness human three dimensional (3D) skin model, using the bacterial enumeration assay and scanning electron microscopy (SEM). Here, we first evaluated the toxicity of fAgCNTs by the MTT assay on human 3D skin which was developed using keratinocytes and fibroblasts, cultured on Alvetex scaffolds. For antibacterial assays, the human 3D skin was incubated with minimum essential medium (MEM) containing 5 μg/ml of fAgCNT for 2h, followed by incubation with MEM containing 1 × 104 colony forming units (cfus) of bacteria for 2h. The MTT assay revealed no toxicity of fAgCNTs to the skin cells at 5μg/ml with 98% cell viability. Additionally, the bacterial count was increased from 104 to 108 cfus in the untreated skin model, whereas skin samples treated with fAgCNTs showed a much smaller increase in bacterial count from 104 to only 105 cfus. SEM analysis further showed the presence of bacteria on the non-treated skin as opposed to the treated skin. The 1000 fold bacterial cfus difference between the treated vs untreated skin confirmed that fAgCNTs may help to reduce the infection due to their antibacterial activity. These findings will aid in to development of novel antibacterial skin substitutes with antibacterial components.
The Effects of Fibronectin, Collagen, and Green Tea on the Proliferation, Morphology, Migration and Integrin Expression of Human Foreskin Fibroblast Cells
1. Bainbridge, P. “Wound healing and the role of fibroblasts.” Journal of wound care 22.8 (2013).
Low-level Laser Therapy (LLLT) 670nm And Vitamin A Topical Treatment In The Time Course On Wound Healing In Rats
Physiotherapy, Federal University of São Carlos (UFSCar), São Carlos, BRAZIL.
Biomedical Engineering, Drexel University, Philadelphia, PA.
Chronic wounds remain a major clinical challenge. Macrophages, the primary cell of the inflammatory response, modulate normal wound healing through their transition from a pro-inflammatory (M1) phenotype to an anti-inflammatory/pro-extracellular matrix deposition (M2a) phenotype over time. However, in chronic wounds, macrophages are stalled in a chronic M1-like state. Human placental membranes, which contain fibroblasts, mesenchymal stem cells, epithelial cells, and endogenous growth factors, have been used treat chronic wounds for over 100 years. Despite the variety of commercially available placental membrane products, the mechanisms of action remain unknown. Therefore, the purpose of this study was to characterize the effects of human cryopreserved viable placental membrane (hCVPM) on macrophage behavior in vitro and determine if the effects were mediated by hCVPM-secreted signals or from the membrane itself. Primary human M1 macrophages co-cultured with hCVPM (n = 3 donors) either by direct seeding or through separation with a transwell insert. RNA was extracted from cell-seeded constructs or non-cell-seeded controls after 24 hours for multiplex gene expression analysis of 42 macrophage phenotype- and angiogenesis-related genes by Nanostring. Multivariate analysis showed that cell-seeded constructs clustered further from M1 controls than transwell-cultured macrophages, suggesting that direct contact with the matrix had more significant effects on macrophage behavior than cell-secreted factors. Differentially expressed genes between cell-seeded constructs and controls were associated with M1 and M2a phenotypes, suggesting promotion of a complex, hybrid phenotype. These results provide important insight into the ability of placental membrane-derived materials to modulate macrophage behavior that regulate healing and angiogenesis in vivo.
Skin grafts are the gold standard of extensive burn wounds1, however, a growing concern is lack of healthy donor sites. Cell-spraying is a proven grafting alternative2, but the issue of cells falling off wounds persists. We propose adding collagen or collagen/poly (ethylene glycol) (PEG) to the cell spray to act as a biological glue to adhere cells to wounds. 15mg/mL fibrillar bovine collagen in media resulted in an effective glue concentration. BJ fibroblasts with collagen, collagen/1%PEG or media were sprayed or deposited by pipette onto tissue-culture plastic and cultured for three days. Cells were stained with calcein-AM/ethidium homodimer-1 to assess for viability. Fluorescence imaging showed greater than 95% fibroblast survival with no significant differences amongst groups, indicating that spray forces, collagen or PEG do not affect viability. A noticeable difference in cell morphology was observed in collagen and collagen/PEG as fibroblasts within collagen/PEG exhibited more spread over the substrate. This study validates collagen as an effective biological glue for spraying fibroblasts onto a wound. The addition of PEG may provide a more suitable extracellular matrix for the fibroblasts, as suggested by their spread when compared to collagen alone. Further work will be needed to determine how a collagen/PEG spray affects wound healing.
1. Gardien KL, Middelkoop E, Ulrich MM. Progress towards cell-based burn wound treatments. Regenerative Medicine, 9, 201, 2014.
2. Cervelli V et al. Use of a novel autologous cell-harvesting device to promote epithelialization and enhance appropriate pigmentation in scar reconstruction. Clinical and Experimental Dermatology, 35, 776, 2010.
Intracellular TGFβ Signaling is Potentiated by Substrate Stiffness
Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA.
Hematology and Stem Cell Laboratory, Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul, Porto Alegre, BRAZIL.
Available treatments for skin regeneration are insufficient for promoting healing. The current study has aimed to produce a cutaneous substitute uniting mesenchymal stem cells, keratinocytes, and a PDLLA biomaterial constructed by electrospinning to use in nude mice. Six groups were tested: (1) only PDLLA; (2) only PDLLA/Lam, a hydrolyzed scaffold with the binding of laminin; (3) PDLLA with cells; (4) PDLLA/Lam with cells (n = 6/group) and (5) animals injured without scaffolds (lesion control group) and (6) healthy control group (n = 4/group). All the animals had 1 cm2 defect performed on their backs, removing all the skin. The biomaterials(or scaffolds) were implanted in the mice for up to 9 days. Part of the defect was taken for histology and another for gene expression. Group 2 presented the best appearance with the softest wound. Gene expression analysis showed a considerable increase of TGFβ1 expression, increased VEGF and balance of the BAX/Bcl-2 ratio for the biomaterial groups when compared to the lesion group. Histological analysis showed well-formed tissue in the groups where the biomaterials and biomaterials plus cells were used. In some animals, in which biomaterials and cells were used, the epidermis was formed throughout the length of the wound. In conclusion, these biomaterials are capable of providing support for the growth of cells, indicating that they can be suitable biomaterials for use in tissue engineering.
Histogen, Inc., San Diego, CA.
Hypoxia-induced mulipotent stem cells secrete a human extracellular matrix (hECM) that contains components associated with stem cell niches and scarless healing including laminins, decorin, hyaluronic acid, SPARC, tenascin, fibronectin, and fibrillin-2. hECM is manufactured reproducibly under GMP conditions and has been shown to support proliferation of hESCs and MSCs, without stimulating human dendritic cell activation. Ex-vivo assays have shown the ability of the hECM to significantly reduce inflammation by down regulating interleukins and a variety of metalloproteases while upregulating genes for new matrix production. CAM assays have also demonstrated the pro-angiogenic properties of the hECM. The high incidence of infections seen with severe thermal burns and chronic wounds led us to test the hECM along with antimicrobial peptides (AMPs), a potential alternative to antibiotics that may overcome antibiotic resistance problems. AMPs are short, positively charged peptides found in the innate immunity of hundreds of species, and demonstrate broad spectrum antimicrobial activity even against antibiotic resistant strains of bacteria. AMPs can be modified to bind selectively to collagen. hECM/AMP solutions were prepared and lyophilized to create flexible wafer-like scaffolds that liquify when in contact with the wound bed. The scaffolds have shown antimicrobial effectiveness in vitro, and the ability to induce angiogenesis in the CAM model, and are being studied in large full-thickness wounds in mice.
The Development of a Gelatin/Galectin-3 Scaffold for Chronic Wound Healing
University of Western Ontario, London, ON, CANADA.
Chronic wounds are a significant burden on the healthcare system, as they are a leading cause of non-traumatic lower limb amputations and are associated with high mortality. Despite the lack of a curative therapy, the use of electrospun scaffolds loaded with matricellular proteins is promising, as it offers a way to provide both the structural integrity of the extracellular matrix and signaling proteins which can influence repair by modifying resident cell phenotypes. In this study, both the biocompatibility and the influence of galectin-3 loaded gelatin scaffolds on normal and impaired models of wound healing were investigated. Biocompatibility was assessed in vitro using human dermal fibroblasts (HDFs). To investigate wound healing, wild type and diabetic mice were surgically inflicted with 6 mm excisional wounds and treated with gelatin scaffolds containing varying galectin-3 concentrations, or left empty. Wound area was measured over a 17 day period. In vitro analysis revealed that galectin-3 loaded gelatin scaffolds were biocompatible, with these scaffolds showing no significant differences in HDF proliferation relative to the tissue culture plastic control. Wound healing studies showed that there were no significant differences in wound closure kinetics across treatments in both wild type and diabetic mice. These results demonstrate that although scaffolds are biocompatible, they do not accelerate wound repair. However, early histological analysis suggests differences in re-epithelialization, which could indicate differences in the quality of repair. Re-epithelialization is a process critical to wound healing; therefore, identifying proteins capable of repairing epithelial defects has important implications for treating chronic wounds.
We evaluated the in vivo efficacy and mechanism of action of InvossaTM in a monosodium acetate (MIA)-driven rat OA model. InvossaTM was injected into an arthritic knee and the effect on pain and cartilage regeneration was assessed. Pain-related behavior was analyzed by the von Frey filament test, and cartilage regeneration was analyzed by histological stainings. InvossaTM showed clear pain relief efficacy. The pain relief started at day 15 and maintained until 56 days post treatment. InvossaTM also demonstrated cartilage regeneration efficacy. Cartilage layer with higher type II collagen content was observed in InvossaTM treated animals. We also observed IL-10 level was increased in InvossaTM treated animals. InvossaTM attracted more arginase 1-positive cells (M2 macrophage). On the other hand, the number of CD86-positive cells (M1 macrophage) was comparable to that of the control group. Quantitative RT-PCR analysis showed that M2 macrophage related markers such as Arginase 1 and CD163 were highly up-regulated in InvossaTM treated animals. Therefore, In the clinical trials, InvossaTM improved the OA symptoms and induced structural changes within OA patients knee joints. We observed that the efficacy of Invossa shown in the clinical trials could be reproduced in the animal models. InvossaTM relieved pain and induced cartilage regeneration in treated animals. When we assessed the effect of InvossaTM on the environment of arthritic knee, Invossa induced the anti-inflammatory environment such as IL-10 and M2 macrophages. We suggest that M2 macrophage induction by InvossaTM may have contributed to the pain and cartilage improvement in the clinical trials.
Low Level Light Therapy Enhances Vasculogenesis in vitro
Ludwig Boltzmann Institute for Traumatology, Vienna, AUSTRIA.
Low level light therapy (LLLT) with light-emitting diodes (LED) receives increasing interest in the fields of wound healing and angiogenesis. The aim of this study was to compare the effects of three different wavelengths on endothelial cells and vasculogenic processes in vitro. Proliferation and migration of HUVEC were investigated in several 2D and 3D cell culture models. Cells were treated with either blue (475 nm), green (516 nm) or red (635 nm) LED light. Control cells were not illuminated. 2D proliferation was determined by manual counting. 2D migration was assessed by scratch assays, 3D migration by Cytodex bead assays. Vasculogenic potential of HUVEC in 3D co-culture with adipose-derived stem cells (ASC) in response to LLLT was determined by analysing the network formation after 4 and 7 days. Stimulation with both red and green pulsed LED light significantly increased HUVEC proliferation. Green light also increased 2D migration potential. 3D migration was significantly enhanced by green and red light. In 3D co-culture HUVEC elongation as precursor of vasculogenesis was enhanced by green and red light during the first 4 days. Both red and green light enhanced proliferation, migration and vasculogenesis processes while blue light was ineffective. Several parameters showed that green light was even more potent to stimulate regenerative processes than well-established red light therapy. Further studies have to focus on intracellular signaling induced by different wavelengths in order to optimize this promising, alternative application in tissue regeneration and wound therapy. Supported by FFG Grant 3925263.
Departamento de Ingenierias Quimica, Electronica y Biomedica, Universidad de Guanajuato, León, MEXICO.
In this work is reported the preparation and characterization of collagen hydrogel biomaterials crosslinked with oligourethanes derived from a triol ethoxylated glycerol functionalized with hexamethylene (HDI) or L-lysine diisocyanates (LDI) and modified with silica derived from an orthosilicate sol-gel process. The physicochemical aspects of the material, such as fibril's formation kinetic, crosslinking degree, biodegradation resistance, water absorption capacity, topography, chemical composition and mechanical properties, were evaluated. In addition, the RAW264.7 macrophage response to these novel hydrogels was analyzed by the viability, morphology, production and expression of the cytokines monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-β1 (TGF-β1), inducible nitric oxide synthase (iNOS) and resistin like molecule-α (Relmα). The crosslinking process of collagen with oligourethanes tri-functionalized resulted in an enhanced resistance to degradation, water absorption capacity and mechanical properties. The materials supported the growth of macrophages and induced an enhanced TGF-β secretion and Relmα expression. The macrophages showed a cell size different to the phenotype M1 macrophages. These results could be associated with an anti-inflammatory response (M2 macrophages) induced by the biomaterial characteristics. Therefore, the manufacture method reported here produces biomaterials derived from collagen hydrogels with physicochemical and biological characteristics that seems promising in the design of dressings for chronic wounds such as diabetic foot ulcers.
Vitamin E Renders Culture Expanded Keratinocytes and Fibroblasts Resistant to Burn Injury
National Center of Excellence in Molecular Biology, Lahore
Biotechnology Laboratory, National Institute of Rehabilitation, Mexico DF, MEXICO.
Research Institute, WINNOVA, Seoul, KOREA, REPUBLIC OF.
Bone morphogenic protein-2 (BMP-2) regulates a variety of cellular processes such as proliferation, differentiation, bone/cartilage morphogenesis, apoptosis and wound healing. In our previous studies, OP10 peptide derived from sequences of amino acids in BMP-2 has the effects of wound healing. It has up-regulated skin regeneration and wrinkle improvement better than the control in protein levels such as MMP1, TIMP1, ERK and collagen synthesis. Based on these results, OP10 peptides were conjugated with trolox which is well-known anti-oxidant ingredient. Trolox, a water-soluble analog of vitamin E, was synthesized in two forms due to racemic compound; Trolox-A and Trolox-B respectively. The effects of conjugated OP10 peptides were carried out using normal human dermal fibroblast (NHDF) by the evaluation of cytotoxicity, proliferation, migration, protein expressions and anti-oxidant assay. Conjugated peptides have noticeable effects on an anti-oxidant experiment, as well as maintain wound healing, resulting in intrinsic properties of OP10. It shows similar trend to ascorbic acid which is well-known strong anti-oxidant in DPPH assay. We suggest that trolox conjugated peptides can effectively play a role in the recovery of skin damage with both effects of anti-oxidant and wound healing and have potential as wound repair medicine and functional cosmetic ingredients.
Lab. of Tissue Eng., KIRAMS, Seoul, KOREA, REPUBLIC OF.
This study was to investigate the potential of chitosan nanofiber mats containing gelatin for use as wound dressings with enhanced hemostatic functions. We also investigated the potential of synergy between the hydrophilic gelatin and sonicated nanofiber mats to improve hemostatic function.
1. Thesis Plastic Surgery Specialization: Enrique Ortiz, 098276482, UNAM, 2016.
GDF-11 Has no Effect on the Regeneration of Skeletal Muscle in Aged Rats After Injury
Regenerative Medicine, Wake Forest University, Winston-Salem, NC.
Bone marrow mesenchymal stem cells (MSCs) has good potential to repair articular cartilage defects. However, delivery MSCs in bulk hydrogel results in void spaces in implantation site due to volume shrinkage and the implants have poor mechanical properties. Here we report successfully generating volume stabilized and mechanical-property-improved human MSC micro-tissues (MSCMTs) using 3D live cell bioprinting repairing a defect in articular cartilage through self-assembly. The size and architectures of the MSCMTs were precisely controlled during printing. Microscopically the cells were distributed evenly throughout the micro-tissue surrounded by rich extracellular matrix. The MSCMTs remained viable over the entire period of the experiment. The chondrogenic capability of the MSCMTs was improved comparing with conventional techniques. Live imaging shows that the MSCMTs have strong capability of outgrowth and self-assembly. The MSCMTs could be directly injected through a 1.4 mm I.D. needle. In an in vitro cartilage explant-defect model, the injected MSCMTs show fusion and viable during the three weeks of culture. No volume shrinkage or void space was observed in the MSCMTs implanted group comparing with over 70% volume shrinkage in MSCs-bulk-gel implanted group. We have successfully developed a novel approach, using 3D live cell bioprinting, to generate MSCMTs that demonstrate promise in repairing cartilage defects via a minimally invasive approach.
Angiotensin-(1-7) Treatment Aids in the Recovery of Skeletal Muscle Function After Acute Injury in Rats
Age and Gender Differences in Skeletal Muscle Regeneration After Compression Injury in Rats
Stem Cell-derived Extracellular Matrix Enhancement of Autologous Chondrocytes Implantation for Articular Cartilage Repair
The Effect of Intramuscular VEGF Treatment on Skeletal Muscle Regeneration After Compartment Syndrome Injury
Compartment syndrome is a result of increased pressure within the fascicles of skeletal muscle resulting in cessation of perfusion leading to tissue necrosis and death. The current standard of care involves fasciotomy, which is time limited and requires early detection for clinical efficacy. After 8 hours of tissue ischemia, there is little efficacy to fasciotomy, thus further treatment modalities are needed, as the sequelae of compartment syndrome can include amputation and death. Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor that has been shown to establish perfusion in tumor and embryological models. VEGF represents a possible treatment modality for post-acute compartment syndrome.
Bone Morphogenetic Proteins' Influence On Intervertebral Disc Cells' Nutrient Consumption And Protein Production: Implications for Regenerative Therapy
Neurosurgery Research, Barrow Neurological Institute, Phoenix, AZ.
Aerospace and Mechanical Engineering, University of Oklahoma, Norman, OK.
One of the great challenges of using stem cells in cartilage repair is promoting their chondrogenic differentiation in a materials-only approach. Our approach focuses on developing high throughput methods of finding cell-binding peptides with the ability of stimulating chondrogenic differentiation in stem cells. Reducing the binding sequences to a small peptide is attractive because of the advantages peptides have compared to large molecules; peptides are easy to synthesize in large quantities, and their small size reduces the chance of any non-specific binding. Incorporating these peptides in a biocompatible and biodegradable construct that provides the requisite structural integrity for cartilage applications can lead to improving chondrogenic differentiation of scaffolds and promoting clinical translation of arthritis treatments. In our approach, by identifying overlapping sequences in target molecules of interest, as well as exploring the literature for a sequence known to bind to a target receptor, we have identified two proprietary peptide sequences. The chondrogenic potential of these peptides was explored by culturing rat bone marrow-derived mesenchymal stem cells either on surfaces coated with the peptides, or on uncoated surfaces with soluble peptide in the medium. Different concentrations of the peptides were explored to elucidate dose effect versus negative (no peptides) and positive (RGD sequence or TGF-β3) controls. The preliminary gene expression results have indicated that the proprietary peptides have the potential to enhance chondrogenic differentiation of rBMSCs and are promising candidates for designing bioactive scaffolds with the capability of improving stem cells chondrogenic differentiation.
Biomedical Engineering, Washington University in St. Louis, St Louis, MO.
Intervertebral disc disorders may originate within the nucleus pulposus (NP) region when NP cell density decreases with age, and lose the ability to maintain and repair the extracellular matrix. We have previously shown that NP cells interacting with laminin-conjugated soft substrates have increased matrix synthesis and healthy NP marker expression. Cell sensitivity to substrate stiffness may be regulated by a co-activator of the serum response factor (SRF) transcription factor, myocardin related transcription factor (MRTF), as shown for other cell types including fibroblasts. Here, primary NP cells were cultured upon soft and stiff laminin-rich substrates synthesized using basement membrane extract (BME®, Trevigen). NP cells on soft substrates formed rounded, multi-cell clusters and cytosolic MRTF-A persisted up to 4 days, as well as minimal SRF activation. NP cells on stiff substrates displayed flattened, elongated morphology with MRTF-A nuclear localization beginning at 24 hours post-attachment. These behaviors were associated with higher phosphorylated SRF levels. These findings demonstrating MRTF-A translocation to the nucleus upon “stiff” but not “soft” substrates suggest that multi-cell cluster formation can inhibit MRTF-A co-activation of SRF in NP cells. This work reveals one mechanism by which substrate stiffness acts to regulate NP cell activity that may allow for restoration and maintenance of a healthy, NP phenotype as a therapeutic strategy for disc disorders.
Perniconi, 2014, Wang, 2013 & Allison, 2011.
The Development and Effect of BMPs Loaded Double Layer Sponge Based on Chitosan and Collagen as a Delivery Carrier for Bone Regeneration
Research Institute, WINNOVA, Seoul, KOREA, REPUBLIC OF.
Hexanoic glycol chitosan (HGC) was water-soluble polymer which is synthesized by hexanoic anhydride through the reaction of N-acylation in glycol chitosan. The HGC was hydrophobically modified, showing reversible sol-gel transition from normal physiological condition to body temperature. It is non-toxic as well as biodegradable and can be easily combined with drug of growth factors as drug delivery system for clinical applications. It can be formulated in a variety of physical forms, including porous sponges, microspheres, and films. In this study based previous results, collagen sponge was introduced to be coated with HGC hydrogel incorporated with BMP-2 and lyophilized. It has double layer structure which is collagen sponge in the center and BMP-2 loaded HGC in the outside of collagen sponge. Its release behavior was evaluated for induction of osteogenesis followed by alkaline phosphatase activity (ALP), calcium deposition (alizarin red s staining). The BMP-2 released from double layer sponges leaded effectively osteogenic differentiation from bone marrow human mesenchymal stem cell compared to control. These results were confirmed by RT-PCR and in-vivo mouse calvarial defeact model showing similar trend above in upregulated bone related gene expression and increased new bone formation. These results implied that structure may serve as a candidate for tissue engineering and regenerative medicine.
Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL.
Approximately 10% of bone injuries in the United States result in non-union, costing an estimated $10.4–26.1 billion annually for treatment. While there are several orthopedic strategies to address non-unions in long bones, all have drawbacks and risks of complications. To advance new tissue engineering treatments that are improvements over current options, we have developed a porcine long bone segmental defect model for testing osseous non-union repair using 3D printed biodegradable scaffolds. A 3.5 cm mid-diaphyseal defect was generated in the radius of the right forelimb in 6-month old pigs. A polycaprolactone (PCL) scaffold implant designed from radius CT scans and produced using solid form fabrication was implanted into the defect and stabilized with a stainless steel plate. Animals are able to stand and place weight on the limb at six hours post-surgery, and ambulating without a noticeable limp by one month. CT imaging post-surgery show bone fill in the defect space by 2 weeks, significant defect closure by 8 weeks, and full union by six months. This healing has taken place on a PCL scaffold with no other treatments in conjunction with the implant. The porcine radius segmental defect model provides a viable platform for testing printed scaffolds and biological composites for efficacy of healing segmental long bone defects.
Wnt Directed Stem Cell Renewal In A 3D Periosteal Model
ISTM, Keele University, Stoke on Trent, UNITED KINGDOM.
The timing, location and level of Wnt signaling are highly regulated during embryonic development and for the maintenance of adult tissues such as bone and cartilage. Consequently the ability to provide a defined and directed source of Wnt proteins can provide a major advance in our understanding of its role in tissue development and to engineer musculoskeletal tissues. Here we describe a method to immobilize Wnt3a proteins on a basal surface which is capable of directing and maintaining a bone marrow derived mesenchymal stem cell population in a 3D periosteal model. We show that this Wnt platform is able to maintain stem basal populations and promote directed migration within a polymer/collagen composite. Our data has shown enhanced mesenchymal stem cell proliferation and STRO1+ expression in the basal layer with increased migration, differentiation and enhanced osteocalcin expression in the middle and top layers of the 3D constructs in vitro. We have tested this Wnt stem cell bandage in an in vitro calvarial periosteal repair model. Our goal is to use this platform for recapitulation of specific stem cell niches for regenerative medicine of musculoskeletal tissues.
Bioengineering, Ege University, Bornova/Izmir, TURKEY.
The appropriate combination of a scaffold, cells, and biological signals can create a living implant that will help damaged tissue to regenerate. To create a bone tissue engineering construct, we combined a cell-adhesive and enzymatically degradable hydrogel based on polyethylene glycol (PEG) and bioactive peptides; human periosteum-derived cells (hPDCs), progenitor cells with mesenchymal stem cell characteristics; and calcium (Ca) and phosphate (P) supplementation, which promotes hPDC proliferation and mineralization. Hydrogels were cultured in growth medium with/without supplemental Ca and P. In some cases, culture in growth medium was followed by Ca- and P-supplementation. Cell proliferation, morphology, viability, and differentiation were measured, and the deposited mineral was characterized via nanofocus computed tomography (nanoCT) and XRD, prior to ectopic implantation in nude mice. Ca- and P-supplementation led to precipitation of a mineral layer on the surface of hydrogels with and without cells. Intriguingly, nanoCT revealed a punctate, radio-opaque phase only inside the hydrogels that contained cells and were incubated with Ca- and P-supplementation, suggesting cell-mediated mineralization. XRD spectra were consistent with hydroxyapatite, and cells remained viable, proliferated, and adopted a spread morphology. After 8 weeks in vivo, more bone was formed by these constructs compared to control hydrogels, as evidenced by additional mineralized tissue formation (nanoCT) and extensive collagen production (histology). In this study, we have demonstrated that molecularly engineered hydrogels can not only support the proliferation of hPDCs but can also promote cell-mediated mineralization when cultured with Ca- and P-supplementation, leading to a construct that can form bone in vivo.
University of Virginia, Charlottesville, VA.
Volumetric muscle loss (VML) or VML-like injuries are common in the civilian and military populations and result in permanent functional and cosmetic deficits for which there are no available therapeutic solutions for complete functional recovery. We seek to develop new cell delivery-capable biomaterials to improve treatment of VML injury. Previous work has shown that utilization of anisotropic scaffolds or bioreactor preconditioning can both increase unidirectional orientation and myotube cell length following seeding of muscle progenitor cells. However, the combination of these two approaches has not yet been explored. To this end, we utilized established directional freezing methods for production of custom-made, 3D silk scaffolds with aligned channels, in conjunction with established bioreactor protocols for myoblast cell alignment. Both pure and hybrid silk/collagen scaffolds (1.5 × 0.5 × 0.5 cm) were manufactured and seeded with myoblasts. Initial results revealed that bioreactor preconditioning produced a dramatic enhancement in cellular coverage and orientation, as well as an increase in the number of multinucleated myotubes, when compared to static silk-seeded control scaffolds. In addition, the resulting bioreactor-derived scaffolds had sufficient suture retention for successful implantation in a biologically relevant rodent tibialis anterior (TA) VML injury. Parallel studies with unseeded silk scaffolds (n = 8) produced no detectable functional recovery following implantation in vivo. The degree of functional recovery remains to be determined based on ongoing in vivo studies. The progress to date demonstrates that we have already overcome major development barriers to utilization of tunable silk scaffolds for TEMR-based VML injury repair.
Although there is a limit to which endogenous mechanisms can recover lost bulk muscle tissue, as can occur in traumatic injuries, satellite stem cells are the classic mediators of the regenerative process. We have developed a tissue engineered muscle repair (TEMR) technology platform to enhance endogenous muscle repair by creating a more favorable microenvironment for muscle regeneration. The TEMR construct consists of rat muscle progenitor cells seeded onto a porcine bladder acellular matrix and preconditioned in a customized bioreactor. We have previously applied the TEMR construct to two biologically relevant, surgically-created rodent models of traumatic injury to the tibialis anterior and lattisimus dorsi muscles, respectively. In both models, TEMR-treated animals exhibited significant functional recovery (60–70%), as well as observable muscle regeneration. The goal of these studies is to further extend the TEMR technology platform to evaluate the impact of the inclusion of satellite stem cells on the extent of functional recovery. Briefly, we have shown via cell sorting techniques that satellite cells can be isolated from bulk rat skeletal muscle harvests, and further, that they will adhere to the cell-seeded TEMR construct during bioreactor preconditioning. We determined via flow cytometry that the satellite cells so isolated are Pax7+ and MyoD-, and account for approximately 5% of the cell population. The degree of functional recovery remains to be determined based on ongoing in vivo studies. However, this work has broken new ground for development of methods to include satellite cells in the existing TEMR platform.
Hydroxyapatite Coated Microribbon-based Hydrogels Induce Robust Osteogenesis And Mineralization Of Mesenchymal Stem Cells In 3D
Hydrogels are attractive injectable matrices for stem cell delivery, but are often mechanically too weak for bone repair. Hydroxyapatite (HA) is a important composition of bone mineral matrix and often great mechanical strength. However, previous HA-based scaffolds are not injectable, and are often too brittle, leading to premature failure of implants. To overcome above limitations, our group has recently reported development of gelatin-based microribbon (μRB)-like hydrogels, which combines injectability with macroporosity with enhanced mechanical strength. The goals of this study are to develop methods for effective coating of μRB hydrogels with HA; and to examine and compare the efficacy of HA coated μRB-based hydrogels for guiding human mesenchymal stem cells (MSCs) osteogenesis in vitro. Gelatin-based μRBs were synthesized as previously reported using wet spinning. Two methods were developed to incorporate HA onto the surface of μRBs including (1) nucleation (4 weeks) in modified simulated body fluid (mSBF) or (2) direct deposition of HA based nanoparticles (HAnp). SEM and EDS assays show both methods allow effective coating of HA onto μRB-based hydrogels. The macroporosity within the μRB-based hydrogels led to robust and interconnected collagen deposition. While HA is not required for collagen deposition, HA coating is critical for inducing maturation of MSCs towards bone lineage including mineralization and upregulation of mature bone markers. Our results suggest that Hanp incorporation would be a preferred method for incorporating HA into μRB-based hydrogels. HA-coated μRB-based hydrogels can serve as a promising injectable biomaterials to enhance stem cell-based therapy for repairing large bone defects.
Matrix Encapsulated Autologous Chondrocyte Implantation versus Microfracture at the Knee: 5 Years Clinical and T2 Mapping Evaluation
Sports Orthopaedics and Arthroscopy, National Rehabilitation Institute, Mexico City, MEXICO.
The purpose of this study was to evaluate the clinical and sequential imaging follow-up results at a mean of sixty-months after Matrix Encapsulated Autologous Chondrocyte Implantation (MECI) versus Microfracture (MFx) technique for the treatment of articular cartilage lesions in the knee. Fifty consecutive patients with symptomatic knee articular cartilage lesions were randomized into two groups: MECI or MFx. In MECI group twenty-six patients were included and two osteochondral biopsies were harvested in the first surgery. Isolated chondrocytes were expanded in monolayer culture during four weeks. A construct was formed with a collagen type-III scaffold enveloped in chondrocytes monolayers. In the second surgery the construct was fixed with an all-arthroscopic novel technique in all cases. In MFx group 24 patients were treated with arthroscopic microfractures as traditionally described by Steadman. The patients were evaluated clinically using clinical validated scores and T2 mapping by magnetic resonance evaluation. At five years follow up there was no significant statistical difference in the clinical evaluation with the scores Lysholm (78 ± 24.37, 77.25 ± 22.74: p = 0.168), Tegner (5 ± 2.55, 4.18 ± 1.97: p = 0.095), IKDC (72.97 ± 18.10, 65.17 ± 22.74: p = 0.438) between MECI and Microfracture, respectively. However, T2-mapping evaluation at repaired tissue, showed significant difference between MECI and MFx, respectively (38.05 ± 6.25, 45.41 ± 10.49; p = 0.040) favoring MECI group. Patients with MECI and Microfracture technique obtained better clinical results than preoperatively with no significant differences between groups at five-years follow-up. However, MECI group patients had better tissue quality measured by T2 mapping evaluation.
Copper Doped Hydroxyapatite Gelatin Micro Patterned Nanocomposite Scaffolds for Bone Graft with Enhanced Angiogenesis
Microbial infection and slow healing are two major problems most of the bone grafts suffer. While first one is becoming difficult to recover due to development of antibiotic resistant superbugs the other one is creating problem mostly due to lack of angiogenesis in bone grafts. Copper as a micro-element is observed to solve both the problem via significant antimicrobial activity as well as enhanced angiogenesis via activation of HIF-α pathway. In current study, we developed Cu-doped calcium phosphate nanorods (CaP) for controlled release of Cu ions. The powder was characterized by TEM, EDAX, SEM, and FTIR. Further it was mixed with gelatin to make nanocomposite and via Laser machining micropattering was performed. It was characterized by stereozoom microscopy, SEM and surface profilometer. Further the antimicrobial property was measured against both (Gram +ve) and (Gram-ve) bacteria. The cell culture study was performed using Wharton's jelly derived MSCs. Along with biological assays like MTT, ALP and microscopic evaluation like fluorescence microscopy and live dead assay, in vitro assays like PCR studies showed significant osteogenic and angiogenic gene expression proving efficiency of the material to serve as a bone graft or an implant coating material.
InvossaTM (tissuegene-c) In Patients With Osteoarthritis: A Phase Iii Trial
InvossaTM (TissueGene-C) is a cell and gene therapy for osteoarthritis that contains non-transformed and transduced chondrocytes by the ratio of 3:1. The transduced cell employs ex-vivo gene delivery via a retrovirally transduced chondrocytes that overexpress transforming growth factor-β1 (TGF-β1). The randomized double blind, multi-center, placebo-controlled phase III trials were conducted to determine both safety and efficacy in patients with knee osteoarthritis. Participants (n = 156) with a confirmed diagnosis of knee osteoarthritis by X-ray and MRI were randomized into the treatment group (Invossa™, n = 78) and the control group (saline, n = 78). The primary evaluation parameters were International Knee Document Committee (IKDC) and VAS. The secondary parameters were evaluated by Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and Knee Injury and Osteoarthritis Outcome Score (KOOS), Joint Space Width (JSW) with X-ray, Whole Organ Magnetic Resonance Imaging Score (WORMS) with MRI, and biomarkers from serum and urine samples. The observation period was one year after a single injection.
The primary parameters, IKDC and VAS, showed statistically significant improvement in 1 year follow up after a single injection of InvossaTM. The secondary parameters, WOMAC and KOOS, also showed statistically significant improvement in 1 year follow up after a single injection of InvossaTM.
In summary, Phase III study indicated that InvossaTM treatment improved pain, sports activities, and quality of daily life in patients with knee osteoarthritis when compared to the placebo control.
The Effect of Culture Substrate on the Structure and Function of Tissue Engineered Skeletal Muscle Units
Biomedical Engineering, University of Michigan, Ann Arbor, MI.
This study aimed to translate our tissue-engineered skeletal muscle units (SMUs) from a rat to a sheep model while reducing construct variability. Currently, our SMUs are cultured on a laminin-coated-Sylgard substrate that may produce inconsistencies through uneven laminin coverage. We have developed a “pin-plate” consisting of a polystyrene culture dish with embedded stainless-steel constraint pins as a more consistent alternative. We hypothesized that increased substrate stiffness and more uniform adhesion of the cells to the “pin-plates” would enhance SMU structural and functional properties. To test this hypothesis, sheep semimembranosus muscle isolates were seeded onto either laminin-coated-Sylgard plates or “pin-plates”. Immunostaining for MyoD coupled with a BrdU assay was used to evaluate myogenic proliferation. Differentiation and structural maturation of myoblasts (myotube size and density) were measured with immunostaining for myogenin and alpha-actinin, respectively. By day 5, the cells on “pin-plates” showed a trend towards higher proliferation of myogenic cells. By day 7, the “pin-plates” had a significantly higher number of differentiating myogenic cells/mm2. On day 13, monolayers exhibited a robust myotube network on the “pin-plates”, while the laminin-coated-Sylgard plates showed patchy monolayers and premature delamination. Monolayers fabricated on “pin-plates” self-delaminated to form three-dimensional constructs, while no three-dimensional constructs were formed on laminin-coated-Sylgard plates. Thus, we demonstrated that SMUs could be fabricated using sheep satellite cells seeded on “pin-plates”. It is possible that the concentration of laminin needed to promote even adhesion and delamination using sheep cells is different than that of our standard rat protocol. Supported by NIH/NIAMS-1R01AR067744-01 and 3R01AR067744-02W1.
During maturation, articular cartilage transforms from an immature growth phenotype to a stable adult phenotype. Compressive mechanical forces may induce maturational changes in cartilage composition, structure, and function due to compaction of local tissue regions. β-aminopropionitrile (BAPN) inhibits formation of collagen crosslinks and promotes cartilage swelling, whereas chondroitinase ABC (ChABC) depletes glycosaminoglycan (GAG) and facilitates collagen network stiffening. The aim of this study was to determine the effects of BAPN and ChABC on cartilage properties with compaction. A total of 156 calf articular cartilage disks were prepared to 1.1mm thickness, containing either the intact superficial (SZ) or middle/deep (MDZ) zones, were incubated in medium with BAPN for 3 days, and then analyzed for the effects of ±BAPN and ±ChABC before and during subsequent compaction. Following treatment and 16 hrs free-swelling, tissue thickness (h), tissue wet weight (ww), GAG content, and unconfined compressive modulus (E) were examined. Effects were assessed by ANOVA, and also compared to free-swelling controls by Tukey test. ChABC led to lower h (SZ, MDZ) and ww (MDZ), with both h and ww being greater in the MDZ than SZ. ChABC and BAPN both led to lower GAG/ww and lower E. Thus, in immature cartilage, the GAG content and state of the collagen network affect how mechanical compression modulates tissue geometry and mechanical properties. These principles may be applicable to cartilage tissue engineering and regenerative medicine.
The Effects Of Engineered Skeletal Muscle Units On Volumetric Muscle Loss In The Tibialis Anterior Muscle Of Rats After 3 Months In Vivo
Volumetric muscle loss (VML) is the traumatic or surgical loss of skeletal muscle that results in impaired muscle function. Current VML repair treatments are limited by donor site morbidity and graft tissue availability, necessitating exogenous muscle graft sources. To address this need, our lab is investigating the use of tissue-engineered skeletal muscle units (SMUs) for repair of VML in rat models. Previous results showed that after 28 days in vivo, VML in the tibialis anterior (TA) muscle repaired with our SMUs recovered significantly more muscle force production than non-repaired TAs, but failed to fully recover native muscle force production1. We hypothesized that longer time in vivo would allow for greater force recovery. Therefore, the purpose of this study was to examine the long-term effects of our SMUs in a 3-month TA VML rat model and to assess if peroneal nerve redirection to the SMUs would enhance recovery. 3 months post-surgical implantation, the TA muscles from the experimental groups (control-no VML, VML+nerve cut, VML+nerve redirect, VML+SMU+nerve cut, and VML+SMU+nerve redirect, n = 5–6) underwent in-situ force testing before being explanted for histological analysis. The maximum force and specific force results showed no significant differences between surgical groups and the control. Cross-sectional staining of H&E showed increased vascularization and cellularity as well as immature myotubes in the repair sites of both SMU repair groups. These findings suggest that after 3 month in vivo, our SMUs are being incorporated into the surrounding skeletal muscle tissue and are remodeling.
Ti6Al4V Lattice Structure by Extrusion Printing for Skeletal Tissue Healing
Acoustic Tweezing Cytometry (ATC) Dictates Stress Induced Differentiation in Human Embryonic Stem Cells (hESCs)
Biomedical Engineering, University of Michigan, Ann Arbor, MI.
The function of chemical cues and adhesion signals, have been studied intensively to understand how cells in the embryo differentiate into specific lineage. However, not to much attention has been put studying mechanical stresses to which cells are exposed to in understanding the role of forces in guiding hESC differentiation. In this work, we investigate the role of external forces in hESCs by utilizing from ATC. Undifferentiated single hESCs were seeded on matrigel coated dishes and cells localized in the area where ATC is applied. The next day, microbubbles functionalized with RGD were linked via integrin to dissociated single hESCs. Ultrasound pulse (1 MHz center frequency,10 Hz pulse repetition frequency, and 50% duty cycle) with ramping amplitudes (0.025, 0.04, 0.06 to 0.08MPa) was applied to the cells at a 45° angle beneath the petri dish for 30min at each amplitude. Surprisingly, stress induced shuttling Oct3/4 and Nanog from nucleus to cytoplasm, while Sox2 was not affected after the onset of ATC application. The percentage of Nanog positive (+) cells were 67.5 ± 12.6 in (−)US(+)MB and 10.2 ± 4.46 in (+)US(+)MB. The percentage of Oct3/4 (+)cells were 74 ± 13.2 in (−)US(+)MB, 6 ± 3.2 in (+)US(+)MB. While the percentage of Sox2 (+)cells were 96.8 ± 3.1 in (−)US(+)MB, 78.57 ± 12.7 in (+)US(+)MB. Shuttling of Oct3/4 and Nanog may indicate that these transcription factors are mechano-sensitive. Nucleo-cytoplasmic Nanog and Oct4 may direct cell programing and determine the cell lineage in early embryo development. These findings might help us to better understand the effect of mechanical forces during development and help us correct defects and to treat diseases
Huntington's disease (HD) is an autosomal dominant, neurodegenerative disease that affects post-mitotic neuronal cells through trinucleotide repeat (TNR) expansions that produce a toxic huntingtin protein. The comprehensive mechanism by which TNR expansions occur is unknown; however, in vitro studies have identified DNA repair pathways to be possible effectors1. We have previously demonstrated that differential methylation patterning within the promoter regions of these DNA repair genes seems to mediate the expression of these the first vascular substitute was developed1, newer approaches have been performed2. Poly (lactid-co-glycolic acid) (PLGA) and gelatin were employed owing to the possibility of regulate the degradation rate and for offering the cell attaching Arg-Gly-Asp sequence (RGD) respectively. In this preliminary report, a fully constructed PLGA-gelatin tubular scaffold is presented aiming its future applications for vascular substitutes. Considering such as constructing as analyzing methods.
Weinberg CB, Bell E. A blood vessel model constructed from collagen and cultured vascular cells. Science. 231, 1986.
Ravi S, Chaikof EL. Biomaterials for vascular tissue engineering. Regenerative Medicine, 5(1), 107, 2010.
Where there's a Wnt there's a Way: Mending Broken Bones with a Novel Wnt-Surrogate
Orthopaedic Surgery, UCSF, San Francisco, CA
15 million bone fractures occur within the United States annually. Of these, 10–20% exhibit delayed or non-union, a ratio that increases to 46% when accompanied by concomitant morbidities such as decreased vascular perfusion, diabetes, or smoking. Fractures heal through two different mechanisms, intramembranous and endochondral ossification. It has been established that canonical Wnt signaling promotes intramembranous ossification, which has prompted the development of several Wnt-related therapies. However, these therapies activate the Wnt pathway indirectly by inhibiting negative regulators. Direct activation of the Wnt pathway through the use of Wnt ligands is translationally irrelevant due to their palmitoylation, which causes Wnts to be hydrophobic and requires time-consuming and costly isolation procedures that often result in inactive protein and low yield. We have developed a novel water-soluble and mass-producible Wnt-Surrogate that activates canonical Wnt signaling by inducing Wnt receptor clustering. We hypothesize that Wnt-Surrogate promotes both intramembranous and endochondral bone repair and have tested this in vitro using a variety of cell lines and tissues explants (MC3T3-E1 preosteoblasts, BM-hMSCs, cartilage fracture callus explants). Preliminary data demonstrate that Wnt-Surrogate promotes both forms of healing as quantified by alkaline phosphatase activity, matrix mineralization, and osteogenic gene expression. Ongoing studies are evaluating the effects of Wnt-Surrogate on fracture healing in vivo and will optimize its delivery using Alginate hydrogels. This study is one of the first to explore the role of canonical Wnt signaling in endochondral bone repair and provides support for the use of Wnt-Surrogate as a therapeutic for bone regeneration.
Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA
The ability to maintain contractile intestinal smooth muscle cells in vitro is crucial for understanding diseases affecting intestinal motility. The intestinal smooth muscle in conventional culture conditions often suffer from incomplete maturity with limited functions, and the contractions of those cultured cells are transitory and irregular, relying on external stimuli. Here we develop the first serum-free culture medium that consistently maintains periodic contractions of cells isolated from intestinal smooth muscularis (ISMC) for a long time. The contractions of ISMC in the serum-free condition are 1) spontaneous, 2) visibly distinct, 3) periodic, 4) maintained for at least 56 days, and 5) with a frequency closely resembling that of native smooth muscle. For the first time, mature smooth muscle cells, interstitial cells of Cajal, neurons and glial cells all survive under this serum-free condition and form intestinal smooth muscle organoids. The contractions could be controllably induced or inhibited by changing the culture condition. We further found that the drug response of contracting ISMC closely approximated that of native intestinal smooth muscles, which may potentially benefit the development of pharmaceuticals for motility disorders. The serum-free medium also supports the growth of intestinal epithelial stem cells, thereby providing a condition for engineering both the epithelium and muscularis of the intestine. Finally, periodic contractions were observed when a similar medium was applied to human ISMC, which may eventually advance this approach to clinical applications.
Böttcher-Haberzeth, Sophie, Thomas Biedermann, and Ernst Reichmann. “Tissue engineering of skin.” Burns 36.4 (2010): 450–460.
A Small Molecule BIO (6-Bromoindirubin-3′-oxim) Induces Adult Inner Ear Supporting Cell Proliferation
Hair cells in the inner ear are essential for hearing and balance that are lost due to aging, medications or loud sounds. The adult mammalian inner ear fails to regenerate hair cells, resulting in permanent deficits. During development, supporting cells within inner ear sensory epithelia can self-renew to give rise to new hair cells; however they become quiescent postnatally. Previously, we have shown that in vivo gene delivery of c-Myc (c-MycT58A) into the inner ear stimulated the proliferation of supporting cells in adult mice utricles leading to formation of few new hair cells. The goal of our current research is to reprogram differentiated inner ear supporting cells into otic progenitors by in situ induction of c-Myc using small molecules. Using Luciferase based cell assay developed in our laboratory, we screened several small molecules for enhanced expression of c-Myc. BIO (6-Bromoindirubin-3′-oxim) induced c-Myc expression significantly. Further, BIO was tested in vitro on adult mice utricle organ cultures. Supporting cell proliferation and hair cell regeneration were analyzed using EdU (Ethynyl-2′-Deoxyuridine) along with supporting and hair cell specific markers, SOX2 and MYO7a. BIO treated adult mouse utricles showed supporting cell proliferation as evidenced by EdU and SOX2 co-labeling. There were small number of EdU positive hair cells suggesting transdifferentiation of supporting cells into hair cells. BIO is being further tested in vivo for its ability to induce reprogramming and differentiation of supporting cells in adult inner ear. These studies offer promising therapeutic potential to treat hearing and balance impairments.
Specific Targeting of hASCs and RF Mediated Osteogenesis Using Dumbbell Shaped AuFe3O4 Nanoparticles Conjugated with Anti-CD146 Antibody and miR148b mimic
Spatial and temporal control for deep tissue regeneration is largely dependent on the precise targeting of therapeutics to progenitor cells at the site of the defect as well as properties of drug delivery vehicle used to affect the intended area. Here we present our efforts to develop a targeted RF stimulated siRNA delivery tool to modulate tissue regeneration via spatiotemporal control of post-transcriptional gene regulation. To this end we have developed a heterodimeric Au-Fe3O4 nanoparticle composed of gold and iron oxide subunits that allow for discrete chemical attachment sites and chemistry for dual functionalization with targeting molecules and miRNA mimics, in this case human CD 146 antibody and miR-148b mimic respectively. To control activation (uncaging) of the miRNA mimic we have developed a stimulus responsive Diels-Alder linker which tethers the oligo to the surface in an inactive state. The Diels Alder linkage provided an effective spatial and temporal control switch to modulate gene expression via radio frequency (RF) activation while CD 146 antibody provides specific targeting of heterodimer NPs to hASCs. Regarding targeting of therapeutics to progenitor cells, MALDI demonstrated effective conjugation of the CD146 antibody to the iron oxide portion of the AuFe3O4. Flow cytometry confirmed attachment of the CD146 antibody to the hASCs. Osteogenic hASCs exhibited upregulation of alkaline phosphatase (ALP) at day 7, as well as calcium deposition at 14 and 21 days when stained with Alizarin Red and further supported by ICP-OES analysis for the magnesium, phosphorous, and calcium.
Nucleic Acid Release in Biological Applications via Photo-cleaveage and Enhanced by Plasmonic Metal Nanoparticles
Hypoxic Preconditioning of Mesenchymal Stem Cell Spheroids Enhances Viability and Proangiogenic Potential
Department of Biomedical Engineering, University of California, Davis, Davis, CA
Mesenchymal stem cell (MSC) therapies hold great potential in tissue engineering, yet they are limited clinically due to poor survival and engraftment in vivo. Preconditioning MSCs under hypoxic conditions can promote cell viability. We hypothesized that preconditioning MSCs in hypoxia before formation into spheroids and entrapment in alginate gels would increase cell survival and proangiogenic potential. MSCs were preconditioned under hypoxia (1% O2) in monolayer culture for up to 3 days (PCX = preconditioned for X days), formed into spheroids of 3 different sizes (3,000; 10,000; 15,000 cells/spheroid), and then entrapped in RGD-modified alginate hydrogels. Groups were assessed for cell viability and vascular endothelial growth factor (VEGF) secretion over 4 days in vitro in serum-deprived/hypoxic conditions to mimic in vivo conditions of a large bone defect. Caspase 3/7 activity, an indicator of apoptosis, decreased with increasing spheroid size. In spheroids, caspase activity significantly decreased in both 1 and 3 day hypoxic preconditioned groups compared to unconditioned controls. Live/dead imaging of spheroids in gels confirmed increased cell viability in preconditioned versus unconditioned groups. Cell adhesion and migration from spheroids in both preconditioned groups were markedly increased. VEGF secretion, a key indicator of proangiogenic potential, was enhanced in PC3 spheroids. These data demonstrate that preconditioning of MSCs prior to spheroid formation and entrapment in alginate hydrogels promotes cell viability, spheroid migration, and proangiogenic potential. Transplantation of hydrogel constructs into murine femoral defects is ongoing. These findings have important implications for the use of MSC spheroids in cell-based approaches for tissue repair.
Bioengineering and Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL
The aim of this project was to evaluate effects of cryopreservation on the metabolism of adipose-derived stem cells (ASC). Our hypothesis was that as cell passage number increases metabolism increases to a point and then decreases with senescence. We evaluated and compared passages (P2, P3) of fresh and cryopreserved ASC for Kreb's cycle intermediates. Swine ASC were isolated and cultured as described (1). Spent media was analyzed via Varian VNS-750 NB (750 MHz) spectrometer. Data were analyzed with ANOVA using the Generalized Linear Model procedure. Tukey's post hoc test was used to perform multiple comparisons with an alpha level of 0.05. We evaluated 8 samples per passage by NMR. The results showed no difference in the formate or acetate concentrations. Pyruvate concentration was higher in fresh than frozen ASC at P3 (P < 0.001). Glucose concentration decreased with increasing passage number for fresh ASC, as expected. However, glucose levels in cryopreserved cells increased with higher passage (P < 0.01 E-12). The fresh ASC results showed expected metabolism profiles (Kreb's cycle modulation) while the frozen ASC had an increase in all the sugars. In conclusion, our data suggests that the Kreb's cycle of frozen/thawed ASC may be different from fresh ASC and that cryopreserved ASC may need time to adapt their metabolism after thawing.
1. Monaco E. et al., PlosOne 7(3) 2012 2. Rubessa M. et al., Reproduction fertility and Development 28(2) 2016
Induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) have gained considerable attention for their potential in regenerative medicine, because of their functions in differentiation, immunomodulation and advantages in infinite cell source and autologous transplantation. Although iPSC-MSCs have been emerged as an alternative source for tissue engineering, their variable differentiation capacity has hampered the development of iPSC-MSCs related therapeutic approaches.
The objectives of this study were to evaluate the differentiation potential of iPSC-MSCs and to clarify the effects of long term passage on cell multipotency. Two characterized iPSC-MSC lines were chosen. Cells were harvested from different passages (Ps) for examining their mesodermal gene expression pattern (MGEP). Furthermore their biological properties over time were evaluated.
FACS analysis and gene analysis together showed that although iPSC-MSCs had similar surface profile as bone marrow (BM)-derived MSCs at P5, they didn't share similar MGEP. BM-MSCs displayed a gene profile of paraxial mesodermal (PM), whereas iPSC-MSCs showed high-level expression of lateral plate mesoderm gene (KDR) but low-level expression of PM genes (PDGFRα, Mesp2). The difference in gene profiles corresponded to distinct differentiation potential. Result showed that iPSC-MSCs had lesser chondrogenicity than BM-MSCs. Furthermore, long term maintenance leaded to irregular variations in surface profile and down regulations of all tested multipotency genes in both iPSC-MSC lines. Forthcoming data will include evaluation of the effects of long term culture in differentiation efficiency of iPSC-MSCs.
In conclusion, the differentiation potential of iPSC-MSCs could be predicted based on their MGEP. Long term passage could induce loose of multipotency of iPSC-MSCs.
Optimization of Skeletal Myocytes Expansion for Potential Application in Cell Therapy for Urinary Incontinence
Wake Forest Institute for Regenerative Medicine, Winston Salem, NC
Damaged cartilage has a limited capacity to heal due to its avascular nature, and can lead to severe musculoskeletal morbidity. Advances in cell-based therapies using mesenchymal stem/stromal cells have demonstrated promising results to increase the efficacy of cartilage repair treatments. Additionally, increasing evidence highlights the importance of miRNA regulation in the development and maintenance of mature articular cartilage. MiRNA are noncoding oligonucleotides capable of regulating post-transcriptional gene expression. Gene therapy with miRNA can be integrated with current mesenchymal stem cell therapies to increase chondrocyte proliferation and enhance chondrogenesis in order to increase the regenerative potential of articular cartilage. This study examines the effects of miRNA 140-5p and miR-21 mimics on proliferation and chondrogenesis of adipose derived mesenchymal stem cells (hASCs). hASCs were cultured in chondrogenic medium in Transwells for up to 3 weeks and supplemented by chemical transfection of miR-140-5p and miR-21. For each sample group the number of cells was quantified over time, and the extracellular matrix was analyzed. Expression of ACAN, COL 2A, and SOX9 was quantified via qRT-PCR. Overexpression of miRNA increased cell proliferation and upregulated the expression of chondrogenic markers in hASCS, resulting in increased ECM production. Future studies are needed to optimize the timing and method of transfection for effective therapeutic use of miRNA.
An in vitro System to Model the Effects of Fibrosis on Liver Development
Directed Differentiation of Cardiac and Vascular Lineages from Pluripotent Stem Cells on Decellularized Myocardial Matrix
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD
Low Intensity Ultrasound Prolongs Lifetimes Of Transplanted Mesenchymal Stem Cells In Situ
An Implantable Device for Screening Hematopoietic Stem Cell Niche Factors in vivo
Development of an Automated Bioreactor for Continuous Loading and Monitoring of Anatomically Shaped Meniscus Constructs
Cornell University, Ithaca, NY
Bioreactors are widely used for culturing a broad range of tissues. For example, mechanically loaded tissue engineered cartilage has been successfully used in humans and is currently in phase III clinical trials. The challenge with bioreactor culture is generating many isolated and individually tailored tissue samples. We previously developed a bioreactor system for stimulating whole tissue engineered menisci with anatomic geometries. However this system is vulnerable to contamination and is labor intensive. Our objective was to develop a scalable, automated bioreactor that enables individual culturing and monitoring of samples. A bioreactor was designed with a rotational stage, stepper motor, and load sensor. Individual tissue culture bags were designed with custom loading plates. Culture bags contained ports for filtered air exchange and media changes. The bioreactor was controlled using a custom LabVIEW interface programmed with an automated loading scheduler. Real-time load data was recorded for each sample. Tissue-engineered menisci were cultured for 2 weeks. The automated scheduler rotated the stage and loaded samples at specified times. Media was changed 3 times a week and all menisci remained sterile throughout culture. The bioreactor designed in this study also evaluated tissue properties throughout culture. This device is scalable and amenable to customization to specific tissues. The maturation of individual samples can be monitored throughout the culture period. Furthermore, the LabVIEW user interface is easily programmed to support different loading regiments and schedules. This device demonstrates the feasibility of scalable, commercial bioreactors to support clinical application of engineered tissues.
Chemical and mechanical stimulation positively influences the differentiation of in vitro cultured stem cells, and increases the quality of deposited extracellular matrix (ECM) when properly utilized. For tendon tissue engineering studies, cyclical mechanical stimulation is the primarily utilized mode of inducing tenocytic differentiation. In order to better understand the mode of force transduction at a cellular level; this study validates the cyclical mechanical stimulation regimen previously used by in house studies, on mesenchymal stem cell (MSC) seeded human umbilical veins (HUV). Mechanical stimulation is well documented in the literature as having a positive influence on tenocytic differentiation, but little work has been done to optimize the process. We hypothesize that stretching the constructs continuously for 1 hour/day causes cellular fatigue, eliminating any beneficial effects of stimulation at some time point during the 1 hour. Breaking up the stimulation into shorter, but more frequent, regimens should mitigate this fatigue and any possible refractory period in the currently unknown pathways that lead to tendonogenic differentiation. This study takes the 1 hour duration previously used, and separates it into smaller amounts throughout the day, 30 min 2/day, 15 min 4/day and 5 min 12/day. After extended bioreactor culture, the tendon constructs are removed from their bioreactor at both 7 and 14 days to examine cellularity, tensile strength, collagen fibril alignment and tendon gene expression compared to static controls. Through this optimization, we hope to engineer a more tendon like construct than has previously been obtained.
A New Mechanical Micro-bioreactor for Cartilage Tissue Engineering
Pasteur Institute of Iran, Tehran, IRAN, ISLAMIC REPUBLIC OF
In the field of cartilage tissue engineering, the hydrostatic pressure and the shear stress have a critical role in the chondrocyte differentiation and maintaining their phenotype in cell culture period in vitro. On the other hand, it is shown that cell aggregation could help cells to differentiate to chondrocytes. In this study a mechanical micro-bioreactor is designed and fabricated to combine mechanical stimulations and bottom-up methods. This bioreactor contains an array of micro-wells to control cell aggregation. Furthermore it could control the hydrostatic pressure and the shear stress simultaneously and independently to mimic mechanical stimulations that is sensed by cells in a normal cartilage. The frequency and amplitude of both mechanical stimulations could be controled by the bioreactor. This device is capable to produce pressures up to 6MPa and the maximum frequency is 0.2 MPa/s. The minimum volume of the medium that this bioreactor could exchange is 2.5 μL. Total volume of cell culture chamber is 518 μL that could be totally exchanged by the bioreactor automatically. The cytotoxicity test shows that up to 90% of the KG1 cells in the cell culture chamber were viable after 7 days of culture. Based on our knowledge it is the first micro-bioreactor that is able to produce hydrostatic and shear stress simultaneously and independently with this order of amplitude for hydrostatic pressure. Because of very low medium that is required for cell culture chamber it is a low cost device for optimizing medium and mechanical conditions for tissue engineering of cartilage.
Osteochondral Tissue Morphogenesis via Magnitude-Dependent Differentiation of Human Mesenchymal Stem Cells under Dynamic Compressive Strain
Bioactuator Using Tissue-engineered Skeletal Muscle
The living muscles have excellent characteristics of lightweight, high flexibility, and remarkable efficiency for energy. Therefore, a muscle cell-based bio-hybrid actuator has the potential of being flexible and highly efficient on a micro to macro scale. The aim of this work is to develop the bio-actuator made of cultured skeletal muscle cells in vitro. We have constructed a three-dimensionally cultured skeletal muscle with collagen gel as cell substrate. Two artificial tendons made up of acellular porcine blood vessel were incorporated at the both ends in order to fix and handle the gel firmly. After insertion of two artificial tendons into pins on the culture substrate, the cell suspension of 100 μl in collagen solution was applied between two tendons by a micropipette. The cell-collagen gel composite started to shrink gradually and a small dumbbell-like muscle was obtained having approximately 15 mm in length and 0.5 mm in diameter at the 14 days after gelation. It was applied for driving a micro-object prepared by the micro stereolithography apparatus. The artificial tendons were tied firmly to the base objects and the electrical pulse stimulation to cause tetanus contraction was added to the muscle. The bio-actuator had contracted about 15% and be able to drive the lever object for the rotation about 10°. There are still several issues to be solved for getting large and powerful bio-actuator which works long term with proven reliability, however it must be done in the near future by intensive studies.
Extracellular Matrix in Spermatogonial Stem Cell Culture
Surgery, University of Pittsburgh, Pittsburgh, PA
Successful Human spermatogonial stem cell (SSC) culture could enable therapies restoring fertility in males subjected to chemotherapy or radiation therapy. Conditions for expanding rodent SSCs in culture are established and robust, but human SSC cultures are in early development and no human SSC culture system has yet been independently verified. Mammalian extracellular matrix (ECM) contains signaling molecules promoting mitogenesis, migration, and/or differentiation of various stem/progenitor cells which we hypothesize will improve human SSC cultures. Human testicular ECM (htECM) and porcine small intestinal submucosa ECM (SIS) were prepared and solubilized as a media supplement for culture experiments. SSCs were cultured on STO or C166 feeder cells, Matrigel, murine or human laminin, or human laminin with htECM or SIS. Cells were passaged at day 7 and stained for the SSC marker UTF-1 at 0, 7, and 14 days to identify the percentage of UTF1+ cells relative to day 0. By 7 days the wells with feeder cells, Matrigel, or murine laminin showed ∼45% the number of starting UTF1+ cells, whereas wells with htECM or SIS showed 77% and 187%, respectively. At 14 days, wells with feeder cells, Matrigel, and murine or human laminin showed ∼20% the number of starting UTF1+ cells, whereas wells with htECM or SIS showed 59% and 114%, respectively. ECM appears to improve SSC survival in culture and establishes a foundation for development of robust human SSC cultures that will be valuable for fundamental investigations and may have application for treating some cases of male infertility.
Tokyo Women's Medical University, Dep. of Obstetrics & Gynecology, Tokyo, JAPAN
Cyclic strain and fluid flow are well known to affect cell behaviour. Also, isotropic and anisotropic strain can affect cells differently. While in-vivo cells experience varying degrees of anisotropy (d.o.a.), in-vitro anisotropic strain studies have mostly focused on uniaxial strains. Here, we determined the effects of varying d.o.a., in combination with fluid flow shear stresses, on endothelial cells (HUVECs) using a newly developed device. The device has 100 units producing various anisotropic strains. This is achieved by stretching a polydimethylsiloxane (PDMS) membrane over circular pillars into surrounding ellipse trenches. The dimensions of the ellipse determine the d.o.a., which is defined as the ratio of maximum to minimum principal strains. The presence of fluid flow channels at varying angels to the ellipses allows for the determination of combined effects of anisotropic strains and flow induced shear stresses. HUVECs aligned along the minimum principal strain direction when only strain was applied. An increase in d.o.a. resulted in increased cell alignment. Cells aligned along the flow direction when only flow was applied. When flow and strain were combined, alignment was predominantly in the direction of the flow, but an offset towards the minimum principal strain direction was detected. The variations in response of cells highlight the need to study the effects of strains of varying d.o.a. on cells. Our device permits such experiments with an increased throughput, making it an important tool to better understand these mechanobiological principles.
Novel Liquid Marble Micro Bioreactor Derived Embryoid Bodies For Cardiac and Neural Differentiation of Induced Pluripotent Stem Cells
Hudson Institute of Medical Research, Melbourne, AUSTRALIA
Directing the spontaneous differentiation of induced Pluripotent Stem cells (iPSCs) to cardiac and neural lineages via formation of 3D aggregates called Embryoid Bodies (EBs) is a major challenge. Our novel micro bioreactor system, Liquid Marbles (LM), used iPSCs to form viable EBs and is the first study of its kind. 2 × 104 feeder free MEF-iPSCs#6 were suspended in 300μl of differentiation medium without mLIF. A drop of cell suspension was placed onto a polytetrafluoroethylene (PTFE, 35μm) powder bed and incubated in a humidified incubator for 7 days. Each LM generated >15 EBs (day 5) and their progressive loss of GFP (from 76.51% at Day 0 to 34.16% at Day 7) based on FACS indicated that the EBs had started to differentiate. Day 5 EBs were plated onto 0.1% gelatinized 6-well dishes in MEF medium. 6 days after EB attachment, neural cells (ectodermal lineages) with extending processes were observed in 60% of the EBs whereas 40% of the EBs that attached went down the meso-endodermal lineages forming cardiomyocytes. RT-PCR data indicated an upregulation of Nkx2.5 (cardiac) and Nestin (neural) genes, which was further confirmed by our immunostaining studies where positive expression of Nestin (neural) and Nkx2.5, cTnT (cardiac) was observed. Based on the molecular and protein analysis of the differentiated EBs, LM generates functional EBs expressing the three germ layer markers for ectoderm (Nestin), mesoderm (Brachyury, Nkx2.5) and endoderm (Gata4, FoxA2). LM is a viable and economical method for generating functional EBs from iPSCs.
Infertility and premature loss of ovarian endocrine function is a concern among many patients facing cytotoxic treatments. None of the clinically available fertility preservation options are suitable for children and young adults, who cannot produce mature oocytes. Ovarian tissue cryopreservation prior to the cytotoxic treatments followed with maturation in vitro or transplantation can provide the patient with mature oocytes years later. We engineered poly(ethylene glycol) (PEG) hydrogels with tunable physical properties, which allow co-encapsulation of ovarian follicles with support cells, such as mouse embrionic stem cells and adipose stem cells. Ovarian follicles co-encapsulated with support cells in 5% PEG hydrogels crosslinked with proteolyically (MMP-1) sensitive peptides had an improved survival and growth, compared to conditions without the support cells. The proteolytical degradation of PEG hydrogels not only mirrored follicle growth, but provided a substrate for the support cells to interact with the encapsulated follicles through paracrine and juxtacrine signaling in vitro. In vivo, PEG hydrogels served as a matrix for engraftment of engineered artificial ovary. Histological evaluation of the explanted ovarian grafts 30 and 60 days after implantation revealed multiple fully developed antral follicles and newly formed vasculature, which corresponded with normal physiological levels of circulating follicle stimulating hormone (FSH) and estrous cycles. In summary, our results demonstrate that synthetic PEG hydrogels successfully supported folliculogenesis and steroidogenesis of the artificial ovary in vitro and in vivo.
Chitosan Microgel Pastes as Injectable Micro-Porous Scaffolds in Tissue Regeneration
Chemical and Biological Engineering, Colorado School of Mines, Golden, CO
Injectable, microporous hydrogels are superior in regenerative medicine due to minimally-invasive delivery and interconnected pores within which cells can migrate and proliferate [1]. In this work, an emulsion crosslinking method was developed to produce chitosan-genipin microgels (diameter ∼100 μm) which could be densely-packed and, to our knowledge for the first time, remain condensed/aggregated after injection into PBS; acting as an injectable and microporous scaffold. Chitosan biopolymer was characterized with respect to pH by light scattering and aqueous titration. Microgels were characterized through swelling, light scattering, and rheometry of densely-packed microgel solutions. The results suggest that as chitosan becomes increasingly deprotonated above the pKa, repulsive forces diminish and intermolecular attractions (i.e. hydrogen bonding and hydrophobic interactions) drive pH-responsive chain aggregation; leading to microgel-microgel aggregation as well. The microgels with the most (i.e. 6 wt%) chitosan and least cross-linker (5 mM genipin) showed greatest mechanical strength when condensed at pH 7.4, with a shear modulus (G’) of 2 kPa for the condensed microgel paste. Two growth factors with opposite overall charge could be encapsulated into the microgels and co-cultured endothelial cells were able to migrate onto the 3D microgel scaffold. Viability of co-cultured endothelial and mesenchymal stem cells was assessed and cells were able to span microgels to create biological crosslinks upon migration and proliferation. Enzymatic degradation by lysozyme was faster than previous reports of chitosan microgels. Current research is examining this microgel scaffold for minimally-invasive applications in orthopedic regenerative medicine.
[1] Griffin, Segura, et al., Nature Materials, 7, 737–744, 2015, doi:10.1038/nmat4294.
Composite Effects of Fibronectin, Collagen, and VEGF on Human Foreskin Fibroblasts: Proliferation and Migration
Macro porous blend membrane of Chitosan and Polyurethane diol as skin graft
Vashist A, Shahabuddin S, Gupta Y. K., Ahmad S. J. Mater. Chem. B, 168, 1,2013.
Analysis of VEGF, PDGF and bFGF Production in Adipose Stem Cells Incorporated in Blood Fibrin Clots
Molecular, Cellular, & Developmental Biology, University of California Santa Barbara, Santa Barbara, CA
Expression of Antimicrobial Peptides by Macrophages Exposed to Solubilized Extracellular Matrix
Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA
Extracellular Matrix (ECM) scaffolds are associated with resistance to persistent bacterial infection [1, 2], with partially understood mechanisms. The biologic activity of the enzymatically degraded products from the ECM, i.e. liberated matricryptic peptides and antimicrobial peptides (AMP), have been shown to facilitate a constructive macrophage response [3, 4] and provide antimicrobial activity [5, 6], respectively. The AMP cathelicidin LL-37 is an important endogenous component of the innate immune response, with direct microbicidal activity [7] and tissue remodeling regulatory functions [8]. The secretion of cathelicidin LL-37 from macrophages following exposure to the degradation products of ECM has not been previously investigated. The present study evaluated the expression of cathelicidin LL-37 of macrophages exposed to solubilized degradation products of ECM-bioscaffolds.
Murine primary bone marrow-derived macrophages were exposed to degradation products of ECM-bioscaffolds composed of small intestinal submucosa (SIS), dermal-ECM, or urinary bladder matrix (UBM) in vitro. Transcriptional activation of the CAP18 gene was determined by qPCR. Immunolabeling and ELISA were used to quantify the secreted product. Degradation products of all three ECM-bioscaffolds significantly promoted the in vitro expression of cathelicidin LL-37.
These results advance the understanding of ECM-bioscaffolds and the potential role of AMP in the context of biomaterial-mediated tissue repair; especially in the context of surgical site infection. The fact that the ECM degradation products provide not only a source of active AMP, but also can induce the expression of a new set of these AMP by infiltrating cells, provides a plausible explanation for the infection resistance of ECM-bioscaffolds.
Bioengineering, Hacettepe University, Ankara, TURKEY
In Vivo Engineering of Bone Organoids with Hematopoietic Functions
University of California, San Diego, La Jolla, CA
Synthetic biomimetic matrices with osteoconductivity and inductivity have been developed to regenerate bone tissues. However, whether such matrices harbor hematopoietic functions in vivo remains unknown. We devised a strategy to engineer bone organoids with functional bone marrow (BM) compartment in vivo by employing a synthetic biomaterial. Specifically, we have developed a synthetic matrix recapitulating the dual-compartment structures of native bone tissue by modular assembly of mineralized and non-mineralized macroporous hydrogels. Our results show that these matrices incorporated with BM cells (BMC) or BM flush (BMF) transplanted into recipient mice matured into functional bone displaying cardinal features of both skeletal and hematopoietic compartments. The skeletal tissue was characterized by the presence of osteoblasts and osteoclasts, whereas the marrow compartment was characterized by the presence of different hematopoietic cell populations. Furthermore, the implants supported long term mixed chimerism and hematopoietic cell mobilization as evident from donor cells in the circulation of non-irradiated recipients. Such engineered bone organoids could potentially be employed as a tool to study hematopoiesis, donor-host cell dynamics, tumor tropism, and hematopoietic cell transplantation.
Western Washington University, Bellingham, WA
Conducting polymers (CPs) such as poly(pyrrole) and poly(3,4-ethylenedioxythiophene) show great promise for use in biomedical applications due to their electrical conductivity, biocompatibility and ability to electrochemically actuate (expand and contract) when low potentials are applied. These actuators are anticipated to serve as implantable dynamic tissue scaffolds (ex. artificial muscles) as well as soft surgical implements. However, CPs are difficult to process and have poor mechanical characteristics. Our group has previously demonstrated that these weaknesses can be ameliorated by creating composites of CPs with silk fibroin. Silk fibroin is a strong, flexible biopolymer that can be used to produce fibroin-CP composites that are processable into a variety of 2D and 3D structures that retain the mechanical characteristics of silk and the conductivity of the CPs. Here, several strategies were explored to produce novel actuation devices from silk-CP composites. Computational modeling of surface topology and dynamics was used to design devices with directed motion, and a laser cutter was used to cut and emboss the desired patterns into silk films. Additionally, selective deposition methods were employed to deposit CP in defined regions of silk films. Device designs were validated by assessing the actuation performance in biologically-relevant electrolyte solutions, and biocompatibility of the materials was confirmed by in vitro cytotoxicity assays. This multi-tiered approach to actuator design should allow for rapid development of electroactive tissue scaffolds capable of defined, dynamic movements (ex. cardiac patches).
Tissue engineering, Cell Therapy and Regenerative Medicine Unit, National Institute of Rehabilitation, Mexico City, MEXICO
Mesenchymal stem cells (MSCs) have the ability to differentiate into functional lineages according to the stimuli from the microenvironment and can generate hyaline cartilage (HC) under specific conditions. Several commercial biocompatible materials are available to produce HC restoration, which can be classified as biologic and synthetic scaffolds. No substantial evidence exists to support the use of one scaffold over another one. The aim of this study was to analyze the effect two biocompatible scaffolds, one biologic composed of collagen and one synthetic composed of polyglycolic acid (PGA) have on growth and differentiation of MSCs derived from equine bone marrow and which were stimulated to generate HC. Isolation of MSCs was performed looking for the presence of specific surface antigens by flow cytometry, which were performed at days 0, 14 and 21 of culture without stimuli. Differentiation trials were performed to detect multipotency using immunofluorescence. MSCs were seeded on a collagen scaffold and a PGA scaffold. Cell proliferation tests were performed comparing the two polymers. After two weeks of culture with stimuli, stains were performed to evaluate whether cells produced glycosaminoglycans and to compare the histological structure of native HC (control). The molecular profile of the cells was evaluated by RT-PCR. We found a similarity of positive areas of glycosaminoglycans. It was evidenciated that the PGA polymer favors the expression of markers of HC as compared with the collagen scaffold. The synthetic polymer provides a better microenvironment for the differentiation of MSCs into HC based on the histological and molecular findings.
In-vivo, cells reside within a 3D network of hierarchically organized fibrous matrix that provides both structural support and micro and nanoscale cues. It is believed that these cues, combined with soluble biochemical signals, guide cell behavior, such as proliferation, migration, survival, morphogenesis and differentiation. Recapitulating the micro and nanoscale cues of the cellular microenvironment of the heart, remains a key challenge in cardiac tissue engineering. We initially sought to evaluate the fibrous structure of the natural ECM. SEM images of decellularized hearts revealed fibers with various diameters, structures, orientations and mechanical properties. Furthermore, we identified a unique population of coiled fibers responsible for tissue contraction and relaxation. Next, according to our findings, we have synthetically engineered scaffolds comprised of similar morphologies and evaluated the role of each fiber population on cell maturation, morphology, fibroblast proliferation and cardiac tissue assembly. Based on these findings we were able to fabricate cardiac patches with superior functional properties, including patches with low excitation threshold that generate a strong contraction force. We envision that the presented data and the aforementioned scaffolds will be essential for engineering cardiac tissues that could be used to improve heart function after myocardial infarction.
Electrospun Nanofibrous Scaffolds made by Animal Fiber Protein
Keratin is a fibrous protein that constitutes the intermediate filaments in epithelial cells of vertebrate animals and is a chief constituent of hair, nail, horn, and beak. A key characteristic of keratin is its amino acid composition and high cysteine content that permits the formation of many disulfide bonds. These bonds maintain a firm steric structure and make the protein water-insoluble. Keratin also contains amino acid sequences such as RGD and LDV that are involved in cell adhesion and have similarities to fibronectin, which is a known cell-adhesive protein. Thus, much like fibronectin, high cell affinity is expected for keratin if it is used as a biomaterial. In this study, keratin protein extracted from human hair was formulated into nanofiber sheets in order to evaluate the potential as a scaffold material. Spinning solution of keratin could be spun to the nanofiber by electrospinning. The physical properties of keratin nanofibers were evaluated for hydrophilicity, mechanical strength, and surface structure. Cultivation of fibroblasts on nanofibers was examined in order to evaluate biocompatibility of human hair keratin. Cells were attached well and grown steadily on nanofibers, though the rate of cell growth on hair derived keratin was different from that on wool derived one. Keratin nanofibers were then subcutaneously implanted in rats. Recipient cells were well attached on nanofibers without eliciting a major inflammatory response.
Effect of Cell Density and Material Stiffness on Human Bone Marrow Stem Cell Chondrogenesis at Physiologically Relevant Mechanical Strengths
Aligned Electrospun Grafts with Gradient Properties for Interfacial Tissue Engineering
Despite numerous advances in recent years, electrospun scaffolds are not yet able to mimic the innate transition of properties at the tendon:bone interface. We have developed a multivariate electrospinning design that permits control over gradient properties in the direction of fiber alignment. In addition to providing grafts that better mimic the structure of the tendon:bone interface, these unique scaffolds may also enable spatial control over cellular behavior. In this study, scaffolds with parallel mechanical, biochemical and topographical gradients were fabricated and assessed as tissue engineering scaffolds. Biodegradable polyurethanes (B-PUR)s were synthesized with 10–50% hard segment to achieve a range of moduli. A continuous gradient of B-PUR10%HS and B-PUR50%HS + hydroxyapatite was electrospun onto a wheel of copper wires that promoted fiber alignment via air gap spinning. Fiber alignment was quantified from SEM images and biochemical properties were evaluated pre- and post-mineralization in simulated body fluid. Tensile properties were assessed and complemented with finite element modeling. This novel setup enabled fabrication of scaffolds with aligned fibers in the direction of the compositional gradient. Mechanical and biochemical gradients were confirmed via tensile properties and FTIR, with mineralization occurring only in the designated bone-like region. Cellular alignment that matched fiber alignment was also observed. Current studies are characterizing the individual and synergistic effects of mineralization, tensile properties, and cyclic loading on cellular behavior. Overall, this study demonstrates a novel method to mimic the complexity of native tissue transitions through a combination of topographical, mechanical and biochemical cues that direct cellular behavior.
Powder Coating 3D Printed Polycaprolactone Scaffolds for Tissue Regeneration
Biomedical Engineering, University of Cincinnati, Cincinnati, OH
3D printing can make custom polycaprolactone (PCL) implants that give mechanical strength to healing tissues. It was hypothesized that the tacky nature of PCL at temperatures in the 45oC range could be used to adhere powders to printed structures, enabling incorporation of a regenerative component into the scaffold. Additionally, the low temperature at which PCL becomes tacky minimizes the effects of thermal degradation. Scaffold discs were printed using fused deposition modeling. A custom compressed air spray gun dispersed the chicken collagen type 2 powder. There were four groups: scaffolds sprayed twice every layer (once halfway through and once at the end of the layer), scaffolds sprayed at the end of each layer, scaffolds that were not sprayed, and scaffolds that were sprayed only after they were removed from the print bed and reheated with a heat gun. Eosin stain was used to confirm that collagen powder was incorporated into the constructs. There was stain present in areas of the sectioned scaffolds that were between layers. The once-sprayed group had quality similar to that of the unsprayed group, but uneven powder distribution. The twice-sprayed group was of inferior quality, but uniform distribution. The post-processed scaffolds lost some resolution, but had a denser coating of collagen on the immediate surface than the other scaffolds. Further validation and viability tests are ongoing. The hybrid scaffolds proposed herein have the potential to inspire a new generation of bioactive scaffolds for many disciplines and can be tailored based on the application.
Minced muscle autografts (MMA) promote de novo muscle fiber regeneration and neuromuscular strength recovery in small and large animal models of volumetric muscle loss (VML). A major limitation of this approach is its reliance on a finite supply of donor tissue. To address this shortcoming, this study sought to evaluate micronized acellular Urinary Bladder Matrix (UBM) as a MMA expansion scaffolding. Male Lewis (n = 6 each) received VML injury in the middle third of the left TA muscle and treatment with either: (1) no repair, (2) repair with MMA at dosage of 100% of the defect mass, (3) repair with MMA at a dosage of 50% of the defect mass supplemented with UBM at a dosage of 25% of the defect mass, and (4) repair with UBM at a dosage of 100% of the defect mass. Rats survived to 8 weeks post injury before functional (in vivo neuromuscular strength), histological, morphological, and biochemical analyses. At 8 weeks, the no repair group exhibited a peak isometric torque deficit of 49% relative to sham controls. The 100% UBM control group showed little recovery of muscle function (47% strength deficit) while the 50% MMA +25% UBM, and 100% MMA groups exhibited similar functional recovery (32%, 31% peak isometric torque deficits, respectively). Based on these preliminary functional measurements, UBM shows promise as a myoconductive expansion material for minced graft therapy to VML. Forthcoming histological observations of myofiber number and architecture will shed further light on the nature of this repair.
Characterization of Human Platelet Lysate Loaded Keratin Hydrogels
Burn Injuries, US Army Institute of Surgical Research, JBSA Fort Sam Houston, TX
Human platelet lysate (hPL) has been used as a therapeutic agent in a number of regenerative applications. In order to provide sustained delivery of hPL-derived growth factors (GF), hPL was used to reconstitute keratin into a hydrogel. Keratin from human hair was extracted through an oxidative process to generate keratose, which was then further processed into a lyophilized powder. When rehydrated, keratose spontaneously forms a hydrogel capable of controlled and sustained release of therapeutics. The ability of varying formulations of keratose (15%, 22.5%, and 30% weight:volume) to control the hydrogel degradation and hPL-GF release was assessed using BCA protein assays and enzyme-linked immunoassays against PDGF. Stable hydrogel formation and porosity were evaluated by parallel-plate rheometry and SEM, respectively. Proliferation and viability of adipose-derived stem cells (HAPSC) in the presence of hPL loaded keratin was assessed via MTT assay. It was determined that by increasing keratose weight:volume, biomaterial degradation and GF release rates could be decreased over time. Stable porous hydrogel formation was confirmed by both rheometry and SEM. HAPSC cultured in the presence of hPL loaded keratose exhibited no loss of viability compared to normal cells cultured in hPL or non-hPL loaded kertose hydrogels. Our results indicate that keratose biomaterials are capable of delivering platelet lysate in a controlled manner. Sustained delivery of platelet-derived growth factors may have potential utility in the field of regenerative medicine.
Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA
The development of electrospun scaffolds has potential for vascular tissue engineering. However, regeneration of healthy endothelium onto vascular graft electrospun scaffolds is imperative for prevention of intimal hyperplasia, cell infiltration, and thrombogenesis. To overcome these challenges, we fabricated surface-modified poly (caprolactone) (PCL) nanofibers using radiation techniques for vascular tissue engineering. The PCL nanofibers were prepared by electrospinning method to modify hydrophilicity by grafting 2-aminoethyl methacrylate (AEMA) or acrylamide (AAm) using gamma-irradiation to immobilize heparin. Electrospun radiation-induced nanofibrous scaffolds endowed with controlled delivery of growth factors may allow for improved regulation of cell behavior for vascular tissue engineering. Future directions may include refinement of biomolecule immobilization and assessment of vascular grafts under circulation.
Development Of A Novel Bilayer Scaffold For Bone Defects Regeneration
Here, we show that new strategy for the formation of a bilayer-structured scaffold that can act as a representative barrier membrane for targeted-bone regeneration. Modified-solvent casting and evaporation technique was used to develop such membranes. Hydrochloric acid (HCL) was introduced into the blended solution of polycaprolactone (PCL) and calcium carbonate (CaCO3) which resulted in the in-situ formation of carbon dioxide (CO2) and water. This led to the phase separation between the PCL and calcium-based compounds and successively to the formation of a bilayer membrane. Thus formed membrane was characterized by field emission scanning electron microscopy (FE-SEM) and energy-dispersive X-ray spectroscopy (EDX). Surface morphology, surface wettability, and (EDX) analysis confirmed the formation of bilayered membrane with a PCL-rich thin layer on the upper surface and a calcium-rich porous layer on the lower surface. FE-SEM images revealed that the PC30 membrane showed a smooth upper layer with pores less than 10μm diameter, whereas the lower layer contained many interconnected larger pores up to 1000μm diameter in addition to the visibly identified macrovoids. The cell adhesion assay confirmed that both surfaces of the membrane responded well to the cells. In addition, PCL-rich upper surface prevented the down-growth of the fibroblasts. The initial results suggest a new strategy for the fabrication of the bilayer membrane for regenerative medicine.
Development Of A Novel Bilayer Scaffold For Bone Defects Regeneration
Here, we show that new strategy for the formation of a bilayer-structured scaffold that can act as a representative barrier membrane for targeted-bone regeneration. Modified-solvent casting and evaporation technique was used to develop such membranes. Hydrochloric acid (HCL) was introduced into the blended solution of polycaprolactone (PCL) and calcium carbonate (CaCO3) which resulted in the in-situ formation of carbon dioxide (CO2) and water. This led to the phase separation between the PCL and calcium-based compounds and successively to the formation of a bilayer membrane. Thus formed membrane was characterized by field emission scanning electron microscopy (FE-SEM) and energy-dispersive X-ray spectroscopy (EDX). Surface morphology, surface wettability, and (EDX) analysis confirmed the formation of bilayered membrane with a PCL-rich thin layer on the upper surface and a calcium-rich porous layer on the lower surface. FE-SEM images revealed that the PC30 membrane showed a smooth upper layer with pores less than 10μm diameter, whereas the lower layer contained many interconnected larger pores up to 1000μm diameter in addition to the visibly identified macrovoids. The cell adhesion assay confirmed that both surfaces of the membrane responded well to the cells. In addition, PCL-rich upper surface prevented the down-growth of the fibroblasts. The initial results suggest a new strategy for the fabrication of the bilayer membrane for regenerative medicine.
Multifactorial 3d Niche To Study Cell-matrix Interactions To Improve Maturation Of Pluripotent Derived Hepatocytes
Department of Stem Cell Biology and Embryology, Stem Cell Institute, KU Leuven, Leuven, BELGIUM
Department of Molecular Science and Technology, Ajou University, Suwon, KOREA, REPUBLIC OF
In situ crosslinkable hydrogels have been widely utilized as therapeutic delivery vesicles and therapeutic implants due to easy encapsulation of therapeutic agents and minimally invasive properties. Recently, horseradish peroxidase (HRP) and hydrogen peroxide (H2O2)-mediated crosslinking reaction has become an attractive method to create in situ forming hydrogels. While the crosslinking system has been widely utilized, there are certain issues require improvement to extend their biomedical applications, including creation of stiff hydrogels without compromising cytocompatibility due to initially high concentrations of H2O2. Herein, we report gelatin-based hydrogels formed through a dual enzyme-mediated crosslinking reaction using HRP and glucose oxidase (GOx) as an H2O2-generating enzyme to gradually supply a radical source in HRP-mediated crosslinking reaction. We demonstrate that physico-chemical properties can be controlled by varying glucose and enzyme concentrations, showing tunable phase transition time (from 30 s to 10 min) and mechanical strength (from 3 to 18 kPa). Furthermore, we demonstrate that our hydrogel matrices provide 3D microenvironments for supporting the growth and spreading of human dermal fibroblasts (hDFBs) with minimized cytotoxicity, despite the cells being encapsulated within stiff hydrogels. These hydrogels formed with HRP and GOx have great potential as artificial microenvironments for a wide range of biomedical applications. In addition, these injectable hydrogels can generate therapeutic dose of ROS, resulting in promotion of wound healing and angiogenesis etc.
Biomedical Engineering, Worcester Polytechnic Institute, Worcester, MA
Stem cell therapy has the potential to regenerate cardiac function after myocardial infarction. In this study, we used fibrin microthread technology to develop a contractile fiber from human induced pluripotent stem cell derived cardiomyocytes (iPS-CM). iPS-CM seeded onto fibrin microthreads began to contract seven days after initial seeding. A digital speckle tracking algorithm was applied to high speed video data (>60fps) to determine contraction behavior including beat frequency, contractile strain, and the principal angle of contraction of iPS-CM contracting on the microthreads over 21 days. At day 7, cells seeded on tissue culture plastic beat at 0.83 ± 0.25 beats/sec with a contractile strain of 4.23 ± 0.3%, which was significantly different from a beat frequency of 1.11 ± 0.38 beats/sec and contractile strain of 2.82 ± 0.34% at day 21 (n = 6, p < 0.05). iPS-CM seeded on microthreads beat at 0.84 ± 0.15 beats/sec with an average contractile strain of 2.94 ± 0.20%, which significantly increased to 1.03 ± 0.19 beats/sec and 3.49 ± 0.28%, respectively, at 21 days (n = 6, p < 0.05). At day 7, 27% of the cells had a principle angle of contraction within 20 degrees of the microthread, whereas at day 21, 65% of iPS-CM were contracting within 20 degrees of the microthread (n = 17). Utilizing high speed calcium transient data (>300fps) of Fluo-4AM loaded iPS-CM seeded microthreads, conduction velocities between 0.71–4.88 cm/seconds (n = 3) were found. iPS-CM seeded microthreads exhibited positive expression for Connexin 43, a gap junction protein, between cells. These data suggest that the fibrin microthread is a suitable scaffold for iPS-CM attachment and contraction and that extended culture allows cells to contract in the direction of the thread.
Peripheral nerves have the capacity of regener[[Unsupported Character - Codename ­]]ating after injury, but this spontaneous nerve repair may not be sufficient to achieve proper functional recovery. Current treatment for nerve injuries is nerve grafts. A promising alternative to conventional grafting is the use of artificial nerve grafts made from biodegradable and biocompatible materials and supportive cells. Electrospinning is a powerful technique to create aligned scaffolds that improve the growth of axons within the conduits. In this study, a poly (lactic-co-glycolic acid) (PLGA) conduit of aligned nanofibers was produced by the electrospinning method and its properties were characterized by scanning electron microscopy (SEM), contact angle measurements and dynamic mechanical analysis. Human mesenchymal cells were seeded onto the conduits and their viability and proliferation was analyzed by the live dead- and by WST8-assay. An 18% PLGA solution was electrospun and the fibers were collected on a rotating mandrel to achieve alignment. The SEM images show that the scaffolds presented uniform aligned nanofibers with an average diameter of 880 ± 330 nm and a contact angle of 112.5° ± 0.12. The nerve conduits were made by rolling 1cm2 pieces of scaffold on a 0.8 mm diameter needle. 5 × 105 mesenchymal cells were seeded either on the surface, into the lumen or on both conduit sides and were found to proliferate and maintain viability over 7 days in vitro. These first results prove that nerve conduits may be a promising substitute for autologous nerve grafts.
In Vitro Characterization of Human Mesenchymal Stem Cell Growth within Hierarchical Bone Scaffolds
Biology, Miami University, Oxford, OH
Department of Chemical and Biological Engineering, Tufts University, Medford, MA
New approaches to repair cartilage and subchondral degeneration are needed due to population longevity, obesity, and arthritis. Current treatment options include autografts and prosthetic implants, but these systems have achieved limited success. The objective of this work was to develop silk protein-based elastomers, combined with versatile and tunable spatial gradients of mineralization domains to mimic osteochondral interfaces. We hypothesized that the silk elastomers with spatially patterned minerals would provide a route to support cell distribution and differentiation, and ultimately architectural support for osteochondral interfacial tissues. To generate the gradient systems, silk-elastomer proteins were combined with continuous gradients of silica-binding peptides to induce silicification. Silk hydrogels embedded with gradient distributions of silica nano-/micro-particles were synthesized and confirmed by scanning electron microscopy and energy-dispersive X-ray spectroscopy. Mechanical studies indicated gradients in compressive modulus along the hydrogels. Silicified silk elastomers successfully promoted human mesenchymal stem cell attachment, spreading and proliferation after 3-week cell culture. The results suggested that silk-based mineralization gradients can be generated and will be further explored for needs in regenerative medicine involving tissue interfaces.
Kodali A, Lim T, Leong D, Tong Y. Macromol. Biosci., 14, 10, 2014.
Detergent Decellularization Affects the Surface Molecular Functionality of Biologic Scaffolds
University of Nottingham, Nottingham, UNITED KINGDOM
Generation of extracellular matrix (ECM) biologic scaffolds utilizes detergents to solubilize cell membranes and dissociate DNA from proteins. The deleterious effects of detergents are well known; yet the effect of detergent treatment upon the surface ligand landscape of biologic scaffolds has not previously been characterized. The objective of this study was to use time of flight secondary ion mass spectroscopy (ToF-SIMS) to determine the effect of different agents upon the surface molecular functionality of urinary bladder matrix (UBM). The treatments comprised: 3% Triton-X 100, 4% sodium deoxycholate, 1% and 0.1% sodium dodecyl sulfate (SDS), 8 mM CHAPS and 0.1% peracetic acid. Surface molecular functional was correlated with in vitro cell behaviour. ToF-SIMS analysis showed that Triton X-100, sodium deoxycholate and SDS residual fragments remained after treatment. It is likely that residual SDS fragments, alongside matrix alterations, contributed to poor phenotype, viability and reduced confluence of human urothelial cells cultured upon SDS treated UBM. Increased SDS concentrations, from 0.1% to 1.0%, intensified residual SDS fragments and adverse cell outcomes. ToF-SIMS analysis detected cellular remnants, attributed to nuclear and membrane material, in PAA and CHAPS treated UBM. Detection of the phosphate and phosphocholine ions facilitates assessment of decellularization efficacy; PAA and CHAPS treatment were ineffective at completely removing cellular material from the UBM samples. This study demonstrates the importance of maintaining a balance between cell removal and detergent disruption of matrix architecture and surface ligand landscape.
Chow et al. Mitigation of diabetes-related complications in implanted collagen and elastin scaffolds using matrix-binding polyphenol. Biomaterials. 2012.
A Study of PLGA Scaffolds with Different Porosity for Bone Tissue Engineering Applications
Silk: New Material Solution for Orthopedic Devices
Tufts University, Medford, MA
Current materials options for orthopedic devices include nondegradable metals and degradable polymers such as polylactic acid (PLA), polyglycolic acid (PGA) and poly-lactic-co-glycolic acid (PLGA). Yet, metal orthopedic devices are associated with problems such as stress shielding, infections, poor bone remodeling and second surgical removal, while the resorbable devices are associated inflammatory foreign body reactions, osteolysis and incomplete bone remodeling. We recently demonstrated the feasibility of silk proteins as a molding and machineable biomaterials systems for the preparation of devices for craniofacial repairs, based on the use of an organic solvent-based process (1,1,1,3,3,3 hexafluro-2-propanol (HFIP). To obtain water-stable structures, HFIP was required to facilitate silk solubility and methanol to induce the formation of β-sheet structure. However, HFIP can limit utility due to cost, risk for residuals in the devices, and difficulty in incorporating labile biomolocules in the fabrication process. Therefore, we further developed a biomimetic, all-aqueous process, to obtain regenerated silk-based bulk materials for the fabrication of orthopedic devices. The silk materials generated in the aqueous-based process replicate the nano-scale structure of natural silk fibers and the resulting materials and devices possess excellent mechanical properties. Further, the materials are machineable, providing a path towards the fabrication of a new family of resorbable orthopedic devices where organic solvents are avoided, thus allowing functionalization with bioactive molecules to promote bone remodeling and integration. Therefore, silk provides a new material solution to bridge the gap between current materials options for orthopedic repairs.
National Cerebral and Cardiovascular Center Research Institute, Osaka, JAPAN
In the field of soft tissue engineering, hydrogels have been focused on not only as fillers but also as biodegradable scaffolds to enhance tissue regeneration. Silk fibroin can be processed into physical gels, but cell infiltration and vascularization into the gels cannot be expected because fibroin has no cell-adhesive sequences. Here, we produced a fusion peptide composed of fibroin H-chain repeat, REDV, MMP-2 cleavable, and VEGF mimic peptides. This peptide was introduced into fibroin gels to improve cell infiltration and vascularization. As the peptide enhanced HUVEC adhesion and proliferation in vitro, it was mixed with fibroin to form peptide-modified fibroin gels. The resultant gels were embedded subcutaneously in rats for eight weeks. H&E staining showed the peptide promoted cell infiltration and vessel formation in the gels. Particularly, at eight weeks post-implantation, the percentage of vessel area relative to the total gel area in the peptide-modified fibroin gels was twice as much as that in fibroin gels without the peptide. Immunostainings of CD31 (endothelial cell marker), CD68 (macrophage marker), and P4HB (fibroblast marker) revealed that endothelial cells infiltrated into the peptide-modified fibroin gel to lead cell groups including macrophages and fibroblasts from one to four weeks post-implantation. In contrast, cell groups were absent and collagen fibrils were observed in the peptide-modified fibroin gel at eight weeks. Therefore, the peptide-modified fibroin gel led not to chronic inflammation but to the regeneration of dermal tissue, showing its availability for soft tissue angiogenic therapy.
Department of Endodontics, Tianjin Medical University School of Stomatology, Tianjin, CHINA
Injectable porous microspheres provide a new method for cell delivery, with characters of patient friendly and few procedures to follow. In this study, gas foaming method in a double emulsion-solvent were used to fabricate the well interconnected porous PLGA microsheres. Several factors were adjusted to acquire proper cell delivery construct. In order to promote cell adhesion, we use desolvation to coat silk protein onto the surfaces of the as-prepared microspheres. Gingival stem cells were seeded on the silk modified PLGA microspheres to evaluate cell viability and osteogenic potentials. Visualization and biochemical analyses of the microcarrier-cell constructs were performed to demonstrate cell proliferation and phenotypic expression. EdU analysis displayed better proliferation of the stem cells on the silk modified microspheres. The calcium nodules were observed on the microspheres after 28 days' osteogenic induction. Deposition of the calcium on the spheres was proved by alizarin staining. EDS analysis demonstrated that calcium and phosphate was deposited on the microspheres, and the XRD results proved that the calcium nodules were formed by hydroxyapatite crystals. In conclusion, injectable silk modified PLGA microspheres are promising cell delivery constructs and for other applications, especially for bone regeneration.
Biotechnology, Universidad Autónoma Metropolitana, Mexico City, MEXICO
Polyelectrolyte complexes (PECs) of Aloe vera (Aloe barbadensis Miller) (AV) and alginate (ALG) were formulated at pH 6 with and without Ca ions using commercial chitosan (QCH) prepared by conventional thermochemical methods and chitosan obtained from biologically extracted chitin (BCH). The blends presented polyanionic behavior with the zeta potential (ζ) determination allowing a stable system, this is important for biological applications due to interactions with cell membranes are minimized and avoiding permeation disturbance. Cell viability was evaluated using lymphocytes culture through MTT and RN assays, which were up 80% in both tests. Fluorescence microscopy analysis evidenced the formation of microgels in the systems due to the fluorescence produced by the interaction of CH from PECs with calcofluor white reagent. Scanning electron microscopy analysis showed disordered morphology with rough and lamellar structures. The PECs-BCH with Ca+2 ions showed a relatively smooth porous structure, whereas PECs-QCH with Ca+2 had a macro rough porous network with pore sizes between 10 and 20 μm. Moreover, the structure of the PEC was strongly influenced by the amount of Ca+2 ions, thus leading to a more compact, homogeneous and less dense structure. This difference between PECs at pH 6 with and without Ca2+ ions might be due to degree of acetylation (DA) of CH, since PECs-QCH-6-Ca presented higher DA than PECs-BCH-6-Ca. PECs showed antimicrobial activity on Staphylococcus epidermidis as it was determined in the live/dead fluorescent assay.
Ludwig Boltzmann Institute for experimental and clinical Traumatology, Vienna, AUSTRIA
Human plasma derived fibrin matrix is one of the most versatile biomaterials for tissue engineering and regenerative medicine. Working for over 30 years in this field we have gained notable experience and we can demonstrate the advantages and limitations, application techniques and the special use for growth factor and cell delivery as well as a gene activated matrix. Another aim of our group is to use, medical garbage” for regenerative purposes. Therefore, we use cells from liposuction, from umbilical cord and placenta derived (PD) - substances (e.g. collagen) and PD structures (e.g. amnion) as well as PD cells (e.g. amnion MSC). “Living” amnion is used either directly (cryopreserved) by using a clinically approved process, e.g. for wound healing and antifibrosis, or in a new process where the stem cells residing on and in amnion (“sessile” cells) are differentiated in toto (osteo, chondrogenic direction). Isolated stem cells are cultured with platelet derived factors (from outdated platelets) to avoid animal products and used directly or pre-differentiated, in autologous or allogeneic fashion. Allogeneic is possible, because the mesenchymal stem cells have minor antigenicity and, in addition, immunosuppressive properties. This talk aims to provide an overview of the use of the above mentioned procedures within the Austrian Cluster for Tissue Regeneration.
UMinho, Braga, PORTUGAL
More than 200,000 peripheral nerve repair procedures are performed annually in the USA. But, the adequate functional recovery of the peripheral nerve is still a significant clinical challenge. The direct suture repair without the use of grafted materials may be used in cases where a short (<5 mm) nerve gap has to be overcome. However, larger defects repaired by neurorrhaphy, exhibit excessive tension over the suture line and offer poor surgical results. The nerve autograft is recognized as the “gold standard” technique but it is limited by tissue availability, donor-site morbidity, secondary deformities, as well as potential differences in tissue structure and size. In our group, we have been developing a series of natural-based biomaterials to be used as nerve conduits that became an alternative to synthetic polymers such as PCL, PLGA and polyurethane. Among the natural materials, chitosan, Gellan gum, keratin and silk fibroin have been used. The relevant in vitro and in vivo studies that have been performed will be presented. Their final properties (e.g., compressive modulus, storage modulus, stiffness, swelling behavior, durability, degradation profile, porosity, permeability, suture ability) can be tuned for specific uses, by means of using different concentrations and processing techniques, i.e. fibres, membranes and tubes can be produced. Furthermore, the inner diameter, thickness of the wall and length of the nerve guidance conduit can be tuned according to the final needs (e.g. permeability and biodegradability), thus opening up new possibilities to address the current challenges in PNR, especially in the treatment of long gaps.
Biodegradation of Magnetic Nanoparticles During Stem Cells Chondrogenesis: Implications for Regenerative Medicine Applications
Laboratoire MSC, CNRS - University Paris VII, Paris, FRANCE
For regenerative medicine, the nanoparticles can be used to magnetically engineer organized tissues. Recently, we have shown that magnetic compaction promotes chondrogenesis within or without scaffolds. However, the long-term fate remains an unexplored issue. Here we explored the degradation of internalized nanoparticles over months, using two tissue models. Stem cells were labeled with iron oxide then magnetically seeded into polysaccharide scaffolds or compacted into spheroids, and placed in chondrogenic medium. The magnetic moments of the so-formed tissues were determined by magnetometry measurements, using a vibrating sample magnetometer. The total iron mass was quantified using an ICP spectrometer. QPCR was performed to determine relative expression of specific genes. Electron microscopy was achieved to localize nanoparticles within the tissue models. In both models, we observed that magnetism decreased from the first day to the end of chondrogenesis. By contrast, the mass of total iron was unchanged. We identified that endosomes were the site of degradation, and that freed iron were loaded into ferritin. We have also shown that iron homeostasis was not affected by the intracellular release of iron. In our tissue models, we evidenced using magnetic methods that nanoparticles experienced a near-complete degradation over a month of tissue maturation, showing that tissues can purge themselves of intracellular nanoparticles. Moreover, the release of iron did not trigger biological disorder and preserved the multipotency of stem cells. These findings demonstrate that the fate of nanoparticles within engineered tissues is not a critical issue. This is reassuring for medical applications, particularly for regenerative medicine.
Shape-Controlled Disc-Type Cartilage Reconstruction Using Chondrocytes Isolated From Elastic Cartilage
College of Life Science, Kyung Hee University, Yongin-si, Gyeonggi-do, KOREA, REPUBLIC OF
There are three different types of cartilage in the body, and each type of cartilage tissue shows distinct tissue morphology and matrix composition. The fact that each distinct type of cartilage tissues are composed of single type of cell, chondrocytes, underscore the importance of cell source in cartilage tissue engineering. We previously identified xiphoid process cartilage as an elastic cartilage, and proved that the in vivo-constructed disc-type cartilage with those chondrocytes stably exhibit phenotype resembling elastic cartilage-specific histological morphology. As an approach of scaffold-free construction of cartilage tissue, we tested various number of chondrocytes for thickness-control during disc-type cartilage construction to see if cartilage tissue of various thickness can be materialized. Xiphoid process cartilage, being the only elastic cartilage tissue source that can be obtained without destroying external shape or function, is a source of elastic chondrocytes that show superb in vitro expansion and reliable differentiation capacity. These results indicate that chondrocytes from elastic cartilage tissue could be a valuable cell source for reconstruction of elastic cartilage and scaffold-free in vitro chondrogenesis protocol can be developed to build cartilage tissues in various thickness and shape.
Scalable Expansion Of Pluripotent Stem Cells In A Three-dimensional Culture Using Hollow Fibers.
Hematopoietic Differentiation Of Pluripotent Stem Cells In A Three-dimensional Culture Using Hollow Fibers
Evaluation Of The Pluripotent State Of mESCs Cultivated In MEF Free Conditions With The Association Of PD0325901 And CHIR99021
Analysis, Federal University of Rio Grande do Sul, Porto Alegre, BRAZIL
A strategy for pluripotency maintenance is the use of small molecules, such as PD0325901 and CHIR99021. The aim has been to evaluate the pluripotent state of mESCs cultivated in MEF free conditions with the association of PD0325901 and CHIR99021. The experimental groups were: mESC/MEF, mESC/gelatin and mESC/gelatin/2i. The pluripotency was evaluated after the 2nd, 6th and 10th passages. The mESCs were derived/characterized in the laboratory and cultivated in DMEM high glucose, fetal calf serum, penicillin/streptomycin, recombinant mouse leukemia inhibitory factor, sodium piruvate, L-glutamine, b-mercaptoethanol and non-essential amino acids; kept at 5% CO2 and 37°C in a humidified atmosphere. The 2i supplementation was performed as follows: CHIR99021 3 μM and PD0325901 1 μM. The evaluations were colony morphology by phase contrast and fluorescence (DAPI/Phalloidin) and immunofluorescence (Oct3/4, SOX-2 and Nanog). The mESCs/MEF and the mESCs/gelatin/2i presented compact colonies with very well defined edges. The mESCs/gelatin showed a completely different morphology with undefined edged colonies and, after ten passages, the compact colony structure was completely lost and the cells were organized in a monolayer form. The experimental groups presented similar results concerning the expression of the pluripotency markers and it is important to note that the morphological differences were not related to their expression. Considering the use of mESCs as a model for the development of tissue engineering protocols, the maintenance of the pluripotency state when the cells are submitted to a variety of environments, such as different biomaterials, is an important tool for allowing the development of a controllable differentiation process.
Reactive oxygen species (ROS) overproduction serves as a critical factor for the reduced osteointegration of titanium implants (TI) under diabetic conditions, at least partially by influencing the osteoblasts function and hinders the local bone regeneration. In the bone defect repairing process, vascularization also plays a significant role, in which vascular endothelial cells (VECs) provide oxygen and cytokines to other cells supporting bone regeneration. We hypothesized excess ROS may impair the VECs' function, leading to the abnormal osteointegration of TI. In vitro, human umbilical vein endothelial cells (HUVECs) cultured on Ti sheet were subjected to different treatment: normal serum (NS), diabetic serum (DS), DS + NAC (a ROS inhibitor) and NS + H2O2. In vivo, we designed a new scaffold with a bore whose diameter can be adjusted to control the rate and amount of blood vessel grown into the scaffolds. The scaffolds were transplanted into the ilium of diabetic rabbits to observe the growth of bone and vessel. Results showed that diabetes induced significant dysfunction and apoptosis of HUVECs, which was ameliorated by scavenging ROS with NAC. Importantly, we found a dose threshold of ROS, higher than which the function of HUVECs was impaired obviously. These results further our knowledge about the relationship between ROS with the HUVECs' function in diabetes and indicate that anti-oxidative treatment can be a promising strategy to improve the vascularization and osteointegration of TI in diabetic patients.
Analysis of Adipose Derived Stem Cell Metabolism Using NMR Spectroscopy
Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL
Recently 1H NMR spectroscopy has been applied to characterize specific stem cells. This technique can be used to monitoring the stem cell culture (1). The aim of this project was to evaluate the stem cell sugar metabolism during the early passages (P2 to P6) of adipose-derived stem cells (ASC). Our hypothesis was that metabolism increases to a point and then decreases with senescence. ASC were isolated and cultured as described previously (2). Spent media was analyzed on a Varian VNS-750 NB (750 MHz) spectrometer (3). Data were analyzed by ANOVA using the Generalized Linear Model (GLM) procedure. Tukey's post hoc test was used for multiple comparisons. The alpha level used was 0.05. We evaluated 8 samples per passage by NMR. Our results showed a constant decrease in formate concentration with increasing cell passage. However, acetate concentration only decreased after P3 (P < 0.0001). Glucose concentration increased (P < 0.0001) at P4 and thereafter. Lactate concentration decreased but was only different at P6 (P < 0.01). The pyruvate concentration peaked at P3 then decreased to P6 (P < 0.01). These results show that the consumption/production of sugars may indicate ASC adaptation from in vivo to in vitro metabolism and that the greatest change in metabolism occurs after P3.
1. MacIntyre D. et al., PlosOne 6 (2) 2011
2. Monaco E. et al., PlosOne 7 (3) 2012
3. Rubessa M. et al., Reproduction fertility and Development 28(2) 2016.
Department of Orthopedic Surgery, National University of Singapore, Singapore, SINGAPORE
Physical stimulation in various forms of mechanical stimulation has been shown to be pivotal in modulating the chondrogenic differentiation outcomes of MSCs through different trans-membrane ion channels. Pulse electromagnetic field (PEMF) exposure has been shown to evoke a mechanotransduction signaling cascade in progenitor cells, and has been implicated in modulation of mesenchymal stem cell (MSC) chondrogenesis. In this study, using a custom-design electromagnetic field pulsing device that produces precisely specified PEMFs, we characterized MSC chondrogenic differentiation to PEMF exposure, by varying the pulse intensity, pulse duration and pulsing dosage. The extent of MSC chondrogenesis was examined by mRNA anaylsis, histology, and quantification of cartilage-extracellular matrix macromolecules sGAG and type II collagen formation. The effect of PEMF on mechanotransduction implicated transient receptor potential (TRP) channels in MSC chondrogenesis was investigated. Our results show that MSCs subjected to a short term (10 minutes), low intensity (2 mT) PEMF optimally enhanced chondrogenesis. In addition, administration of a single pulse at the early stage of chondrogenic induction was more effective than multiple pulses, administered either within a single week, or weekly for 3 weeks. Analysis of TRP channels indicates an association of TRPC1 and TRPV4 response to PEMF in relationship to MSC chondrogenesis. The effectiveness of low electromagnetic pulsing amplitude, brief pulsing period on MSC chondrogenesis; as well as single pulse exerting better effect than multiple pulses, has wide implication for translational clinical application of PEMF for stem cell-based cartilage regeneration.
Plasmonic nanoparticles possess unique resonance, generated by free oscillating electrons, when activated by electromagnetic radiation, enhancing the scattering and absorption properties of the particle. Absorbed light can be converted to heat energy in plasmonic nanoparticles at or near their absorption peak wavelengths. Heat generated by plasmonic resonance can be coupled with thermally cleaveable linkers via a retro Diels Alder chemical reaction to deliver oligonucleotides for gene therapy. Thermal-based drug delivery applications offer promising potential for in vivo release at longer wavelengths for deeper tissue penetration. The distinct plasmonic resonances associated with composition and morphology of nanoparticles provide the ability to multiplex the release of different siRNA at distinct wavelengths. The proposed study investigates the ability to silence GFP and RFP expression temporally in vitro when the siRNA nucleotides are separately functionalized on gold and silver nanoparticles, and activated at their respective wavelengths sequentially. HEK293-GFP/RFP were used in this study. Q-RT-PCR and fluorescent microscopy was used in order to measure the expression level of GFP and RFP after the activation of NPs at different wavelengths. The silencing of GFP and RFP served as a proof of concept that we are able to temporally regulate two genes within the same tissue volume.
Human Adipose Stem Cells Differentiated on Braided Polylactide Scaffolds Is a Potential Approach for Tendon Tissue Engineering
Adult Stem Cells Group, BioMediTech, Tampere, FINLAND
We aimed to find an efficient strategy to produce a potential tendon tissue engineering construct in vitro. The preliminary tests included the optimization of the tenogenic differentiation medium (TM) for the human adipose stem cells (hASCs), and comparing suitable tissue engineered scaffold structures. The optimized TM enhanced significantly proliferation and tenogenic differentiation of the hASCs. The braided 8-filament poly-L-D-lactide 96/4 (PLA) scaffolds supported hASC adhesion, proliferation and tenogenic differentiation compared to the foamed poly(L-lactic-co-ɛ-caprolactone) 70/30 (PLCL) scaffolds in the TM condition. The PLA 96/4 scaffolds supported the formation of a uniform cell layer in the TM condition compared to the maintenance medium (MM) condition at 2 weeks. The total collagen content and gene expression of tenogenic marker genes of the hASCs was significantly higher with the optimized TM after 14 days. The elastic modulus the braided PLA 96/4 scaffold was more similar to that reported for the native human Achilles tendon tissue. Our study showed that the optimized TM condition supports in vitro efficient tendon-like matrix production of the hASCs. The braided PLA 96/4 scaffolds combined with the optimized TM significantly enhanced tenogenic differentiation of the hASCs. The proposed tendon tissue engineering applications represent an emerging approach with potential feasibility.
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Linz/Vienna, AUSTRIA
Human adipose tissue is an attractive and abundantly available source of adult stem cells applicable in regenerative medicine and tissue engineering. Hence, prerequisite for the translation into clinics is the production of the stromal vascular fraction (SVF) under good manufacturing practice (GMP). A number of companies developed systems aiming for a closed, sterile, safe and reproducible cell isolation process. However, many of these systems are based on enzymatic digestion which is the most expensive part of the isolation process, complicates regulatory authorization and may have negative impacts on cell potency and efficacy. Therefore we compared classical enzymatic cell isolation methods with reduced enzyme concentration methods and a new non-enzymatic isolation method regarding cell yield, identity and potency. Our results demonstrate that enzyme treatment is necessary for applications which require large cell numbers, since reduction of enzyme concentration to 30%, 10% and 0% result in smaller cell yields. In contrast, the viability of the isolated cells as determined via cellular ATP decreased with increasing collagenase concentration. Furthermore, reduction of collagenase concentration showed significant higher adipogenic, osteogenic and chondrogenic differentiation potential. We found that our non-enzymatic method is suitable to isolate therapeutically relevant subpopulations such as endothelial progenitor cells (CD45−/CD31+/CD34+), pericyte-like cells (CD45−/CD31−/CD146+), and supra-adventitial cells (CD45−/CD31−/CD146−/CD34+) with high cellular ATP content and elevated differentiation potency. Cells derived from our new non-enzymatic method showed stronger potential to form tube-like structures. Our findings support the concept of using non-enzymatic closed systems which allow the isolation of therapeutically active cells in a one-step procedure.
Although human Cardiac Primitive Cells (CPC) when injected in infarcted heart are not retained by host myocardium, they improve cardiac function. Emerging evidence supports the hypothesis that exosomes may be responsible for beneficial effects induced by stem cells delivered in the infarcted myocardium. Exosomes are nano-sized vesicles naturally secreted by almost all cells and ubiquitously found in cell culture supernatants and biological fluids. Transporting and transferring peptides, lipids, and nucleic acids, exosomes have the potential to modulate signaling pathways, cell growth, migration, and proliferation of recipient cells. Accordingly, CPC may deliver chemoattractive, pro-survival and differentiating signals to resident cells through exosomes. To test our hypothesis, we isolated exosomes released in culture by CPC isolated from adult human myocardium (Exo-CPC) and analyzed the composition of their cargo. Specifically, we searched for the presence of specific factors known to regulate CPC migration, survival and differentiation, as HGF, IGF, TGF, Nkx2.5, Tbx, and Mef2c. Additionally, we tested in vitro the potential of Exo-CPC of either regulating CPC proliferation and programmed cell death, and modulating interstitial fibrosis, extracellular-matrix (ECM) synthesis and deposition. Interestingly, on one hand, signals delivered by Exo-CPC affected proliferation and survival of CPC and, on the other hand, regulated ECM proteins production. Therefore, we might speculate that Exo-CPC have potential effect on both resident CPC and fibroblasts when injected in cardiac wall.
A Novel Reporter System For Monitoring Of Skeletal Muscle Cell Differentiation
Ludwig Boltzmann Institute for experimental and clinical Traumatology, Vienna, AUSTRIA
Human mesenchymal stem cells (hMSCs) residing in bone marrow stem cell niches are regulated by components of the niche. In this study, we identified soluble factors abundant in the bone marrow niche, characterized their effects on hMSCs, and utilized the soluble factors for hMSC-based tissue regeneration. Our results showed that treatment of bone marrow extract increased hMSC proliferation and reduced cellular senescence. Human MSCs pretreated with bone marrow extract showed improved osteogenesis and chondrogenesis compared to control hMSCs. We then identified 6 most abundant proteins in bone marrow and further determined that the combination of lipocalin-2 (LCN2) and prolactin (PRL) was able to closely resemble bone marrow extract in regulation of hMSC activities. To evaluate the potential of the selected soluble factors for hMSC-based tissue regeneration, we implanted scaffolds seeded with LCN2 and PRL-pretreated hMSCs at calvarial defects of immunocompromised mice. Micro-CT scanning showed more bone formation in the defects implanted with LCN2 and PRL-pretreated hMSCs than in the defects implanted with control hMSCs. Histological analysis showed new bone formation in the scaffold seeded with LCN2 and PRL-pretreated hMSCs but cartilage formation in the scaffolds seeded with control hMSCs. Our findings demonstrating effects of bone marrow-extracted soluble factors on hMSC activities in culture provide insight into how the cell is regulated by chemical cues in bone marrow niches and may also be used to develop a viable approach to maintain hMSC properties Ex Vivo for orthopedic applications.
High Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells
Gene-edited human cells are important resources for drug target identification and regenerative medicine. Recently, use of the CRISPR/Cas9 system drastically cut the time required to produce gene-edited cell lines down to a few months. Targeted gene disruption in a population of human cells followed by selection and next-generation sequencing can identify drug targets, however many of these methods destroy all mutant clones, so a subsequent gene-editing experiment is required to obtain living mutant cells for downstream analysis. Overall, there is a need to increase the speed, multiplexing and precision in generating CRISPR/Cas9 mutants. ArrayEdit, a simple approach utilizing surface-modified multiwell plates, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This allows for real-time observation of mutations causing phenotypic differences such as proliferation or other high-content observable qualities that can be measured in in vitro tissues and organoids during differentiation as opposed to defined end points. Furthermore, this technique allows for the screening of gene-edited human primary cells for undesirable off-target mutations that may be cancerous or cause other, downstream adverse effects. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.
Lack of Vimentin Impairs Endothelial Differentiation of Embryonic Stem Cells
Stem cells are likely to be the source for generating endothelial cells needed for emerging regenerative medicine approaches to treat cardiovascular pathologies. As a highly mechanoresponsive phenotype, functional endothelial cells rely on the cytoskeleton, including the intermediate filament vimentin. Little is known, however, of the need for vimentin during differentiation towards the endothelial phenotype. The objective of this study was to determine the role of vimentin on differentiation of embryonic stem cells (ESCs) to the endothelial phenotype in vitro.
Vimentin knockout (VIM-/-) and wild type (WT) ESCs were evaluated for differences in spontaneous differentiation as embryoid bodies (EBs). The VIM-/- EBs displayed an altered morphology compared to WT controls. Cross-sectional areas of the VIM-/- EBs were quantifiably and significantly smaller than the WT EBs, consistent with the observation of fewer Ki67+ proliferating cells. Furthermore, the knockout EBs had an altered morphology, with a rippled outer surface (SEM) and a disrupted outer ECAD+ layer (immunohistochemistry). Gene expression (RT-PCR) of markers along the pathway of endothelial differentiation (Brachy-T, FLK1, TIE2, PECAM, and VE-CADHERIN) were all significantly lower for the VIM-/- EBs during 7 days of culture. Over this period, WT cells increased expression of endothelial markers by 4-90X, a ∼5-fold greater change than that in VIM-/- cells. These differences were corroborated by decreased protein expression after 10 days of differentiation. Thus, absence of vimentin impairs differentiation of ESCs to the endothelial phenotype.
This study indicates that the proper acquisition of cytoskeleton proteins is critical in generating functional phenotypes for cell-based therapies.
Directed Embryonic Stem Cell Derived Vascular Endothelial Sub-phenotypes
A well-formed and robust vasculature is critical to the health of most organ systems in the body. However, endothelial cells (EC) can exhibit a number of distinct functional sub-phenotypes like arterial or venous EC, as well as angiogenic tip and stalk EC. Our laboratory investigates the directed the differentiation of endothelial cells phenotypes from mouse and human embryonic stem cells (ESC) in vitro using staged and chemically-defined methodology. Using these methods, we discovered highly angiogenic tip/stalk-containing ESC-EC are distinct from less proliferative and less migratory phalanx ESC-EC. We found that both sub-populations are more than 80% VE-cad+/ CD31+ without FACS purification. These derived EC exhibited distinct mRNA gene expression and surface marker expression profiles, as well as increased sprouting capacity in a mosaic fibrin gel assay. The tip/stalk EC are Flt4+/Dll4+/Flt-1-/Notch-1- reflecting a migratory more VEGF responsive phenotype indicative of tip cell surface expression pattern. The phalanx EC are more homogeneous and less responsive to VEGF signaling – comprising of higher levels of Flt-1 and Notch-1. Furthermore, tip/stalk EC overexpressed 9 key angiogenesis associated genes compared to the phalanx EC. The ability to generate functionally distinct vascular ESC-EC in vitro is an important development in regenerative medicine.
Development of an in vitro Model of Blastema Formation for Epimorphic Regeneration
Mammals have the potential for epimorphic regeneration (ER), the regrowth of a digit or limb after amputation, though largely limited to the distal digit tips in mice and humans. Studies in the murine model allows for important insights into the sequence of events that lead to ER, but the complex in vivo milieu complicates mechanistic studies. Recent isolation of stromal cells from regeneration-competent (P3 bone) and regeneration-incompetent (P2 bone) regions of the digit allow for in vitro comparative studies.
One of the first processes in ER is the formation of a blastema, a stable cell mass that differentiates to synthesize tissue. We have recently shown that suspension culture of cell clusters is an appropriate model to study the blastema. While P3 cells spontaneously form these spheroids, P2 cells fail to aggregate. The objective here was to establish a method to overcome the limitation of P2 cells to form cell clusters.
P2 cells were forced to aggregate in microwells with inoculation densities of 100–1000 cells/microwell. For all densities, cells compacted within 24 hours with no significant increase in size over 5 days of culture. Release of spheroids from the microwells into suspension culture, however, led to an immediate increase in size and continued growth. While all released spheroids contained Ki67+ proliferating cells, none were observed in spheroids maintained within the microwells. Thus, forced aggregation of P2 cells in microwells followed by release into suspension allows for cell spheroid culture, an appropriate in vitro model for mechanistic studies of ER processes.
Modifying Commercial Parenteral Fluids and Drugs as Alternative Medium for Cell and Tissue Transplantation
Department of Biomedical Science and Technology, Institute of Biomedical Science and Technology, Konkuk University, Seoul, KOREA, REPUBLIC OF
In recent years, the need for a safe and physiologically compatible fluids for use in biomedical, livestock production and veterinary medicine is becoming more important. We produced a fluid-based culture medium (FCM) using existing injectable and fluid drugs to develop a safe alternative for culturing cells and tissues prior to transplantation. Using animal (Rat H9c2 cells) and human (synovium - derived mesenchymal stem cells) cells, culture conditions to simulate 1) DMEM-high glucose (HG), an original commercial medium, and 2) optimized modified culture medium-HG were prepared and the differences in cell proliferation rates, conditioned medium pH values, and conditioned medium osmolalities were analyzed using the Student t-test, with significance defined at P < 0.05. The morphologies and proliferation rates of H9c2 cells and FS-MSCs cultured under FCM-HG showed no significant differences compared to those grown in DMEM-HG. It is important to investigate the effects of serum-free FCM on cell proliferation, cell morphology, cell passage conditions, and cell physiologic characteristics to determine the possibility of the FCM as a xeno-free alternative to the fetal bovine serum (FBS), or combined with human cord bloodserum or albumin instead of FBS. The full potential of the FCM can explored further by investigating its efficiency in culturing other cell types and as injection vehicle for cell transplantation such in-vitro fertilization-embryo transfer (IVF-ET) or artificial insemination. We have established that the FCM is a more economically beneficialand safer than commercial medium and can be used to culture cells intended for human and animal cell and tissue transplantation.
Despite therapeutic advances, cardiovascular disease remains a leading cause of mortality and morbidity worldwide. Embryonic stem cells (ESCs) may offer significant advances in creating in vitro cardiac tissue models for disease modelling, drug testing and development; however, the induction of ESCs to a more adult-like cardiomyocyte (CM) phenotype still remains challenging. In this study, we employed defined physical signals to induce cardiac differentiation and maturation of mouse and human ESC-derived CMs (ESC-CMs). We demonstrate that combining a cyclic surface strain (5%) with continuous medium flow (1.48 ml per minute) induces differentiation and facilitates ESC-CM maturation. Murine and human ESC-CMs showed an altered morphology, increased cardiac gene and protein expression, and calcium handling properties when exposed to the dynamic culture for either 18 or 20 days. Raman microspectroscopy was employed to detect ESC-CM maturation states by previously identified lipid and protein band intensities in the Raman spectra. Our findings show that physical cues are crucial for the maturation of mouse and human ESC-CMs, and offer exciting new opportunities for bioreactor-based pre-clinical human in vitro models, to study cardiovascular diseases and investigate potential drug candidates.
Human Mesenchymal Stem Cell Responses To Hydrostatic Pressure And Shear Stress
CNRS, Paris, FRANCE
The effects of mechanical stimuli to which cells are exposed in vivo are, incompletely understood; in this respect, gene-level information regarding cell functions which are pertinent to new tissue formation is of special interest and importance in applications such as tissue engineering and tissue regeneration. This study investigated the early responses of HuMSC to intermittent shear stress (ISS) and to cyclic hydrostatic pressure (CHP) simulating some aspects of the biological milieu in which these cells exist in vivo. Production of nitricoxide (NO) and mRNA expression of several known mechanosensitive genes as well as ERK1/2 activation in the hMSC response to the two mechanical stimuli tested were monitored and compared. NO production depended on the type of the mechanical stimulus to which the hMSCs were exposed and was significantly higher after exposure to ISS than to CHP. At the conditions of NO peak release (i.e., at 0.7 Pa for ISS and 50,000 Pa for CHP), ISS was more effective than CHP in up-regulating mechanosensitive genes. ERK1/2 was activated by ISS but not by CHP. This study is the first to report that PGTS2, IER3, EGR1, IGF1, IGFBP1, ITGB1, VEGFA and FGF2 are involved in the response of hMSCs to ISS. These findings establish that, of the two mechanical stimuli tested, ISS is more effective than CHP in triggering expression of genes from hMSCs which are bioactive and pertinent to several cell functions (such as cell differentiation, release of specific growth factors, cytokines) and also to tissue-related processes such as wound healing.
Biomedical Engineering, Tulane University, New Orleans, LA
Adipose-derived mesenchymal stem cells (ASCs) are a promising source for cell-based therapies needed for the advancement of regenerative medicine, such as revascularization of ischemic myocardium. The dynamic mechanical environment of the myocardium may regulate the mechanoresponse of transplanted stem cells, affecting cell state as well as certain cellular functions such as proliferation and migration. Separately it has been shown that the age of the donor also affects cellular function and differentiation potential. What remains to be known, however, is if aging affects the cellular mechanoresponse, largely mediated by the cytoskeleton. The objective of this study was to determine the effect of donor age on gene expression of cytoskeletal elements in ASCs.
Human adult ASCs from young (≤25 yo) and old (≥60 yo) donors were evaluated for differences in intrinsic gene expression. Briefly, samples were lysed and RNA was isolated and converted to cDNA for assessment through high-throughput RNA sequencing (RNA-Seq). Using this technology, we detected 43,966 genes. Only 78 genes, however, showed a significantly different (p < 0.05) expression level between the two groups (n = 3). Of these genes, certain cytoskeletal remodeling genes were identified, including but not limited to phosphate and actin regulator (PHACTR4), coiled-coil domain containing 88A (CCDC88A), and dihydropyrimidinase-like 4 (DPYSL4). These observations indicate that aging may induce changes in the gene expression of some elements of the cytoskeletal network, which is a major contributor to cell size, shape, and function.
B2OA UMR CNRS 7052, Université Paris Diderot, Paris, FRANCE
Mesenchymal Stem Cells (MSCs) have captured attention and research endeavors of the scientific world because of their differentiation potential. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are predominantly due to the multitude of bioactive mediators secreted by these cells. Since the paracrine potential of MSCs is closely related to their microenvironment, the present study investigated and characterized select aspects of the hMSC secretome and assessed its in vitro and in vivo bioactivity as a function of oxygen tension, and specifically near anoxia (0.1% O2) and hypoxia (5% O2), conditions which reflect the environment MSC are exposed during MSC-based therapies in vivo. In contrast to supernatant conditioned media (CM) obtained from hMSCs cultured at either 5 or 21% of O2, CM from hMSCs cultured under near anoxia exhibited significantly (p < 0.05) enhanced chemotactic and pro-angiogenic properties, and a significant (p < 0.05) decrease in the inflammatory mediators content. An analysis of the hMSC secretome revealed a specific profile under near anoxia, hMSCs increase their paracrine expression of the angiogenic mediators VEGFA, VEGFC, IL-8, RANTES and MCP1, but significantly decrease expression of several inflammatory/immunomodulatory mediators. These findings provide new evidence that elucidates aspects of great importance for the use of MSCs in regenerative medicine and could contribute to improving the efficacy of such therapies, and most importantly highlighted the interest to use conditioned media in therapeutic modalities.
The success in effective generation of myogenic progenitors from human pluripotent stem cells (hPSCs) offers the opportunity to treat muscular diseases through regenerative medicine. However, it remains unclear how biophysical and biochemical cues in cell microenvironments affect the behavior of these progenitors. We have engineered materials having nanotopographical surface patterns and immobilized biopolymers and peptide ligands and examined how these biophysical and biochemical cues affect cell alignment and myogenic differentiation. The hPSC-derived myogenic cells differentiated robustly and resulted in well-aligned myofibers on the engineered films having nanotopographic features and immobilized biochemical ligands. The alignment was dependent on the feature size and the type of biochemical ligand collectively.
Spatiotemporal Regulation of Self-assembling Human Mesenchymal Stem Cells Specifies Formation of Articular Cartilage with Physiologic Organization and In Vivo Stability
Electrical and Mechanical Stimulation of Co-cultured Skeletal Muscle Tissue Engineered in a Modular Bioreactor
School of Science and Engineering, Univeristy of Dundee, Dundee, UNITED KINGDOM
Advancements in tissue engineering has been hampered by numerous inherent problems; namely the reduced functionality due to lack of external and intracellular architecture of engineered tissues. This research aims to apply combined electrical and mechanical stimulation to engineered co-culture skeletal muscle tissue, using a modular bioreactor to investigate if there are any implications of such treatment on the maturity and function of engineered skeletal muscle. 3-D co-culture constructs will be engineered using mouse fibroblast and C2C12 cells and the parameters of the electrical and mechanical stimulation modules of the bioreactor which create an optimum environment for engineered muscle tissue growth will be established. After stimulation, the co-culture constructs will undergo Optical Coherence Elastography (OCE) to provide cutting edge results concerning the elastic properties of the tissue. Immunohistochemistry (IHC) and Transmission Electron Microscopy (TEM) will also be carried out to identify the presence of contractile proteins and collagen deposition/maturity; allowing the evaluation of tissue structures, architecture, functionality (contractility), maturity of the engineered tissues, as well as to evaluate the progress of the de novo tissue interface created due to increased production of collagen by both C2C12 and fibroblast cells as the evidence suggests. Preliminary results unveil that stimulation of C2C12 cell constructs results in collagen fiber deposition at the bioreactor anchor site of the constructs. The hypothesis is that electromechanical stimulation of co-cultured constructs will further increase collagen deposition at the anchorage site; providing a scaffold which may further optimise the potential of C2C12 and fibroblasts to engineer a tendon/tissue interface.
Challenges remain in articular cartilage tissue-engineering cell sourcing and in generating neocartilage with sufficient mechanical integrity. Using costal chondrocytes as an alternative cell source, this study aimed to enhance scaffold-free neocartilage properties through compressive stimulation. Compressive magnitude was optimized in Phase 1 by applying 0, 3.3, 5, or 6.7 kPa to neocartilage. Both 3.3 kPa and 5 kPa significantly increased neocartilage compressive properties by 42% and 38% over unloaded controls respectively, reaching ∼200 kPa in relaxation modulus. Phase 2 sought to optimize application time by applying stimulation at days 10–14, days 18–22, or both times. The results showed that applying 3.3 kPa at either days 10–14 or days 18–22 increased mechanical properties; however, stimulation at both times did not further improve properties beyond a one-time stimulation. Phase 3 examined the interactions of compressive stimulation with bioactive treatment (TGFβ1, chondroitinase ABC, and lysyl oxidase-like 2). The hypothesis was that the bioactive treatment would increase the tensile properties of neocartilage, without compromising the gains in compressive properties obtained through compressive stimulation. Indeed, with the bioactive treatment, neocartilage tensile modulus reached 4.1 MPa and even 4.9 MPa when compressive stimulation was added. Glycosaminoglycan and collagen content were 4% and 3% of neocartilage wet weight respectively. Functionality index comparing neocartilage against native tissue was 0.58, indicating that neocartilage reached 58% of native tissue mechanical and biochemical properties overall. Thus, this study demonstrates that costochondral neocartilage treated with the identified mechanical and biochemical stimuli is a promising alternative autologous tissue for cartilage regeneration. Acknowledgement:NIHR01AR067821
High-throughput Microfluidic Isolation of Zonal Chondrocytes for Regenerating Natural-structured Articular Cartilage
Singapore-MIT Alliance for Research and Technology Center, Singapore, SINGAPORE
Current clinical approaches for articular cartilage repair have not been able to restore their hierarchically organized architecture, and thus the full mechanical function and durability. Although literatures have suggested that mimicking the zonal organization of articular cartilage in neo-tissue by the use of zonal chondrocyte subpopulations could enhance the functionality of the graft, the engineering of stratified tissue has not yet been realized using zonal chondrocytes due to the lack of specific zonal chondrocyte isolation protocols and limited amount of donor tissue to yield adequate numbers of viable zonal cells. We show that by using a spiral microchannel device, the superficial, middle and deep zone chondrocytes can be isolated from full thickness porcine cartilage in high-throughput manner based on their differences in cell size. RT-PCR analysis of PRG4, Col II, Aggrecan, COMP and Col IX levels reveal their zonal specific characteristics. The isolated zonal chondrocytes are able to maintain their zone specificities after 2-dimensional expansion culture. RT-PCR and histology analysis and protein quantification of the extracellular matrix proteins show that the cartilage regenerated by both freshly isolated and expanded zonal chondrocytes formed cartilage tissue in 3-dimensional hydrogelin vitro bearing respective zonal characteristics. In addition, the cartilages regenerated in hydrogel by zonal chondrocytes have clear advantage in mechanical strength over those regenerated by mixed chondrocytes. This study provides an effective way to obtain zonal chondrocytes in large number, and shed light on potential layer-by-layer implantation of three zonal chondrocytes for repairing articular cartilage with natural architecture.
Department of Biomedical Engineering, University of Michigan, ANN ARBOR, MI
Cincinnati Children's Hospital Medical Center, Cincinnati, OH
Mesenchymal stem cells migrate, proliferate and differentiate in response to specific extracellular matrix (ECM) components. In this work, decellularized cartilage ECM (dECM) was processed and used as a cell culture coating to initially evaluate its effect on viability, homing and chondrogenesis of hMSCs in vitro. Porcine cartilage was decellularized as confirmed by microscopy and DNA analysis resulting in removal of 95% of the initial starting material (t-test, p < 0.01), while the majority of the ECM proteins (72.5%, p < 0.005) were preserved. Retained cytokines in dECM promoted cell migration in coated plates compared to non-coated ones. Further, cell viability was improved on dECM coating compared to fibronectin and non-coated plates. More importantly, chondroinduction of hMSCs was upregulated in coated plates with collagen II, aggrecan and Sox 9 gene expression an order or more in magnitude compared to non-coated plates (p < 0.005). This was further supported by prominent collagen II and reduced collagen I expression of the hMSCs by immunocytochemistry on coated plates compared to non-coated ones. The dECM was then incorporated within poly(lactic acid) microspheres of mean diameter ∼100 μm; encapsulation efficiency of PLA microspheres was 47%, resulting in a bioactive, biocompatible microsphere capable of inducing chondrogenesis that is then integrated within a biocompatible polymer via additive manufacturing to produce a novel biomaterial suitable for the treatment of large osteochondral defects.
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology/Emory University, Atlanta, GA
Rotator cuff tears (RCT) can lead to the development of osteoarthritis in the shoulder (1). Cathepsin proteases are thought to be partially responsible for cartilage degeneration characteristic of osteoarthritis (2). Cystatin-C is the endogenous protein inhibitor of cathepsins that can interact with heparin (Hep) via electrostatic interactions. The goals of this project are 1) characterize arthritic changes in cartilage following RCT in an animal rat model and 2) develop an injectable heparin-based microparticle system to inhibit cathepsins.
Adult male rats (n = 8) underwent suprascapular nerve denervation and infraspinatus/supraspinatus tendon transection. Twelve weeks post-operation, humeral heads were treated with 10% Hexabrix (Covidien) and scanned (45kVp, 200μA, 600ms) via contrast-enhanced microCT (ScancoMedical). For cystatin-C release, fully desulfated heparin (Hep-) and PEG-diacrylate particles were used as controls that should interact less with cystatin-C. Microparticles were fabricated via water-in-oil emulsion and release (n = 3) was tracked over 7 days.
In 4 out of 8 injured shoulders, focal defects (average: 0.12 ± 0.09 mm3) were observed, whereas no focal defects were found in control samples. Hep- microparticles loaded significantly more cystatin-C than the PEG control (PEG:35.4 ± 8ng, Hep:50.4 ± 12.7ng, Hep-:64.3 ± 8.3ng). However, after 7 days, heparin microparticles released significantly more cystatin-C (PEG:6.28 ± 1.48 ng, Hep:9.86 ± 1.18ng, Hep-:3.69 ± 0.10ng).
Cartilage loss indicates the rat model can replicate osteoarthritic changes seen following RCT in humans. Hep microparticles released the most cystatin-C, possibly due to their ability to prevent protein denaturation during release. Hep microparticles achieved sustained released of cystatin-C over 7 days, making this a potential injectable intra-articular therapy to preserve cartilage following RCT.
(1) Kramer JSES, 2013.
(2) Morko ARD, 2004.
Label-free Microfluidic Separation of Isolated Muscle Satellite Cells
Biomedical Engineering, University of Michigan, Ann Arbor, MI
Skeletal muscle satellite cells play an essential role in repairing muscle damage and have tremendous therapeutic potential in cell therapy or skeletal muscle tissue engineering. Obtaining a sufficiently pure satellite cell population, however, presents a significant challenge. We hypothesized that the size difference between satellite cells and fibroblasts, two primary cell types obtained from muscle dissociation, would allow for label-free, inertial separation in a microfluidic device and that purified satellite cells could be used to engineer skeletal muscle. Immediately after dissociation and sorting, aliquots of cells were fixed via Cytospin. These cells were stained for DAPI, a combination of Pax7 and desmin to identify myogenic cells, and PDGFRα to identify fibroblast progenitors. Remaining cells were cultured to form a 3D skeletal muscle construct. Images of the monolayer were recorded on Day 14 to evaluate myotube density, and isometric tetanic force production was measured in 3D constructs on Day 16. Cell dissociation yielded a myogenic cell purity of 44.6 ± 3.79%. This purity was enriched to 75.6 ± 2.60% by sorting. Myotube density in the sorted cell population was 35.1 ± 3.13 myotubes/mm2, significantly greater than the 12.64 ± 3.13 myotubes/mm2 in unsorted controls. Constructs fabricated from sorted cells also produced significantly greater tetanic forces (117.2 ± 5.65 μN) than unsorted controls (57.1 ± 9.43 μN). These results demonstrate the promise of microfluidic sorting in purifying isolated satellite cells. This unique technology could assist researchers in translating the regenerative potential of satellite cells to cell therapy and tissue engineering therapies.
Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA
Repair of large bone defects is critically dependent on adequate vascular support. We hypothesize that co-delivery of vascular precursors, either as multicellular microvascular fragments (MVF) or as component single cells (SVF, stromal vascular fraction), with BMP-2 within a collagen sponge, will accelerate bone healing. As a step towards this goal, we first characterized the in vitro release of loaded BMP-2 from cell-laden collagen sponges. MVF and SVF were isolated from Lewis rat adipose tissue. Temporal release of BMP-2 was quantified by ELISA on harvested culture media. Interestingly, while the BMP-2 release from the SVF-loaded constructs was not different than the acellular group (both ∼20% release by 3 weeks), the MVF-loaded constructs displayed significantly greater release (∼75%). MVF and SVF are isolated from the same tissue source and thus composed of the same cell types, yet only MVFs induced more BMP-2 release. Microscopy revealed that MVFs dissociate into single cells and reform microvascular networks rather than displaying typical sprouting angiogenesis; SVF begin as single cells and assemble into similar network structures. We hypothesize that MVFs augment release of BMP-2 from collagen sponge during dissociation either by heightened protease activity or by directly producing BMP-2. Future work will include quantification of protease activity and temporal gene expression in MVF- and SVF-seeded collagen sponges. This treatment approach may increase the extent of early vascularization within large bone defects and also improve BMP-2-mediated bone repair while minimizing the required dose of exogenous growth factor.
College of Creative Studies, University of California, Santa Barbara, Santa Barbara, CA
Fibrin clots (FCs) capture and retain platelets (PLTs) [1], bind and release growth factors contained in PLTs [2] and have been clinically demonstrated to aid tissue healing. To our knowledge, no study has determined the degree that PLTs are captured and concentrated during the formation of a FC. Our hypothesis is that FCs highly capture and concentrate available PLTs. The purpose of this study was to determine the level of platelet capture and concentration by the formation of a FC. From 10 volunteers, 35 ml of whole blood was obtained. Five ml was used to determine pre-clot cell count. A FC was formed using the remaining 30 ml of blood using a clotting device. Five ml of post-clot plasma was sent for post-clot cell count. PLT's decreased significantly from a pre-clot level of 187.80 to a post-clot level of 4.40 (%lt.01, 2 tail T-test). Using this technique, FC formation captured and concentrated 92% of the available PLTs. FC formation captures and concentrates 92% of available PLTs.
1. Inchingolo F et al. Trial with Platelet-Rich Fibrin and Bio-Oss used a grafting materials in the treatment of the severe maxillar bone atrophy: clinical and radiological evaluations. Eur Rev Med Pharmacol Sci 2010:14: 1075–10842.
2. Visser, LC et al. Platelet-Rich Fibrin constructs elute higher concentrations of transforming growth factor-b1 and increase tendon cell proliferation over time when compared to blood clots: a comparative in vitro analysis. Vet Surg 2010; 39:811–817
1. Criswell T et al., Am J Pathol. 180, 787, 2012.
2. Zhou Y et al., Am J Pathol. 184, 2226, 2014.
Human Multipotent Adult Progenitor Class Cells Upregulate Angiogenesis During Bone Repair
The promotion of vascularization in a bone defect is an important precursor to new bone formation, and defects lacking adequate circulation can suffer from fracture non-unions. The use of MSC therapy for angiogenic and osteogenic applications has been extensively investigated; however the angiogenic potential of multipotent adult progenitor class cells (MAPC®) within osteogenic applications remains largely undiscovered. We hypothesize that the ability of MAPC-class cells to thrive under hypoxic conditions contributes to increased angiogenesis and subsequent osteogenesis observed both in vitro and in vivo, and we speculate this is occurring through a hypoxia inducible factor 1-alpha (HIF-1α) mediated pathway. In vitro studies were conducted on cells cultured indirectly with demineralized bone matrix (DBM) in a transwell. A ten-fold increase in vascular endothelial growth factor (VEGF) and a two-fold increase in Angiogenin were observed compared to the controls. HIF-1α shRNA or control shRNA were introduced into MAPC-class cells using lentiviral transduction and the angiogenic and osteogenic potential was evaluated in vitro. When MAPC-class cells seeded onto DBM were implanted into a rat fibular defect, a statistically significant 3-fold increase in blood vessel ingrowth after 14 days was observed using IHC with von Willebrand Factor (vWF) antibody, compared to DBM alone. The results demonstrate the angiogenic properties of MAPC-class cells, making them a promising cell source for orthopedic regeneration in poorly vascularized defects.
Impaired Bmp-2 Mediated Bone Regeneration In A Severe Musculoskeletal Trauma Model Is Rescued By A Regenerative Muscle Therapy
[1] Hurtgen et al. J Musculoskelet Neuronal Interact. In Press.
[2] Pollot et al. J Trauma Acute Care Surg. In Press.
Heparin Microparticles to Modulate Chondrocytic Differentiation
Cell-based therapies for cartilage injuries are hampered by difficulty in controlling differentiation during in vitro expansion (Somoza, Tissue Eng B, 2014). To better temporally control differentiation, we employed heparin microparticles to modulate endogenous growth factor presentation to cells via localized sequestration.
Heparin methacrylamide microparticles (Hettiaratchi, Biomaterials, 2014) and PEG-diacrylate microparticles were fabricated via emulsion and free-radical initiated polymerization. ATDC5 cells, a model chondrocytic progenitor cell line, were cultured with heparin and PEG microparticles in transwell or 700 cell aggregates (spheroids) for 12 or 18 days, respectively, providing platforms to investigate microparticle function with or without cell contact.
Chondrocytic gene expression was significantly down-regulated in heparin microparticle groups, compared to PEG and no microparticle controls (3.6 ± 0.95, 3.0 ± 1.2, and 4.0 ± 1.4-fold decrease for collagen II, aggrecan, and collagen X in heparin microparticle transwell, respectively; 7.4 ± 1.8 and 16.5 ± 6.18-fold decrease for collagen II and aggrecan in heparin microparticle spheroids, respectively, compared to no microparticle control, day 6). Chondrocytic matrix deposition was significantly lower in transwell heparin microparticle groups and observed lower in spheroid heparin microparticle groups as compared to PEG and no microparticle controls (59.7 ± 13% and 73.6 ± 1% GAG and mineral deposition, respectively, for transwell heparin compared to no microparticle control, day 12).
Heparin microparticles delayed ATDC5 cell differentiation with or without physical contact with cells, suggesting that soluble factor sequestration may be the mechanism behind this phenomenon. Overall, heparin microparticles have the potential to temporally modulate chondrocytic differentiation in therapeutic cell types through sequestration of endogenous growth factors, a novel strategy for cell-based cartilage repair.
Development and Optimization of a Tenogenesis Assay for Applications in Tendon Research
Mesenchymal stem cells (MSCs) have shown promise for the treatment of debilitating tendon injuries. In the absence of a facile tenogenesis assay, different types of stem cells for therapy cannot be evaluated in a high throughput manner. To this end, we have evaluated novel assays to culture MSCs in 3D collagen hydrogels under uniaxial strain with tenogenic growth factors using commercially available supplies to generate a 3D construct. We hypothesize that 3D culture of bone marrow MSCs with BMP-12 and IGF-1 maintained under contraction-induced uniaxial strain will result in an effective tenogenesis assay. Rat tail collagen and MSCs at pre-optimized concentrations were mixed together and plated in rectangular dishes, affixed with 2 fixed point anchors. Experimental groups comprised the following: 1) growth media control, growth media supplemented with 2) BMP-12, 3) BMP-12 and FGF2, 4) BMP-12 and IGF-1, and or 5) IGF-1 and TGF-β1. After 10 days, constructs were harvested and analyzed for changes in gene expression, biochemistry, histology and rates of contraction. All constructs contracted over a 10 day period to a longitudinal tendon-like structure. Cellular alignment parallel to the axis of tension was subjectively increased over control in groups: BMP-12, BMP-12/IGF-1 and IGF-1/TGF-β1. BMP-12/IGF-1 group showed increased expression of a panel of tendon marker genes. BM-MSCs grown in our 3D environment supplemented with BMP-12 and IGF-1 yielded the best tenogenic outcomes. This easy to use bench-top assay can be used to screen large numbers of stem cell lines to determine potential to heal tendon.
Magnetic Stem Cell Confinement for Articular Cartilage Repair
Repair of the cartilaginous layer of the condyle in case of disease or trauma remains a significant clinical challenge. An approach recently explored to engineer autologous cartilage replacements is the differentiation of stem cells into chondrocytes. As cell-cell contact is known to initiate chondrogenic differentiation, our lab has developed a new approach to condense cells based on magnetic labeling and seeding. Human mesenchymal stem cells were first labeled magnetically using iron oxide (maghemite) nanoparticles at a concentration of [Fe] = 0.2 mM and seeded within porous polysaccharide scaffolds (7 mm thick) via magnetic condensation. The seeded scaffolds were then cultured for 21 days in a TisXell bioreactor (QuinXell, Singapore) that offers a dynamic mechanical environment as well as improved nutrient and gas diffusion. The constructs were also subjected to various levels of growth factors (TGF-β3, BMP-2, IGF-1) and oxygen tension (normoxia vs. 3% hypoxia) to assess their effect on the synthesis kinetics of extracellular matrix components. Under optimal culture conditions, cartilage tissue production was highly improved, with a 50-fold increase in collagen II expression, an overexpression of aggrecan, and a very low expression of collagen I and RUNX2. Transmission electron microscopy analysis confirmed these results and reveled long collagen fibers that presented the characteristic periodicity of collagen II. Additionally, the expression of collagen X was modulated by hypoxic conditions. This unprecedented cartilage tissue production within porous polysaccharide scaffolds represents a major step forward in producing replacement tissue for cartilage defects.
NEMO-binding domain peptide reduces overloading-induced MMP-3 expression in human Nucleus Pulposus Cells
Mechanical overloading and NFΚB activation are involved in spinal disc degeneration, characterized by an increase in matrix metalloproteases (MMPs) in the nucleus pulposus. Clinically available procedures do not treat these causes. NEMO binding domain peptide (NBD) blocks NFΚB activation. However, the ability of NBD to reduce NFΚB-induced levels of MMPs in human nucleus pulposus cells (hNPCs) under overloading is unknown.
We hypothesized that mechanical overloading results in increased expression of MMPs in hNPCs and that these high levels can be down-regulated by addition of NBD. The aims were to demonstrate an up-regulation of MMP genes/proteins and to show that NBD reduces this effect.
Human NPCs were IL-1β pre-stimulated, embedded in a 3D matrix and exposed to high compression in the presence and absence of 5μM/50μM of NBD. Cell viability was demonstrated in terms of a calcein/ethidium staining/MTS test. MMP-3, -9 and -13 gene/protein expression levels in cell constructs/supernatant were determined in terms of qPCR/ELISA.
Levels of MMP-3, but not MMP-9/-13 were up-regulated in response to mechanical overloading (MMP-3 protein: 173% compared to unloaded control, p < 0.05). Addition of NBD reduced the up-regulated MMP-3 protein levels (5μM NBD: 10%, 50μM NBD: 30% compared to loaded control, p < 0.05). No significant differences in cell viability were detected.
Increased MMP-3 levels may transfer signals of high mechanical loading into degeneration-associated processes. Furthermore, NBD may reduce catabolic effects induced by mechanical overloading and have the potential to decrease loading-induced disk degeneration.
Extrusion-Based 3D Printing of Fibrin for Modular Bone Tissue Engineering
Cortical bone is comprised of repeating functional units called osteons. As traditional scaffolds fabrication methods have relatively low precision, the manufacturing of osteon-like scaffolds have been challenging. Extrusion based 3D printing offers an efficient tool to accurately dispense biomaterials in a tunable manner to generate biomimetic construct. Fibrin is a glycoprotein that have shown potential in bone tissue engineering. Although fibrin is a well characterized material, the challenge is to adapt it for 3D printing. We have developed a 3D printable fibrin bioink by combining fibrinogen and gelatin. This study aimed to 1) optimize printing resolution by varying gelatin and fibrin concentration, 2) characterize parameters affecting scaffold architecture and 3) evaluate cell viability after plotting. The effect of fibrinogen/gelatin concentration on the shape and mechanical properties of constructs was studied using DMA. Low concentrations of gelatin and/or fibrinogen lead to pores collapse. The optimized bioink formulation was 5 w/v% gelatin and 15 w/v% fibrinogen. In order to enable 3D-fibers deposition of fibrin, an evaluation of rheological properties was performed. Fibrinogen concentration and cell density altered the rheological properties, increasing the viscosity and therefore affecting the printing parameters. Parameters affecting the scaffold and pore architecture were assessed. The printing speed, needle diameter, temperature and applied pressure were varied to optimize the resolution of the printed fibers, and ensure a reproducible printing process. The influence of those printing parameters on cell survival was also investigated. This work holds potential to be developed as a competitive candidate scaffold for modular engineering of bones.
Antibiotic-releasing Porous Space Maintainers Mitigate Infection and Restore the Osteogenic Potential of Induced Membranes in Infected Bone Defects
Rice University, Houston, TX
Infection and local antibiotic delivery can complicate the reconstruction of long bone defects. The induced membrane technique is a two-stage reconstructive strategy employing a temporary poly (methyl methacrylate) (PMMA) space maintainer to generate an osteogenic membrane that supports later reconstruction and additionally delivers antibiotics to bacteria-contaminated defects. This study's objective is to evaluate the interaction between local clindamycin delivery and bacterial infection with regards to the prevention of surgical site infection and the regenerative state of the induced membrane. Porous PMMA space maintainers with and without clindamycin were implanted in an 8mm rat femoral defect model with or without Staphylococcus aureus inoculation for 28 days (full-factorial, 4 groups, n = 8/group). All eight animals in the inoculated/no antibiotic group were culture positive for S. aureus at 4 weeks, which was reduced to 1/8 animals in the inoculated/antibiotic group. Quantitative polymerase chain reaction showed that clindamycin treatment restored inflammatory cytokine and growth factor expression to the same levels as the no inoculation/no antibiotic group. Clindamycin can obviate the negative effects of infection and does not itself negative impact membrane quality. Main effects analysis shows that inoculation negatively impacts production of bone morphogenetic protein-5, while clindamycin delivery positively influences its expression. Bacterial inoculation and clindamycin have independent and interacting effects on the gene expression profile of the induced membrane. Using clindamycin as a model drug, these results highlight that antibiotics can independently influence the regeneration of infected defects separately from their antimicrobial properties.
Department of Health Science, Pontifícia Universidade Católica do Paraná, Curitiba, BRAZIL
Despite the fact that titanium has good mechanical properties, corrosion resistance, and biocompatibility, the bone binding ability of titanium still needs to be improved. Nanostructured material surfaces provide an enhanced interface for interactions between implant surfaces and cells in the tissue [1]. Studies with the anatase or anatase/rutile titania nanotube arrays have shown accelerated growth and mineralization of osteoblasts [2]. In this study, we have investigated the functionality of adipose derived stem cells (ADSCs) on titania nanotube arrays that were annealed at different temperatures. The titania nanotube arrays were fabricated by potentiostatic anodization of titanium in Diethylene glycol/Hydrofluoric acid electrolyte at 60 V for 6 h, then annealed at 300oC and 530oC for 5 h. The resulting nanotube arrays were characterized using SEM, contact angle goniometry and XRD. ADSCs were cultured on titania nanotube arrays at a density of 1 × 104 cells/ml. The cells were allowed to adhere and to proliferate for 1, 4 and 7 days. Cell viability was characterized by the Alamar Blue assay and cell morphology by SEM. Cell adhesion, proliferation and morphology were characterized using fluorescence microscopy. The present study showed that annealing temperature affects the cell adhesion, proliferation as well as the morphology. Future studies are now directed towards evaluating differentiation of ADSCs on different surfaces.
1. Le Guehennec L, Martin F, Lopez-Heredia MA, Louarn G, Amouriq Y, Cousty J, Layrolle P. Osteoblastic cell behavior on nanostructured metal implants. Nanomedicine, 61–71, 2008.
2. Palmieri A, PezzettiF, BrunelliG et al. Anatase nanosurface regulates microRNAs. J Craniofac Surg 19, 328–333, 2008.
Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, ISRAEL
Universidad de los Andes, Bogotá D.C., COLOMBIA
The loss of functional tissue is an expensive health care problem, with limited treatment options. The golden standard for large bone damage is a cadaveric bone with stainless steel support; however, this solution only applies to simple morphologies, the material is limited and presents a variety of long term problems. Therefore, this investigation focusses in designing a scaffold with properties similar to the human bone. Thus, an alginate and coral matrix was created; the coral was chosen because of its chemical composition and the alginate due to its compatibility and mechanical properties. Accordingly, the following methodology was employed: coral preparation, scaffold fabrication and mechanical and biological characterization. The coral was milled with both a horizontal and a ball mill in order to evaluate the morphology of the particles obtained. Then, using a silicon mold, constructs for the mechanical and biological assays were made. Compression tests, density and porosity were calculated and cell cultures with fibroblast were performed. Results showed that Young's moduli were dependent of the pulverization method and the proportion of the materials. The maximum value was 5,4MPa for the 50/50 proportion of alginate and horizontally milled coral. The biological assay showed more extracellular matrix in the scaffolds consisting of more alginate and less coral. The density and porosity were proportional to the amount of coral. These results showed that this composite has potential as a biomaterial. But due to its behavior and small Young's Modulus, the application may be for tissues similar to cartilage.
Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC
Osteomyelitis is a common manifestation of invasive Staphylococcus aureus infection, often resulting in extensive bone loss and destruction. Reconstruction of large bone defects secondary to Osteomyelitis is still a major challenge, mainly due to chances of re-infection of bone grafts and rise of Methicillin-resistant Staphylococcus aureus (MRSA). The objective of this study is to use 3D bioprinting for custom fabrication of bone scaffolds containing silver nanoparticles (AgNPs); where a sustained release of AgNPs from the scaffold would provide antibacterial activity, and the scaffold architecture would allow new bone ingrowth. The bone scaffold was designed and 3D printed using a combination of poly(ɛ-caprolactone) (PCL) and poly(lactide-co-glycolide) (PLGA) containing AgNPs (PCL-AgNP and PLGA-AgNP, respectively). AgNP release kinetic studies showed that almost 100% of AgNPs are released from PLGA-AgNP in 6 weeks; while for only 4% of the AgNPs release from PCL-AgNP. A NaOH pre-treatment method was used to accelerate the degradation of PCL, which resulted in about 30–35% AgNPs release in 2 weeks. Additionally, an in situ hydroxyapatite (HA) plus AgNP deposition method to surface coat the bone scaffold resulted in an enhanced antimicrobial activity in vitro against Staphylococcus aureus and Pseudomonas aeruginosa. In vivo studies have been initiated to evaluate the therapeutical effects of inducted Osteomyelitis in a rat bone defect model. The combination of differentially degrading PCL and PLGA polymers containing AgNPs would enable the sustained release of AgNPs for treating Osteomyelitis infection during the bone regeneration.
Tissue-Engineered Cellular Neotendinous Constructs for Hernia Repair in a Rat
Section of Plastic Surgery, University of Michigan, Ann Arbor, MI
Ventral incisional hernia is a major issue following laparotomies and often results in hernia recurrence after abdominal wall repair. Current soft tissue prostheses have suboptimal mechanical properties and insufficient potential for biointegration, resulting in failure of the repair. Our lab has developed tissue-engineering strategies to overcome these shortcomings found in soft tissue grafts. The purpose of this study was to evaluate the ability of a novel tissue-engineered cellular neotendinous (CNT) construct, designed using our scaffoldless tissue engineering approach, to repair abdominal wall hernias in a rat model. Rats were subjected to a full-thickness chronic one-month abdominal wall defect and were subsequently repaired with either a CNT construct (n = 6), Alloderm biologic prosthesis (n = 6), or Prolene synthetic mesh (n = 6) for comparison. After 7 weeks of recovery, rats were euthanized and abdominal walls were excised for mechanical burst testing and histological analysis. Alloderm, Prolene and CNT repaired abdominal walls were determined to have a significantly lower modulus compared to native abdominal walls, but were not significantly different among themselves. Unlike the Alloderm and Prolene meshes, however, the CNT constructs did not develop notable adhesions and significantly increased in stiffness from in vitro measurements. The use of CNTs as a potential soft tissue prosthesis for hernia repair shows promise clinically with its ability to mitigate acute and chronic hernia failure mechanisms.
Cardiac tissue engineering aims at restoring cell compartment along with myocardial extracellular microenvironment. Even though countless combinations of synthetic or biological scaffolds and stem/progenitor cells of various origin have been tested so far, the most suitable candidate is yet to be found. With the aim to develop a natural injectable self-assembling scaffold able to serve as both three-dimensional platform and stem/progenitor cell delivery method, we assembled fibrin gels incorporating cells and cardiac decellularized extracellular matrix (d-ECM). Cryosections of cardiac ECM were decellularized according to previously published protocol, and dECM was lyophilized and solubilized. dECM solution was mixed with the fibrin solution carrying Cardiac Primitive Cells (CPC) isolated from adult human heart and allowed to gel at 37°C. Fibrin to dECM ratio (F:M) of 1:1, 1:2, 1:3 and 1:4 were tested to determine the ideal composition in terms of time of gelling and three-dimensional architecture. Gelling time varied from one minute to twenty-four hours. Gels were cultured for three days, then fixed and processed as tissues for histological study. Histochemistry revealed the presence of viable CPC in the gels whose architecture varied from densely packed to very loose. Due to time of gelling and to concentration of fibrin, the distribution of CPC in the scaffold was even only in the gel with F:M of 1:2. According to our results, the combination of CPC with fibrin and dECM at F:M of 1:2, being injectable and self-assembling at body temperature, provides an attracting alternate to bioconstructs.
Adipose Derived Delivery Vehicle For Adipogenic Factors
Bioengineering, University of Pittsburgh, Pittsburgh, PA
Injectable hydrogels serving as biomaterial scaffolds for adipose tissue engineering are a common investigative strategy. Hydrogels made from adipose derived extracellular matrix (AdECM) have shown potential in its ability to generate new adipose tissue in vivo. To further these strategies, the current research has developed a composite adipose derived delivery system (CADDS) containing single- and double-walled dexamethasone encapsulated microspheres (SW and DW Dex MS). Previously, our laboratory has published work on the use of Dex MS as an additive to enhance adipogenesis and angiogenesis in adipose grafts. In the current work, AdECM and CADDS are extensively characterized along with in vitro cell culture analysis. Results of the study indicate the AdECM to have minimal cellular and lipid content allowing for gelation of its collagen content under physiological conditions. Adipose stem cell (ASC) culture studies confirmed biocompatibility within the CADDS and adipogenesis was increased in experimental groups containing the hydrogel scaffold. In vitro studies of microspheres demonstrated a controlled release of dexamethasone from SW and DW formulations along with peripheral blood mononuclear cell culture (PBMC) studies confirming bioactivity and drug stability. Preliminary in vivo studies indicated the CADDS is easily injectable into the subcutaneous dorsal aspect of a mouse model. The delivery of Dex MS via an injectable hydrogel scaffold combines two biologically responsive components to develop a minimally, invasive, off-the-shelf biomaterial for adipose tissue engineering
Neurosurgery, University of Pennsylvania, Philadelphia, PA
Peripheral nerve injury (PNI) is a common consequence of trauma or surgery, and may result in permanent motor and/or sensory deficits. The current standard for surgical repair of major PNI is a sensory nerve autograft; however, functional recovery is only achieved in ∼50% of cases, with performance decreasing starkly for large segmental defects >3cm. We previously developed tissue engineered “living scaffolds” consisting of long, aligned sensory axonal tracts in a 3-D, anisotropic environment to direct regenerating host axons. In the current study, we are advancing this “living scaffold” technology to include long motor axons, which we hypothesize will preferentially drive host motor axon regeneration by recapitulating developmental pathfinding where motor axons are preferentially guided to their end targets along existing motor axons. To accomplish this, we have coaxed the formation of dense neuronal aggregates from primary spinal motor neurons, and cultured them in custom built mechano-bioreactors. Once projecting axons formed an interconnected network, they were subjected to controlled mechanical tension at micron-sized increments, resulting in healthy, dense “stretch-grown” axonal tracts spanning up to several centimeters. Axons projecting from motor neuron aggregates exhibited greater fasciculation and resilience upon mechanical tension-induced growth. We are currently testing the efficacy of constructs consisting of purely motor or motor and sensory axons to preferentially promote motor axon regeneration in vitro as well as following PNI in rodents.
Biomedical Engineering, The University of Akron, Akron, OH
Departamento de Biología Celular y Tisular, Universidad Nacional Autónoma de México, Ciudad de México, MEXICO
Weinberg CB, Bell E. A blood vessel model constructed from collagen and cultured vascular cells. Science. 231, 1986.
Ravi S, Chaikof EL. Biomaterials for vascular tissue engineering. Regenerative Medicine, 5(1), 107, 2010.
Chemical Engineering, Florida Institute of Technology, Melbourne, FL.
Coronary artery disease (CAD) accounts for 1 in 7 deaths in the US1. Tissue-engineered vascular grafts (TEVGs) are a promising treatment option but limitations such as intimal hyperplasia and thrombosis hamper their use for the replacement of small-diameter vessels (<5mm). One approach to address the current limitations of TEVGs is to develop a tubular scaffold that mimics the physicochemical properties (composition, structure and mechanics) of the native vessel. In this study, we hypothesized that the electrochemical fabrication methodology can be employed to fabricate a bilayered collagen-elastin scaffold that resembles the native vessel. Collagen solution was loaded between two cylindrical electrodes placed in concentric fashion and the electrochemical process parameters - voltage (1.7 V-20 V) and time (5 min-1 hour) - were modulated to attain a highly compact and bubble-free luminal layer of the scaffold. Elastin incorporated electrochemically aligned collagen (ELAC) fibers2 were wound circumferentially around the tubular lumen to form the bilayered scaffold. Mechanical integrity of the scaffold was improved by employing EDC-NHS crosslinking. Results indicated that application of 3V for 30 min resulted in a highly compact and bubble-free tubular lumen layer. Verhoff-Van-Geison staining and SEM confirmed the morphology and integrity of the bilayered scaffold. Studies assessing the mechanical properties of the scaffold and cellular response are currently ongoing. Together, electrochemical fabrication is a reliable method to fabricate tissue-mimicking scaffolds with potential to be used for vascular tissue engineering applications.
1. Mozaffarian et al. Circulation 133, e38, 2016.
2. Nguyen et al. Biomedical Materials 11, 025008, 2016.
Department of Urology, Faculty of Medicine, Comenius University in Bratislava, Bratislava, SLOVAKIA.
In this study we analyzed collagen-based scaffold for cytotoxicity and biocompatibility under in vitro conditions. Moreover, we evaluated myogenic differentiation of adipose tissue-derived stem cells on these scaffold in respect to potential application in tissue engineering of urethra. Obtained results showed that collagen based scaffold did not produce any cytotoxic effect and was fully biocompatible. Moreover, adipose tissue-derived stem cells underwent myogenic differentiation in vitro. Taken together, the collagen based scaffold is suitable for fabrication of artificial urethra. Supported by grant VEGA number 1/0207/16.
Department of Bioengineering, University of Maryland, College Park, MD.
Keratin is a physiologically biodegradable protein, which provides adequate cell support, and can self-assemble or be crosslinked into hydrogels. We have developed a reproducible method for the 3D printing of keratin scaffolds. Using a riboflavin-sodium persulfate-hydroquinone (initiator-catalyst-inhibitor) photosensitive solution, we produced 3D keratin constructs via UV crosslinking in a lithography-based printer. In this work, the printing resolution and cytotoxicity characteristics of keratin hydrogels (4, 5, 6% wt/vol) were tested to assess their viability for tissue engineering applications. Print resolution was assessed by printing cubes, cylinders, and stepped pyramids with dimensions ranging from 0.5 to 4 mm. Microscopic images were measured and compared against the design values. Swelling capacity and saturation with phosphate-buffered saline (PBS) and minimum essential medium (MEM) over time were measured to evaluate solution uptake. Cytotoxic response was evaluated by direct contact and conditioned media tests on L929 fibroblasts. Specifically, cells were visualized using a calcein-ethidium homodimer live/dead stain and metabolic activity was quantified with XTT assays. Our approach produced scaffolds significantly close to the design dimensions (p < 0.05) with features of <1 mm. Maximum PBS and MEM uptake levels of 1773 and 1582% respectively occurred in 20 min; these results suggest that the constructs are suitable for growth factor uptake and delivery, as well as cell sustenance. Finally, printed constructs showed no significant cytotoxicity, while supporting cell adhesion and proliferation. The novel keratin-based printing resin and methodology allows for the rapid fabrication of patient-specific hydrogels for wound healing applications.
Our lab has developed an injectable bone graft system that is both biodegradable and highly porous via polymerization of high internal phase emulsions (HIPEs). We have previously demonstrated that these injectable grafts have rapid in situ cure, microscale integration with native bone, and compressive properties comparable to cancellous bone. The goal of the current study is to enhance the osteoinductivity of the polyHIPE scaffolds via surface modification with extracellular matrix proteins. A comparison of the effect of different proteins (gelatin, collagen, Designer Collagens) on hMSC adhesion, spreading, and osteogenic differentiation will be used to identify relevant integrin-mediated processes key to bone regeneration. To this end, hMSC attachment and viability was first assessed on gelatin-modified polyHIPE scaffolds. Briefly, gelatin was reacted with 2-isocyantoethyl methacrylate to introduce reactive sites that would anchor the protein to the pore wall. The functionalized gelatin then added to the aqueous phase of the HIPE and cured. hMSCs were seeded onto sterilized polyHIPE scaffolds and cell attachment and spreading was analyzed. Preliminary results demonstrated increased attachment and spreading on these gelatin modified scaffolds. Current studies are extending this work to collagen and Designer Collagens with selected antibody blocking studies to probe the involvement of integrin signaling in scaffold-mediated osteogenic differentiation. This research will provide a deeper understanding of integrin-mediated osteogenesis in order to develop osteoinductive polyHIPE scaffolds for bone regeneration.
Impedance Affinity Biosensor for Detection of Differentiation Markers in Tissue Engineered Salivary Gland Nanofiber Scaffolds
Antibody-based biosensing and protein conjugation in conjunction with techniques in electrochemical impedance spectroscopy and interdigitated gold electrode substrates can be used to develop real-time, protein biosensors for use in Tissue engineering studies. We have developed complex multi-scale scaffolds with micropatterned structures, nanoscale polymer fibers, and various basement membrane biomolecule additives, which creates a wide array of potential scaffold configurations. In order to continuously improve the quality and performance of the scaffold, a biosensor could help identify the best performers among the sample group. Manufactured electrodes with 5 micron spacing and width in an interdigitated arrangement are physisorbed with a layer of Protein A-based IgG specific pre-binding layer. Gel-purified SABPA (salivary gland differentiation marker) specific monoclonal antibodies are adsorbed onto the binding layer, which provides high binding site accessibility and total electrode coverage. When immersed in cell culture media containing, protein binds to the surface, which is detected by a shift in the AC impedance and charge-transfer resistance of the circuit. The biosensor has demonstrated a strong response to control analyte and moderate response to target analyte. Efforts to improve signal strength include tweaking electrolyte ionicity to enhance response to charge transfer characteristics. Large hydrodynamic diameter ionic species including ferrocyanide and ferrous sulphides can be used in small concentrations for this purpose. Real-time continuous detection of differentiation markers in tissue engineered in-vitro scaffold systems will enable a host of novel experimental capabilities, including mid-course drug response assessment and exact onset time of marker secretion.
Keratin Dose-Dependently Rescues Human Cardiac Stem Cells from Hypoxia-Mediated Cytotoxicity
1. Doppler SA, et al. J Thorac Dis. 5, 683, 2013.
2. Reichl S. Biomaterials. 30, 6854, 2009.
3. Shen D, et al. Biomaterials. 32, 9290, 2011.
Polylactic-Co-Glycolic Acid-Gelatin Electrospun Scaffolds Seeded With Human Mesenchymal Stem Cells For Skin Tissue Engineering
Cell Biology and Histology, National Autonomous University of Mexico, Mexico City, MEXICO.
This research was supported by project DGAPA PAPIITIN218315
Cell and Tissue Biology, Medicine School, Universidad Autónoma de México, México, MEXICO.
WINNOVA, SEOUL, KOREA, REPUBLIC OF.
In this study, the regeneration of damaged carilage was researched with GDF-5 loaded injectable hydrogel. Successful cartilage repair relies on three specific critieria: cells, growth factors and scaffolds. The cell population originated from cartilage, or growth factors may differentiate mesenchymal stem cells to a chondrogenic phenotype. Many studies reports that Transforming growth factor beta 3 (TGFβ-3) has critical roles in mesenchymal cell condensation and chondrogenesis in 3D pellet culture system. Among of the growth factors related chondrogenesis, growth/differentiation factor 5 (GDF-5) was introdued to injectable hydrogel, and previously compared its effect of chondrogenic differenation of MSC with TGFβ-3. Based on the histological staining of glycosaminoglysan we found that TGFβ-3 and GDF-5 could induced chondrogenic differentiation of MSCs. This chondrogenic effect of TGFβ-3 and GDF-5 was confirmed by quantification of GAG andthe expression of collagen type II. In practice application in cartilage repair, we introduce of drug delivery system with injectable hydrogel, which is carboxylic anhydride-conjuageted glycol chitosan hydrogel. It is non-toxic and biodegredable, as wellas can be transformed sol-gel according to temperature. Hydrogel loaded with GDF-5 produced proteoglycan in MSCs culture system, displaying silmilar level of the control (only hydrogel). Moreover, gene expression of collagen type II was increased GDF-5 loaded hydrogel more than the control. Now, We are currently evaluate the more chondrogenic effects in hMSC system using GDF-5 loaded hydrogel by RT-PCR for gene expression related to chondrogenesis: type X collagen, aggrecan, and sox9.
Curcumin, a yellow pigment from Indian spice turmeric is known for its ability of anti-inflammatory, anti-cancer and antioxidant. Gellan gum, clear gel like polymer, has been used for cartilage regeneration by its easy handling and biocompatibility. In this study, curcumin loaded gellan gum hydrogel scaffolds were well fabricated at several concentrations for cartilage regeneration. Fabricated gellan gum hydrogel scaffolds with different concentration of curcumin were analyzed for various properties such as SEM, water uptake, degradation, compressive strength, FTIR, MTT assay, sGAG content, mRNA expression, in vivo test, etc. Among all experimental groups, 10 wt.% curcumin loaded with 0.7 wt.% gellan gum solution scaffold showed appropriate morphology, enhanced cell proliferation, sGAG content and mRNA expression. The composite material supports cell growth covered with extracellular matrix (ECM) while maintaining its function. Thus, curcumin loaded gellan gum scaffolds can be envisioned as a potential material to cartilage regeneration, possessing proper mechanical strength and non- toxicity.
Cellular Behavior of Bone Marrow-derived Mesenchymal Stem Cells on Thermo-Responsive RGD-Conjugated MPEG-b-PCL Hydrogels
Institute of Cell & Tissue Engineering, The Catholic University of Korea, Seoul, KOREA, REPUBLIC OF.
Thermo-responsive methoxy polyethylene glycol-b-poly(ɛ-caprolactone) (MPEG-b-PCL) diblock copolymer undergoes a reversible sol-gel transition around body temperature by controlling the molecular weights of the polymers. Due to the merit of MPEG-b-PCL, the polymer can be used as injectable hydrogel scaffolds for tissue engineering applications. Nevertheless, MPEG-b-PCL lacks cell-binding sites, and therefore it may have limitations in use as tissue engineering scaffolds. In the present study, Arg-Gly-Asp (RGD)-conjugated MPEG-b-PCL was prepared for improving the interaction with bone marrow-derived mesenchymal stem cells (BMSCs). Thermo-responsive RGD-conjugated MPEG-b-PCL hydrogels were prepared by controlling the mole percentage of the polymer. In order to evaluate the cellular behavior on the hydrogels, the cells were mixed with the polymer solution. Then, hydrogels were formed by adjusting at 37°C and compared with the results conducted with pure MPEG-b-PCL. Microscopic studies showed that a cluster of BMSCs were observed inside RGD-conjugated MPEG-b-PCL hydrogels. In addition, BMSCs were adhered to the three-dimensional networks of the hydrogels by focal adhesion. RGD-conjugated MPEG-b-PCL hydrogels showed higher survival rates of BMSCs than pure MPEG-b-PCL for up to 14 days of culture. Since 7 days of culture, a remarkable cell spreading was observed in RGD-conjugated MPEG-b-PCL hydrogels cultured with BMSCs, indicating interaction between RGD and integrin of the cell transmembrane receptor. Our findings suggest that RGD-conjugated MPEG-b-PCL hydrogels may serve as clinical scaffolds for tissue engineering applications.
Self-healing exists widely in living organisms, from micro blood clotting to macro skin repair. This special biological process benefits living organisms to restore their integrity and prolongs their lifespan. To mimic such natural healing feature, materials with the capacity to repair by themselves after damage are highly desirable with the increasing issues of environment and energy. A variety of smart materials have been developed on the basis of various strategies. Photo stimulated self-healing of polymers is a promising approach to construct cell-compatible intelligent materials for medicine use, since the self-healing process would minimize the needs of manual intervention and can be conducted unconsciously. We report a novel photo-stimulated, self-healing and cytocompatible hydrogel system. A coumarin methacrylate cross-linker was synthesized to modify the polyacrylamide-based hydrogels. With the [2 + 2] cyclo-addition of coumarin moieties, the hydrogels exhibited excellent self-healing capacity when they were exposed to light with wavelengths at 280 and 365 nm, respectively. The self-healing gels also possess high mechanical strength after healing for 60 min with stress intensity of 2×105 Pa and the elongation at break over 96%. More interestingly, cell attachment property was significantly improved by adding a poly (amidoamine) crosslinker. The hydrogels were used to regulate bone marrow stromal cells differentiation, and were shown to induce a regular cellular pattern. The development of methods and materials in this work may provide a new insight into the fabrication of novel biocompatible self-healing materials for extensive applications.
An In Vivo Study of the Host Tissue Response to Small Intestinal Submucosa Scaffolds Using a Subcutaneous Rat Model: Preliminary Results
Biomedical Engineering, Universidad de los Andes, Bogota, COLOMBIA.
Porcine small intestinal submucosa (SIS) is an extracellular matrix continuously studied for its application as a vascular graft. To achieve this goal further understanding of material's biologic behavior is needed, especially due to induced changes through the fabrication process. The purpose of this project was to study effect of SIS membrane thickness and orientation in the host tissue response, to investigate its in-vivo viability as a vascular graft. SIS membranes were fabricated as described by Sánchez et al1. Four test groups were implanted on a subcutaneous rat model (n = 5), varying the number of layers and the SIS side in contact with the muscle. Each group was explanted after 15 and 21 days. The harvested tissue was examined using H&E and immunohistochemical staining to identify macrophage markers CD68, CCR7 and CD68. Histological analysis showed the formation of a fibrous vascularized capsule surrounding the membrane and no sign of acute inflammatory reaction. In addition, it was found that most cellular invasion occurred on the borders of the samples in the intra-laminal spaces of the layers, where immunohistochemistry preliminary results also showed a higher level of macrophages expression. Although current results do not show distinctive response towards one specific macrophage phenotype, the presence of the fibrous tissue imply scaffold remodeling. In conclusion, this study shows that SIS thickness and orientation do not affect the host tissue response. Also, suggests that SIS physical modifications might enhance vascular remodeling when used as a vascular graft.
1. Sánchez DM et al. J. Biomechanics, 47(11), 2766, 2014.
Biomedical Engineering, New Jersey Institute of Technology, Newark, NJ.
Electro-conductive networks within the native myocardium direct signal propagation and ultimately the contractile behavior of the heart. Graphene, a highly conductive carbon derivative, has recently emerged as a promising material for developing electroactive tissues. To examine the effects of graphene containing scaffolds on mouse stem cell-derived cardiomyocytes (mES-CM), a novel three-dimensional nanofibrous composite scaffold of graphene and poly(caprolactone) (PCL+G) was used. Cardiomyocytes derived from mES cells [1] were seeded onto PCL + G scaffolds and cultured for 6–14 days. MEC-CM adhered well, spontaneously contracted, exhibited a positive inotropic response to β-adrenergic stimulation and expressed classical cardiac markers on PCL + G scaffolds exhibiting phenotypic characteristic of native cardiomyocytes. More importantly, PCL + G scaffolds facilitated functional Ca2+ handling behavior of mES-CM with enhanced fractional Ca2+ release and shortened caffeine induced transient T50. Specifically, mES-CM cultured on PCL + G scaffolds exhibited more efficient Ca2+ efflux out of the cytosol through Na+/Ca2+ exchanger mechanism [2,3]. Enhanced Ca2+ handling behavior of mES-CM leads to advanced contractile function and maturation of mES-CM in vitro. This study demonstrates the functional benefits of graphene containing electroactive scaffolds in the development of functional engineered cardiac tissues.
[1] Hitscherich, P.H., Biotechnol Bioeng, 2016, 113(7):1577–85.
[2] Liu, J., Stem Cells, 2007, 25(12):3038–44.
[3] Sato, D., Biophysical Journal, 2012, 102(8):L31–33.
Injectable, microporous hydrogels are superior in regenerative medicine due to minimally-invasive delivery and interconnected pores within which cells can migrate and proliferate [1]. Previously, our lab has found that densely-packed chitosan microgels are able to create an injectable and microporous scaffold which has potential for broad applications in tissue engineering [2]. The microgels are able to aggregate in a pH-responsive manner through hydrogen bonding and hydrophobic interactions. In the first part of this work, hydroxyapatite micro-whiskers were incorporated with the chitosan microgels to create a 2-component micro-porous paste which exhibited a yield stress of over 160 Pa and retained shape over 3 days after injection into PBS. The chitosan-hydroxyapatite paste is being developed for minimally-invasive applications in bone tissue engineering. Stem cell migration through the scaffold is currently being assessed. In the second part of this work, alginate was incorporated into the chitosan microgel paste to increase mechanical strength and cell viability. Although alginate addition to chitosan microgels interferes with the microgel-microgel hydrogen bonding, the alginate phase is simply crosslinked with soluble calcium and able to serve as an injectable biomaterial which does not dissolve during cell culture. Current research is examining these microgel scaffold composites for minimally-invasive applications in orthopedic regenerative medicine and will be the focus of this iPoster.
[1] Griffin, Segura, et al., Nature Materials, 7, 737–744, 2015, doi:10.1038/nmat4294.
[2] Riederer, Krebs, et al., Submitted 6/17/2016
Macroprous Extracellular Matrix-based Microribbon Hydrogels Enhance Osteogenesis Of Mesenchymal Stem Cells In 3D
Orthopaedic Surgery, Stanford University, Stanford, CA.
Hydrogels have been widely used for fabricating tissue engineering scaffolds. However, most hydrogels are nanoporous, whereas macroporosity is desirable for efficient nutrient diffusion, cell proliferation and matrix deposition. While porogens may be used to introduce macroporosity, they further decrease the already weak mechanical properties of the hydrogels. To overcome these limitations, we have recently reported a method to fabricate gelatin hydrogels into microribbon-like structures, which can be subsequently crosslinked into 3D-macroporous scaffolds for direct cell encapsulation. The goal of this study is to develop a method for fabricating various extracellular-matrix (ECM) compositions into microribbon-like structures for forming 3D-macroporous niche to promote osteogenesis of mesenchymal stem cells (MSCs) for bone repair. Three ECM compositions were used including hyaluronic acid, chondroitin sulfate, and heparin sulfate. All ECM molecules were functionalized with acrylamide and fabricated into microribbon structures. To support cell adhesion, cell adhesive ligand (CRGDS) were incorporated before spinning. The microribbon shape was internally fixed by dithiothreitol, and then preformed microribbons were suspended in PBS and homogeneously mixed with hMSCs before photo-crosslinked into 3D-macroporous scaffold. All ECM-based microribbons supported homogeneous cell distribution in 3D with high cell viability. Unlike conventional nonporous hydrogels, our microribbon-based hydrogels provided interconnected macroporosity and led to enhanced cell spreading and proliferation. After 5-week culture in osteogenic medium, all ECM-based microribbon scaffolds demonstrated substantially enhanced bone matrix deposition and mineralization compare to the hydrogels. We envision these microribbons-based biomaterials could provide novel injectable macroporous matrices for stem cell delivery to enhance osteogenesis and bone regeneration in situ.
Surgery (Plastic), Yale University, New haven, CT.
Chronic wounds may exhibit poor vascular perfusion and limited cellular angiogenic potential, including a diminished population of proangiogenic cells. Cell-based vascularization has widely been used to treat chronic ischemic conditions. Major challenges to clinical translation of such cell-based therapies include identifying the cell population best suited for wound therapy, as well as developing optimized delivery vehicles and/or adjunctive therapies to protect and improve the regenerative capacity of stem cells within the wound environment. With these limitations in mind, we have employed a multidisciplinary approach to achieve rational design of a sophisticated Scaffold-In-Particle carrier for delivery of human induced pluripotent stem cell (hiPSC)-derived endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) as well as stromal derived growth factor-1 (SDF-1), a chemoattractant cytokine, within the wound microenvironment. We hypothesize that a system comprised of a multifunctional biomimetic collagen scaffold and elastin-like peptide-SDF-1 (ELP-SDF1) nanoparticles (Collagen-In-ELP/SDF1) will synergistically promote both cell survival and vascularization to treat chronic wounds. We have already fabricated the biomimetic collagen scaffold and ELP-SDF1 nanoparticles and established an integration-free hiPSC line and abundantly generated highly pure and functional VSMCs and ECs. The vascular cells when embedded in the scaffold showed better cell viability, maintained their phenotype and formed strong vascularization under in vitro conditions. Furthermore, the multifunctional scaffold containing ELP-SDF1 nanoparticles showed chemotactic activity by attracting HL-60 cells. In the future, we will assess the biological activity of the multifunctional vascular scaffolds in chronic wound fluid obtained from diabetic patients and in a splinted back mice wound model.
Hydrogels provide a three-dimensional (3D) framework with tissue-like elasticity for culturing cells in regenerative medicine. Injectable materials delivered in aqueous solution are considered ideal for the introduction of cells and bioactive factors through minimally invasive methods to fill complex 3D shapes. A novel injectable click-crosslinkable collagen-based hydrogel has been created capable of cell encapsulation in vitro/in vivo. The objectives were to prepare a novel cross-linking strategy for the formation of hydrogels with varied mechanical and physical properties to examine differentiation and proliferation with dental pulp stem cells (DPSCs). Collagen was thiolated by a substitution reaction using Traut's reagent at pH 7.4 without the need of an additional base. The degree of functionalization was quantified using a TNBS assay and circular dichroism was used to confirm the ratio of positive to negative peaks (RPN) was comparable to native collagen. The hydrogels were formed in 10 minutes by a thiol-ene photo-click reaction using violet light (405 nm) between the thiolated collagen and poly(ethylene glycol) norbornene. Varied mechanical and physical properties of the gels have been made by varying the molar ratio of SH: norbornene and the degree of thiol functionalization. The effect of degradation on the mechanical and physical properties were studied using both simulated body fluid and collagenase. The prepolymer solution was prepared in PBS and DPSCs and G292 cells were encapsulated in the gel in vitro. Cell spreading, cell migration and cell survival were analysed using ATP assay and LIVE/DEAD staining.
A Gold-Based Stretchable Multielectrode Array for Muscle Stimulation and Recording During Peripheral Nerve Repair
Surfacd Modified Bio-Functional Titanium Implant to Promote Osteogenesis for Dental Application
Department of Dentistry, Graduate School, Kyung Hee University, SEOUL, KOREA, REPUBLIC OF.
In the dental tissue engineering area, surface treated titanium implant (Ti) is a promising tool to promote a reconstruction of dental-associated bone defects for rapid osseointegration and osteogenesis. In this study, we designed a hybrid Ti including heparin modified Ti surface followed by Growth/differentiation factor-5 (GDF-5) loading via carbonyldiimidazole (CDI) and Poly-L-lysine chemistry. After surface modification, our products were characterized by water contact angle, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS). And then, quantitative analysis of functionalized groups was also confirmed. The release behavior of GDF-5 grafted samples was confirmed up to 21 days. As a result, surface modification was successful and effectively immobilization of GDF-5 with sustained release behavior. As an in vitro test, GDF-5 loaded Ti showed significantly enhances the osteogenic differentiation with increased calcium deposition under nontoxic state against periodontal ligament stem cells (PDLSc). Furthermore, an in vivo result showed that GDF-5 loaded Ti had a significant influence on the new bone formation. All of experiments clearly confirmed that our strategy may suggest a useful paradigm by inducing osseo-integration as means to remodeling and healing of bone defects for restorative procedures in dental clinical field.
In recent years, a bacterial infection still remains as a serious problem including a wound infection and osteomyelitis. In this study, we designed a new co-electrospun membrane consisted of gelatin (GE) and Poly(D,L-lactide-co-glycolide) (PLGA) fibrous sheet containing different concentrations (0, 0.1, 0.5, 1 wt %) of silver sulfadiazine (AgSD) was designed for both an antimicrobial effect and improved biocompatibility. Well-defined products were characterized by physicochemical analyses. Mouse osteoblastic MC3T3-E1 cells were cultured on the scaffolds to confirm cell proliferation, followed by an antibacterial activity against gram-negative Pseudomonas aeruginosa (P. aeruginosa), gram-positive Staphylococcus aureus (S. aureus), and Methicillin-resistant Staphylococcus aureus (MRSA). These biological tests showed that GE/PLGA-AgSD scaffolds had good cell viability, as well as effective antimicrobial activity. These remarkable results suggest that GE/PLGA-AGSD scaffolds possess a great versatility for the treatment of osteomyelitis in the field of bone tissue engineering.
Cultivation And Maintenance Of Mesenchymal Stem Cells And Mesenchymal Stem Cells Spheroids In 3d PCL/PLGA Scaffolds
Mesenchymal stem cells (MSCs) can be isolated from most postnatal tissue and organs and have the potential to differentiate into cells of mesenchymal origin. This study has focused on the analysis of MSC and MSC-spheroid growth in poly(lactic-co-glycolic acid) (PLGA) nanofibers under polycaprolactone (PCL) 3D support. MSC-spheroids were generated by hanging drop cultures of 2.5 × 103 or 2.5 × 104 per 35 μl of DMEM for 2 days. PLGA 16% nanofibers were manufactured using the electrospinning technique under PCL 3D support. Nanofiber morphology was analyzed by scanning electron microscopy on ImageJ software. Cell adhesion was analyzed by DAPI and phalloidin staining. Cell viability was analyzed by WST8 assay. Diameter of the 2.5 × 104 MSC-spheroids was higher than the 2.5 × 103MSC-spheroids, 0.517 μm (±0.025 μm) and 0.279 μm (±0.024 μm) (p < 0.0001), respectively. The occupation rate during the 10 day period was similar in both spheroids, being 3.01 ± 0.18 mm and 2.6 ± 0.19 mm, respectively. 2.5 × 103 MSC-spheroids were used for the next experiments. One spheroid or 2.5 × 103 MSCs was plated in 6 mm scaffolds or culture plates as the experimental control. The control MSCs showed a higher number of viable cells (p < 0.01) followed by MSCs cultivated in the scaffolds and control spheroids. Spheroids cultivated in the scaffolds showed the least viability (p < 0.05). After 6 days, the MSCs were disseminated in the scaffolds, while the spheroids were not located. Spheroids were generated from MSCs and occupied the surface of the wells. The PLGA/PCL scaffolds were as successfully colonized only by the MSCs.
The Effect of Large Silk Fibroin Microspheres on Breast Cancer Cells in Vitro
Chemical Engineering, Chulalongkorn University, Bangkok, THAILAND.
The current work studied the effect of large silk fibroin (SF) microspheres on cancer cells to evaluate their potential for using for in vitro chemo-sensitivity testing. Water soluble form of SF extracted from the Thai domestic silk cocoons (Nangnoi Srisaket 1) had similar characteristics to those extracted from Chinese, or Japanese cocoons. However the Thai SF showed slightly higher hydrophobic amino acid contents. The SF was fabricated into microspheres without or with the blending of gelatin (G) at the various ratios of SF: G from 90:10 to 50:50. The microspheres were crosslinked in either glutaraldehyde or in dehydrothermal condition. The average sizes of the microspheres ranged from 400 – 1,100 microns and the water absorption ranged from 120 – 460% (by wt.). Surfaces of the microspheres showed mild negatively to neutral charges. Half-life of their in vitro biodegradability ranged from 0.8 to >14 days. Breast cancer cells (MCF-7) could attach on surfaces of the microspheres at the highest density of 55,000 cells/mg or 70% of the original seeded cells. The cells proliferated to 78,000–110,000 cells/mg within 14 days of culture. The population doubling time of the cells was in the range of 108–228 hours. The cells cultured on microspheres were exposed to an anti-cancer drug, Tamoxifen. The IC50 of the drug was 0.72 micromolar (cells on microspheres), compared to 0.12 micromolar if the cells were cultured on a 2D system. plates.
Laboratorio de Cirugía Experimental, Instituto Nacional de Pediatria, Mexico City, MEXICO.
Bioengineering, University of California, San Diego, La Jolla, CA.
3D printing or additive manufacturing is a method that deposits the materials such as metals, ceramics and polymers to fabricate the complex 3D structures by layer-by-layer. Fused deposition modeling (FDM) is a common method and widely applicate in the medical region. This printer extrudes the materials on the stage through heating block for fabricated the 3D complex structure. FDM type has advantages that is solvent free and used in biocompatible and biodegradable polymers widely. Poly (L-lactide-co-ɛ-caprolactone) (PLCL) is a synthetic biodegradable and biocompatible copolymer and has high elasticity for soft tissue engineering. Self-assembling peptide (SAP) hydrogel offers 3D environment supporting cell attachment and differentiation of cells. Substance P (SP) is an 11-amino acid neuropeptide and recruit CD29+ stromal-like cells for wound healing. Here, we developed PLCL/SAP-SP finger structure by 3D printing for skin regeneration. It is checked morphology by SEM, and porosity and mechanical properties also. This finger structure scaffold implanted in mouse for 1 and 4 weeks with human dermal fibroblast. We stained the scaffold for histology after weeks. We expect that this process can be applied to regeneration of skin for each patient owing to customized structure.
A Comparative Study Of Cortical And Cancellous Bone Grafts From Pigs
Pearce, A. I., et al. “Animal models for implant biomaterial research in bone: a review.” Eur Cell Mater 13,1, 2007.
Development Of Thin Transparent Collagen Film For Corneal Wound Repair
Liu, Yang, et al. “Preparation and characterization of a novel tobramycin-containing antibacterial collagen film for corneal tissue engineering.” Acta biomaterialia 10.1 (2014): 289–299.
Novel Synthesis of Oligo Polyethelyene Glycol Fumarate Biodegradable Hydrogels
1. Kinard LA. Nat Protoc 2012.
Plasma Immersion Ion Implantation Treatment of Silk Biomaterials Enhances Biological Function
Graduate School of Biomedical Engineering, University of New South Wales, Sydney, AUSTRALIA.
Regenerated silk is a pure biocompatible, biodegradable protein purified from silk worm cocoons that can be engineered into an unprecedented range of material formats with robust, tunable mechanical properties. However, B. mori silk has no inherent cell-interactive properties and often requires biofunctionalization to achieve appropriate mammalian biological responses in vitro and in vivo. Plasma immersion ion implantation (PIII) is a process in which positively charged ions from a gas discharge are accelerated through a high voltage sheath and implanted into the surface of the material being modified. In this study, silk was modified with PIII to generate a carbon-rich interface. The study aims to investigate if the PIII functionalized silk support biological functionalization and cell growth. Surface Plasmon Resonance (SPR) analysis with sodium dodecyl sulphate (SDS) washing demonstrated covalent binding of proteins to PIII-treated silk biomaterials. PIII-treated silk biomaterials were biofunctionalized with proteins and ELISA analysis using antibodies against different protein domains showed similar presentation to that on untreated silk. PIII-treated silk biomaterials also supported improved cell interactions compared to untreated silk, even in the absence of biofunctionalization. Endothelial cells seeded on PIII-treated silk biomaterials showed significantly higher adhesion compared to untreated silk. Unlike untreated silk, cells adhered to PIII-treated silk as early as 5 min post-seeding. PIII-treated silk biomaterials showed enhanced biological functions. These new biomaterials are promising new class of bioactive materials for use in tissue engineering and regenerative medicine, as well as diagnostic, industrial and high technology applications.
Investigation of Collagen Maturity and Organization in Multimerized Connective Tissue Growth Factor siRNA Nanocomplex Treated Wound
Hybrid Hydrogel Containing Decellularized Aortic Valve Leaflets Promotes Myofibroblastic Differentiation
Tissue engineering heart valves may provide a solution to the lack of growth potential of current bioprosthetic valve replacements for valvular heart disease by providing a suitable environment for cellular remodeling. Previous studies showed differentiation of mesenchymal stem cells towards different lineages when cultured on solubilized ECM. However, these hydrogels show severe compaction, and it is unknown if the solubilized matrix has a beneficial effect for TEHV applications. We are investigating the role of solubilized acellular valve matrix on human adipose-derived MSC (HADMSC) and human aortic valve interstitial cell (HAVIC) differentiation and maintenance. Porcine valves were decellularized and solubilized into a hydrogel (dECM). Varying concentrations of dECM was mixed with methacrylated hyaluronic acid (MeHA) with HADMSC or HAVIC encapsulated, crosslinked under UV, cultured up to 14 days, and assayed for biocompatibility, gel compaction, and phenotype. Live/dead and MTT assays revealed high cell viability for both cells within all dECM concentrations. Increasing dECM concentration showed more cell spreading and minimal compaction for both HAVIC and HADMSC. Both cell types showed a trend of increasing αSMA, calponin, and MMP expression, indicating a transitioning to a myofibroblastic phenotype. The results show that HADMSC behaved similarly to HAVIC, suggesting differentiation towards a myofibroblastic cell type, which is initially needed for valve remodeling. These results may have implications for future TEHV applications by incorporating dECM within a hydrogel system to provide a better environment for HADMSC to differentiate and begin remodeling the matrix.
3D Bioprinting and Non-invasive X-ray Assessment of dECM Hydrogel-PCL Hybrid Constructs for Cartilage Regeneration
Effects of Amnion Chorion Membranes on Tendon Healing
Tendon injuries are painful and debilitating, often leading to significant scarring and loss of function. Amnion/chorion products have been utilized as both adhesion barriers and as an aid in tissue repair; however, limited data is available on the mechanism. The purpose of this study is to investigate the mechanisms of action that may contribute to tendon healing response seen with dehydrated amnion/chorion membranes (DHACM).
To evaluate the growth factor content within these membranes, a quantitative multiplex ELISA was used to quantify concentrations of growth factors relevant to tendon healing in DHACM from 5 donors. Proliferation assays (14 days) of human tenocytes from multiple donors were conducted using conditioned media (CM) from DHACM. Additionally, migration assays were also completed using conditioned media from DHACM in a standard Boyden chamber assay. Proteomic analysis of DHACM showed DHACM contained physiologically relevant levels of growth factors relevant to tendon healing including IGF-1, TGF-β1, PDGF-BB, aFGF and bFGF. DHACM was found to significantly increased tenocyte proliferation (3.34 ± 0.68 fold over control, p ≥ 0.001 n = 12). Additionally, DHACM conditioned media was found to significantly increase tenocyte migration (127.8 ± 96.9% increase over control, p > 0.0001 n = 12).
The results from the study show that DHACM contains a wide array of growth factors and cytokines relevant to tendon repair. Specifically these molecules promote tenocyte migration and proliferation. Work is ongoing to evaluate the role of anti-inflammatory properties of amnion/chorion membrane in tenocyte repair.
ACL Regeneration and Osteointegration using a New Silk Fiber-Based Scaffold: Results From a Study in Sheep
University of Applied Sciences, Technikum Wien, Vienna, AUSTRIA.
New approaches treating ACL injuries, in particular strategies based on tissue engineering have gained research interest. The optimal scaffold for ACL regeneration is biocompatible and biodegradable to allow tissue ingrowth, but also needs to have mechanical properties to provide immediate stability.
Tissue Engineering and Applied Cell Sciences Department, Tehran university of medical science, Tehran, IRAN, ISLAMIC REPUBLIC OF.
Biodegradable polyurethane elastomers have been utilized in fabrication of elastomeric scaffolds for soft tissue repair and regeneration. Polyurethanes have multiple properties that make them potentially suitable scaffolding materials for soft tissues, particularly for dynamic tissues such as cardiovascular [1]. Scaffold should offer appropriate degradation rate in accordance with development and organization of tissues. Our aim was to synthesize polyurethane elastomer with slower degradation behavior. In this study, we have synthesized poly(ester urethane urea) (PEUU) by one-pot method [2]. Non- porous and porous elastomeric scaffolds were then fabricated using casting and solvent casting-salt leaching, respectively. Scaffolds were characterized for mechanical strength, porosity, degradation rate, contact angle, water uptake and biocompatibility. Human bone marrow-derived mesenchymal stem cells (hBMSCs) were isolated and characterized by flow cytometry and osteogenic and adipogenic differentiation. Fabricated scaffold presented long-term degradation rate and supported growth and attachment of hBMSCs and human Umbilical Vein Endothelial Cells (HUVEC) in vitro. This study illustrates the possibility of fabricating non- porous and porous scaffolds from polyurethane synthesized and also suggests potential of these scaffolds in the repair and regeneration of soft tissues such as cardiovascular tissue engineering.
1. William R. Wagner. Non-invasive characterization of polyurethane-based tissue constructs in a rat abdominal repair model using high frequency ultrasound elasticity imaging. Biomaterials 34, 2013.
2. Yeganeh H, Mirzadeh H. Simple and versatile method for the one-pot synthesis of segmented poly (urethane urea) s via in situ-formed AB-type macromonomers. Polymer International. 60, 2011.
Tissue Engineering and Applied Cell Sciences Department, School of Advanced Technologies in Medicine, Tehran, IRAN, ISLAMIC REPUBLIC OF.
Following a myocardial infarction (MI), cardiomyocytes loss is subsequently replaced by non-contractible scar tissue. Myocardial tissue engineering based on appropriate cells and scaffolds is a promising approach to create constructs that mimic myocardial tissue. Here, we have developed a variety of composite scaffolds based on silk fibroin/chitosan/gelatin that mimic the properties of natural myocardial extra-cellular matrix. Silk fibroin was extracted from the bombyx mori silkworm, then porous scaffolds were fabricated using freeze-drying technique and characterized for surface properties, swelling behavior, contact angle, degradation rate and mechanical properties. H9c2 rat cardiomyoblast were cultured into scaffolds and then evaluated for cell adhesion, cell to cell communication, cellular metabolic activity and histological examination. Scanning electron microscopy showed the scaffolds with proper pore sizes, good interconnectivity and porosity which is suitable for cell growth. The addition of gelatin increased water uptake and degradation rate and reduced mechanical strength but silk fibroin affected reversely on the degradation and mechanical strength of composite scaffolds. MTT-assay was demonstrated growth and adhesion of human Umbilical Vein Endothelial Cells (HUVEC) and L929 fibroblast into scaffolds. Differentiation of h9c2 cells into scaffolds was confirmed by stronger positive markers, such as α-actinin and cTnT. This study demonstrated that cardiomyoblast-seeded scaffolds have potential to be used in cardiac tissue engineering.
1. Kundu Sc, Engel FB, Silk protein Fibroin from Antheraea Mylitta for Cardiac Tissue Engineering. Biomaterials 33, 2012.
2. Suhaeri M, Park K. Cardiomyoblast (H9c2) Differentiation on Tunable Extracellular Matrix Microenvironment Tissue Engineering; Part A, 21, 11and 12, 2015.
Bioengineering, University of Pittsburgh, Pittsburgh, PA.
1. Chung, Recent research on the growth plate. J Mol Endocrinol 53,2014.
A principal limitation of drug delivery to the brain is the blood brain barrier, formed by the cooperation of glial and vascular cells in a functional system termed the gliovascular unit GVU. Both in vitro laboratory and animal models fail to recapitulate the properties of the blood brain barrier BBB seen in humans. Through the process of three-dimensional bioprinting, we seek to develop a standardized laboratory model of the human GVU. Isolated human primary astrocytes, pericytes and smooth muscle cells were integrated into fibrin gel microextrusion bioprinting and human brain microvascular endothelial cells were integrated into a dissolvable gelatin for printing of a pre-planned vessel lumen. Printed structures were incubated in stationary media culture at 37°C. On Day 4 and 7, structures were analyzed and/or processed for staining. Haematoxylin and eosin H&E stained sections reveal that they maintained a well-defined lumen on Day 4 with cell growth into the lumen on Day 7. Immunohistochemistry for the astrocyte marker GFAP and endothelial cell marker CD31 revealed defined cellular localization on Day 4 with an endothelial cell-lined lumen and astrocytes in the surrounding region. Viability assessment on Day 4 showed over 90% of cells to be viable. Current studies are geared towards adapting the bioprinted GVU to a dynamic, microfluidic culture environment and assessing the expression of tight-junction markers characteristic of the BBB. We foresee this model to improve our understanding of human neurologic disorders involving the BBB and assist in the development of novel therapeutics for neurodegenerative and neuro-oncologic diseases.
A 3D Submucosal Microenvironment for Investigation of Topography Induced Effects in Colorectal Cancer Cells
Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC.
Recent investigation of colorectal cancer metastasis has identified the tumor microenvironment as a large proponent of metastasis. Factors such as tissue stiffness, fiber alignment and bundling, and cell-cell interactions have been targeted as affecting cancer cell phenotype, proliferation and drug susceptibility. To study these effects in vitro, this study aims to develop a micro-facsimile of colonic submucosal microstructure using cellularized type I collagen (Col I) hydrogels. Constructs are fabricated using rabbit colonic smooth muscle cells (RCSMCs) suspended in a Col I hydrogel. RCSMCs prolifically remodel Col I structure across concentrations and express functional markers associated with smooth muscle. To assess the effects of RCSMC remodeling on cancer cells, we embedded a foci composed of HCT-116 cells (a malignant colorectal cancer cell-line) into the submucosal construct. HCT-116 cells produced mesenchymal phenotypes in Col I hydrogels, but interestingly reverted to an epithelial phenotype within the submucosal construct. We probed this phenomenon by inhibiting RCSMC remodeling capabilities which resulted in mesenchymal expression of HCT-116 cells. In addition, we probed the drug susceptibility of cancer cells grown in either submucosal constructs or bare Col I and found cancer cells were significantly more resistant to chemotherapeutics when grown inside the submucosa. These results indicate the RCSMC might be producing a more “normal” or healthy environment and thus inducing a more normal phenotype from embedded cancer cells. Future directions include probing the biomolecular effects of fiber alignment to understand the mechanism behind this transition.
Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD.
Microfluidic Device For Motility And Biochemical Assessment In Parallel Drug Testing
Biomedical Engineering, Virginia Tech - Wake Forest School of Biomedical Engineering and Sciences, Winston-Salem, NC.
Studying cancer dynamics and testing drugs on cancer models that mimic in vivo tumor structure can aid in developing approaches for treatment of cancer. 3D cell culture recapitulates the tumor microenvironment more accurately than in 2D model and translates to humans better than animal models. Here, we have developed a photopatterning technique through which 3D cell cultures of arbitrary size and shape can be formed in situ in the chambers of an active fluidic device, enabling complex structures to be produced and allowing independent chemical delivery to parallel models. In this study, multiple replicates of in situ 3D cell culture structures containing human colon carcinoma cells (HCT116) are challenged with various concentrations of the chemotherapeutic drug 5-Fluorouracil in a single microfluidic device. Cancer cell migration and viability under the drug influence are studied over 7 days. Our initial goal is to apply this technique to studying the role of WNT signaling pathway in chemotherapeutic drug resistance. Wnt is an important regulatory pathway involved in growth factor signaling, cell proliferation, specification of cell fate, and tumor initiation. In the future, this tool can be expanded to reproduce the complex, multilayer structures of tumors and will be used as a patient-specific test module for in situ assessment of drug dosing to improve treatment design.
1. Loessner, et al. Biomaterials 31(32):8494–506, (2010).
2. Imamura, et al. Oncol Rep 33(4):1837–43, (2015).
3. Lee, et al. Lab Invest 93(5):528–42, (2013).
4. Cooksey, et al. Lab Chip, doi:10.1039/C4LC00173G, (2014).
Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University in Bratislava, Bratislava, SLOVAKIA.
Periodontal ligaments represent promising source of adult stem cells, which have great potential for tissue engineering. In case of therapeutic application, it is essential to isolate and culture stem cells in vitro to obtain their sufficient quantity. However, pro-longed cultivation provides adequate amount of cells it has been shown that this approach is associated with increased risk of transformation of cultured cells. In this study we isolated stem cells from periodontal ligaments of normal human impacted third molars. They were expanded in vitro up to 30th passage and analyzed for morphological and biological changes. Obtained results showed first significant changes in morphology in 20th passage, cell became bigger and contained endosomes in their cytoplasm. The expression of surface markers, proliferation rate and telomerase activity decreased in the same time. The analysis of Bcl-2 expression showed lowest values after 25 passages while the expression of TP53 was highest. In conclusion, our data indicate that prolonged in vitro expansion of periodontal ligaments-derived stem cells resulted not only in significant changes of morphological characteristics but also affect the proliferation kinetics and cell cycle. On the other hand, there is low tumorigenic potential mainly due to maintaining the normal function of cell cycle and apoptosis regulators as well as lack of telomerase activity. However, further studies focused on stem cells bio-safety have to be carried out prior their clinical application. Supported by grant MZSR no. 2012/4-UKBA-4.
Bioengineering, UCSD, La Jolla, CA.
Cancer cells within a tumor are very heterogeneous, and only a small fraction are able to form secondary tumors. Universal biological markers that clearly identify these cells are limited; we propose that physical properties of the microenvironment, i.e. cell-matrix adhesion, may be differentially regulated in metastasizing versus non-metastasizing cancer cells and could be used as a prognostic indicator. We assessed cell-matrix adhesion strength of metastatic mammary and prostate cancer cells in the presence or absence of cation concentrations that mimic the stromal microenvironment. Metastatic cells exhibit remarkable attachment strength heterogeneity only in stromal-like conditions, unlike their non-metastatic counterparts who exhibit stronger, cation-insensitive attachment. This heterogeneity is the result of increased sensitivity to cation-mediated focal adhesion disassembly in metastatic cells. Less cation-sensitive metastatic cells are less migratory and invasive, similar to non-metastatic cell lines whereas non-shear selected populations maintain an aggressive, highly migratory subpopulation. Attachment strength heterogeneity was observed across multiple cancer cell lines (mammary and prostate) as well as isogenicially with H-Ras conversion of a non-metastatic line, suggesting that weak attachment strength in stromal-like niche may serve as a general marker of highly metastatic cells.
Histology and Embryology Department, Selçuk University, Konya, TURKEY.
Breast cancer is the most common cancer in women. The highly metastatic breast cancer cells are usually difficult to combat with and they usually lead to casualties. Therefore, it may be of value to have a system for early detection of this type of cells. It was aimed to prepare a quartz microbalance biosensor to detect highly metastatic cells, namely MDA-MB 231 cells by targeting their transferrin receptors. The gold surface of the QCM biosensor chip was functionalized by the attachment of transferrin coated Poly(2-hydroxyethyl methacrylate) (pHEMA) nanoparticles. Nanoparticles were characterized by zeta sizer analysis. Nanoparticles without transferrin on their surface were used as control. The QCM sensor systems were analysed by using atomic force microscope (AFM), elypsometer and FTIR-ATR. It was seen that the nanoparticles formed a monolayer over the surface. Following characterization studies the sensors prepared with or without transferrin were tested for their ability to detect invasive breast cancer cells. The binding affinity and kinetics were determined. Adsorbtion kinetics were found to obey Langmuir- Freundlich model. The biosensor was tested with MCF 7 non invasive breast cancer cells and MDA MB 231 cells at prolonged serum deprivation in order to test selectivity. It is concluded that, this biosensor is highly sensitive and selective for vital MDA-MB 231 metastatic human breast cancer cells. Thus, this system may be used to test blood samples for early detection of circulating metastatic breast cancer cells.
Laboratory for Stem Cells and Tissue Engineering, Department of Biomedical Engineering, Columbia University, New York, NY.
Multidrug resistance (MDR) and adverse toxic side effects of chemodrugs are the major challenges to cancer chemotherapy. Herein, we design and fabricate a pH and protease dual responsive mesoporous silica nanoparticles (MSNs) for reversing MDR and reducing systemic toxicity by intracellular delivery of doxorubicin (DOX), whose release can be triggered by low pH and lysosomal proteases. Sericin, a natural protein from cocoon silk fibers, is selected to coat MSNs as a gatekeeper via pH sensitive imine linkages. The sericin coating acts as an excellent gatekeeper against the leakage of encapsulated DOX, enhances MSN cellular uptake via endocytosis by cancer cells, promotes DOX release through the breakage of pH sensitive bonds between sericin and MSNs and the proteolysis of sericin by lysosomal proteases. The sericin coated MSNs (SMSNs) have excellent cytocompatibility, hemocompatibility as well as immunocompatibility. Importantly, the SMSN treatment in MDR breast tumor-bearing mice significantly enhances the therapeutic efficacy of DOX and inhibits tumor growth with drastically reduced toxicity to the major organs (heart, liver and kidneys). Together, the pH/protease dual responsive SMSNs may be a promising delivery system for overcoming multidrug resistance and minimizing chemodrugs' systemic toxicity.
Bioengineering an Improved 3D Culture Model of Human Breast Cancer Cells on Silk Scaffold for Multi-drug Resistance (MDR) Evaluation
Multidrug resistance (MDR) is one of major obstacles to improve the outcomes of chemotherapy in tumor patients. However, limited progresses have been achieved to overcome this phenomenon effectively due to our poor understanding of its underlying mechanism. This is mainly caused by the severely shortage of suitable culture models that could highly mimic MDR of tumor cells in vitro. To address this issue, a more pathologically relevant, three-dimensional (3D) culture system of human breast cancer was developed by inoculating adriamycin-resistant human breast cancer cell line MCF-7/ADM on silk scaffold with collagen incorporation. Distinct growth profile of MCF-7/ADM cells was observed in the 3D cultures. The presence of silk scaffold not only promoted MDR cell proliferation but also induced cellular aggregate formation. A significantly increased expression of drug resistance-related genes and proteins was also detected out in the 3D cultures. Most importantly, a remarkably enhanced activity of drug resistance was found in the 3D cultures of MCF-7/ADM, evidenced by their increased IC50 values to some anti-cancer drugs and improved drug efflux activity. These might be partly explained by their altered cell cycle distribution and improved percentage of breast cancer stem cell (BCSC)-like cells analyzed by flowcytometry. Thus, this in vitro 3D culture of breast cancer cells represent an improved pathologically relevant 3D microenvironment for MDR cancer cells, providing a robust tool to explore the mechanism of MDR and its reverse in those drug resistant cancer cells.
3D Honey Embedded Silk Fibroin Mussel Inspired Aqueous Adhesive Therapeutic Patch for Oral Mucosal Pre-cancer/ Cancer Therapy
Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, INDIA.
In oral and maxillofacial surgeries, closure of surgical wound for functional restoration and regenerative healing after surgical resection of malignant lesion is still a big challenge. There are two ways to close the wound, stitching of two flaps together with sutures or using tissue adhesives. It has been great challenge to create aqueous based tissue adhesive patches that can function fast and controllable on wet internal tissue with better adhesion. In this context, taking inspiration from mussel associated protein 3,4 hydroxyphenylalanine (DOPA), silk fibroin (SF) backbone functionalized with dopamine. To balance out the hydrophobicity of SF, it is blended with honey of different concentrations. Using soft lithography 3D honey/silk fibroin flexible therapeutic patches have been fabricated and physico-chemically characterized. The adhesive bonding of fabricated patches were also investigated, where it was found dopamine conjugates have increased adhesive strength in compare to silk fibroin lacking catechol functionalization. In-vivo bio compatibility and adhesion on wet tissue were tested and confirmed through trials on cutaneous wound on wistar rat models and found fast wound healing with membranes incorporated with honey (2%). Later, membranes were tested with normal, pre-cancer and cancer oral cells and found differential selective behaviour of cells. Proliferation of normal cells was favoured whereas diseased cells were inhibited on same substrate which is further validated by molecular gene expression analysis. Thus, these findings results into a standalone therapeutic patch, which when attached over an open wound can lead to faster wound healing with minimized probability of cancer/pre-cancer recurrence.
Orthopaedics, Massachusetts General Hospital, Boston, MA.
Cancer caused about 25% of all deaths in the United States in 2014. In vitro cancer models are widely used in cancer biology study, the pharmaceutical industry and chemosensitivity tests for patients. Presently, 2D and simple 3D culture models are mostly used and they usually unable to recapitulate the tumor microenvironment, which leads to poor predictions of the in vivo drug effects. Using 3D bioprinting with live cells from the established cell lines or primary cells from the patients and extracellular matrix, we report success in reconstructing the tumor microenvironments and generating the in vitro 3D tumor models which resemble the characteristics of the native tumors. We characterized the 3D bioprinted tumors in many aspects and the bioprinted tumors show the following characteristics: high viability during the relatively long term culture, morphology comparable to the native tumors, similar proliferation pattern to the in vivo tumors, invasion behavior, metastasis behavior, proangiogenesis behavior and more drug resistance. When grafting, the rate and degree of tumorigenicity were also improved comparing with conventional grafting methods. Our 3D bioprinted in vitro tumor models should provide a versatile platform for cancer biology study, cancer drug development and selecting specific sensitive drugs for an individual tumor patient.
Vascular Cell Response and Release Profile for Carbon Monoxide Releasing Molecules Incorporated within Electrospun Scaffolds
Biomedical Engineering, Florida Institute of Technology, Melbourne, FL.
Delivery of the gasotransmitter carbon monoxide (CO) has the potential to improve endothelial function in small-diameter vascular grafts. CO is a cell-signaling molecule produced naturally in the body, and can exhibit vasorelaxant and anti-inflammatory effects with appropriate doses. We have previously demonstrated that CO can be controllably released through incorporation of an organic, photo-activated CO releasing molecule (CORM). However, a limited 1h time frame for CORM-loaded scaffold activation was observed under cell culture conditions. In this study, our goal is to incorporate a more hydrophobic material into our scaffolds to extend the maximum incubation time. Both the previous anthracene-based diketone (control) and an aliphatic chain modified diketone were synthesized. Poly(ɛ-caparolactone) with 0–2% w/w CORMs were electrospun. Scaffolds were activated with 470nm light and the extent of CO release tracked through the generated fluorescence signal. For the previous CORM, rat smooth muscle cells were better spread without CORM-loading, but endothelial cells exhibited a higher cell density on CORM-loaded scaffolds with activation (phalloidin; n = 2–3). Interestingly, these cell-type differences in response to similar CO doses were also suggested in a Motterlini et al. review article. With CORM activation, endothelial cells both proliferated and increased vWF expression (immunofluorescence) from 3–7 days. We also have preliminary release profiles with the more hydrophobic CORM. Importantly, this CORM-loaded scaffold maintains the ability to be activated and release CO after an extended culture period (43.9 and 33.4% of dry release for 1 and 24h incubation, respectively; n = 3). We are currently replicating these results and studying the cellular response.
Biomedical Engineering, Illinois Institute of Technology, Chicago, IL.
Successful repair and return of function following intestinal injury as a result of disease or surgical manipulation can be enhanced or disrupted by the intestinal microbiota. Common treatment involves antibiotic administration which has been shown to disrupt intestinal microbial composition leading to further healing impairment. We have shown that under phosphate deprived conditions, pathogenic intestinal microbes produce high levels of collagenase, a key phenotype involved in impaired intestinal healing. Strategies that allow for targeted delivery of phosphates to the intestinal epithelium while allowing commensal bacteria to proliferate normally have a significant advantage in addressing this problem. We have developed a novel drug delivery approach involving the use of hydrogel nanoparticles that allow for prolonged release of phosphates to be delivered to the intestinal epithelium. Polyphosphate (PPi), was encapsulated in poly(ethylene) glycol diacrylate hydrogel nanoparticles (NP-PPi) formed using inverse miniemulsion polymerization. NP-PPi have an average particle diameter, zeta potential and swelling ratio of 181.7 nm ±52.2 nm, −17.92 mV ±1.05 mV and 2.8 ± 0.08, respectively, and allow for sustained release of PPi over three days. Free PPi reduces collagenase levels in gram-negative pathogens (P. aeruginosa and S. marcescens) while maintaining bacterial survival. Preliminary in vitro studies illustrate that NP-PPi suppressed collagenase levels of S. marcescens. Current studies are being performed to test the effectiveness of NP-PPi in suppressing collagenase levels across gram-positive and gram-negative pathogens.
University of Utah, Salt lake city, UT.
Despite improvements in biomaterials and surgical approaches, non-union of segmental bone defects still remains a clinical problem. We present a bioreactor for repair of segmental bone defects consisting of three main components: a biodegradable poly-l-lactic acid (PLLA) scaffold seeded with adipose stem cells (ASCs), a drug reservoir made of PLLA and a controlled drug diffusion system for sustained release of bone morphogenic protein (BMP-2). We hypothesize that this device can release BMP-2 from the reservoir at a concentration that allows for the differentiation of ASCs into osteogenic cells on the 3-D scaffold. The reservoir and 3-D porous scaffold were manufactured with PLLA and ASCs loaded onto the scaffold after assembling the device. Fluorescently labeled dextran, similar diffusion coefficient to BMP-2, was used to examine device release kinetics. Differentiation of ASCs was determined by evaluating gene expression by RT-PCR and deposition of mineral by alizarin red staining following exposure to BMP-2. Dextran was released in a sustained manner from the device for 30 days. BMP-2 at a concentration of 100ng/mL was able to differentiate ASCs as measured by increased expression of Runx-2 and ALK. ASCs loaded onto the scaffold and exposed to 100ng/mL of BMP-2 exhibited increased calcification compared to cells exposed to growth media and no scaffold. Thus the ASCs loaded onto our PLLA scaffold and exposed to BMP-2 enhanced differentiation of the ASCs into osteogenic cells. Further testing will be done to demonstrate that directly delivering BMP-2 from our reservoir to the ASC seeded scaffold enhances bone regeneration.
Renal disease is a worldwide health issue. Besides transplantation, current therapies revolve around dialysis, which is limited to delaying disease progression through filtering metabolic wastes in blood. However, dialysis is unable to replace other renal functions, such as synthesizing erythropoietin. To address these limitations, cell-based approaches have been proposed to restore damaged kidneys as an alternative to current therapies. Particularly, recent studies have shown that stem cell-derived secretomes could enhance tissue regeneration. This study explores a gel-based delivery system for controlled delivery of trophic factors secreted from human placental stem cells (hPSC) (conditioned medium: CM) and evaluates the effect of trophic factors on renal regeneration. In vitro cell viability and proliferation assays demonstrated that CM treatment significantly enhanced cell proliferation when compared with the control without CM. Platelet-rich plasma (PRP) was used as a delivery vehicle for CM. To test the feasibility of controlled delivery, CM was encapsulated within the PRP, followed by assessing the release kinetics of CM from the gel. The release profiles show that CM can be released from PRP in a controlled manner by altering gel stiffness. An In vivo study using a rat acute kidney injury model showed that CM delivery using the gel system into the injured kidney tissue facilitated less renal tissue damage, leading to rapid functional recovery than that of saline, CM or vehicle only group. These results suggest that the delivery of hPSC-derived trophic factors in a controlled manner may contribute to efficient kidney repair from renal tissue injury.
“Adipose tissue retention via controlled drug delivery microsphere system”
Adipose tissue is essential in reconstructive surgeries, since current materials for soft tissue reconstruction suffer from shortcomings such as suboptimal volume retention or poor biocompatibility. The aim of this study was to examine a controlled delivery system of a potent adipogenic factor, dexamethasone (Dex), to generate stable adipose tissue when mixed with disaggregated human fat in a nude mouse model over 6 months. Dexamethasone was encapsulated in poly (lactic-co-glycolic acid) (PLGA) single walled microspheres also in PLGA and polylactic acid (PLLA) double walled microspheres. Single walled and double walled polymer dexamethasone microspheres were tested separately and combined for the purpose of achieving adipose tissue retention. A high dose group, 50 mg of Dex MS and a low dose group of 27 mg of Dex MS from both microspheres were tested and compared with a group that contained empty microspheres and lipoaspirate only. Three combinations of single and double walled microspheres were used with 3:1, 1:1 and 1:3 ratios with the respect of single walled microspheres. Fat was extracted from animals and followed by volume measurements and histology. Dexamethasone microsphere-containing samples demonstrated greater adipose tissue retention compared to controls. Histological analysis, including H&E and CD31, indicated increased vascularization within the Dex MS-containing samples. Controlled delivery of the adipogenic factor dexamethasone via polymer microspheres can significantly improve retention of adipose tissue, maintaining healthy tissue formation and vascularization, and represents a clinically relevant therapeutic strategy of adipose regeneration.
Slow Release of miRNAs from Injectable Decellularized Extracellular Matrix Hydrogels
Bioengineering, University of California, San Diego, La Jolla, CA.
3B's Research Group, University of Minho, Barco, Guimarães, PORTUGAL.
Therapeutic deep eutectic systems (THEDES) are here proposed as a new class of pharmaceutical active ingredient (API) whose distinctive characteristic is the fact that they are liquid at room temperature. Eutectic systems have been described in the pharmaceutical sciences as an alternative formulation able to enhance bioavailability of the API. THEDES are constituted by one active agent and a coformer or two active agents, which when combined at a particular molar ratio become liquid at room temperature. In this work, we explore the prepararation of the THEDES menthol-ibuprofen. The solubility and dissolution profile in phosphate buffer solution (pH 7.4) was performed at 37°C and compared with those of the pure API. Furthermore, the in vitro permeability of the liquid THEDES and the API in powder form, was evaluated using a Franz diffusion cell and caco-2 cells as model of human intestinal epithelium. The solubility of the API's when in the THEDES system improved as much as up to 12 fold. Furthermore, for this system the permeability was calculated to be 14 × 10−5 cm/s representing a 3 fold increase in comparison with the pure API. The results indicate that both solubilization and permeability were greatly enhanced when the API is complexed in the THEDES liquid form. The results obtained demonstrate the potential of THEDES to overcome the drawbacks of poorly water soluble molecules and increase bioavailability of the APIs and the possibility to develop new delivery systems.
Gene delivery is a fundamental strategy to regulate gene expression across therapeutic and research applications in regenerative medicine. Classic gene delivery strategies utilize plasmid DNA (pDNA) to deliver the gene of interest. However, these methods are not ideal for in vivo applications, because of risks of insertional mutagenesis and low non-viral transfer efficiency of pDNA to non-mitotic populations. Non-viral delivery of in vitro-transcribed mRNA is safe and has high transfection efficiency, but is limited by short-lived timeframes of desired gene upregulation - on the order of hours. Here we present a biomaterials-based approach, whereby biomimetic mineral coatings on microparticles (MCMs) are designed for both efficient non-viral transfection and stable protein delivery. MCMs delivering mRNA encoding for basic fibroblast growth factor (bFGF) resulted in a two-fold increase in cell proliferation and 50% increase in expansion of primary human dermal fibroblasts compared to delivery of mRNA without MCMs. Additionally, the proliferation increase persisted beyond 48 hours, significantly longer than the period of bFGF expression. Lastly, the increase in cell expansion and proliferation with MCM-mediated delivery of mRNA was higher or equivalent to pDNA delivery, despite the overall lower expression of bFGF. Our materials-based mRNA delivery strategy leverages the advantages of higher non-viral efficiency in non-mitotic cell populations with an improved safety profile compared to viral delivery, and potentiates a longer and more robust biological response to a therapeutically relevant cytokine.
Development Of A Controlled Drug Release System Of Cripto For Muscular Dystrophies Therapy
Muscular dystrophies (MDs) are inherited disorders that manifest as progressive muscle wasting and weakness1. Cripto, an extracellular protein, has been recently found by our collaborator, Prof. Minchiotti (IGB Naples, Italy), to have therapeutic value in alleviating muscle injury and diseases by regulating muscle regeneration and satellite cell progression toward the myogenic lineage2. In this study, we establish a highly reliable methodology for producing large quantities of the Cripto protein and further clarify the strategy in which we can encapsulate the Cripto into the microgel carrier for the purpose of controlled release. PEG-fibrinogen (PF) hydrogels provide the substrate for protein production as well as the controlled release in situ delivery vehicle. For efficient production of Cripto, PF microcarriers were designed to enable mammalian cell survival, proliferation, and secretion of large amounts of therapeutic proteins. Cripto overexpressing HEK293T cell lines were encapsulated in the PF microcarriers and cultivated in stirred suspension bioreactors. Subsequently, a PF micro-scale delivery system was developed for intramuscular injection to provide stability as well as immunoprotection for the Cripto. Preliminary studies of the in vitro controlled release of Cripto from the micro-scale delivery system shows a biphasic release profile, characterized by an initial burst release followed by a sustained release phase.
1. Mercuri E, Muntoni F. Lancet 381:845–860, 2013.
2. Guardiola O et al. Proc Natl Acad Sci USA 109:E3231-E32,2012.
Sequential Release of Dual Payloads using Acoustically-Responsive Scaffolds
The Effect of Fibronectin, Collagen, and Ethanol on human Mesenchymal Stem Cell Adhesion, Proliferation and Migration.
1. Frantz, C., Stewart, K. M., & Weaver, V. M. (2010). The extracellular matrix at a glance. Journal of Cell Science, 123(24), 4195–4200. http://jcs.biologists.org/content/joces/123/24/4195.full.pdf
Behavior of Cells Embedded in Fibrin Beads
Researchers need better in vitro wound healing models to solve chronic wound issues. Current wound healing models utilize only one type of cell and the cells were coated outside of the microbeads. In addition, fabrication techniques involved heat and potentially harmful solvents. In this study, we have created a fibrin bead fabrication technique that does not require any harmful solvents or heat. Fibroblasts and keratinocytes are encapsulated within the fibrin microbeads so that researchers can track migration and proliferation of these cells in 2-D and 3-D in novel wound healing models for up to 1 week. Three different models for fibrin analysis were created and examined: fibrin beads containing cells of either keratinocytes or fibroblasts on a cell culture plate, beads on a layer of mixed fibrinogen and thrombin, and beads between two layers of fibrinogen and thrombin. The results for cell migration and proliferation showed that each cell behaves differently on certain surfaces. Fibroblasts and keratinocytes migrate and proliferate best on a plastic surface. Both cells migrate slowest inside a 3D fibrin construct because cells can migrate in all radial directions at random, resulting in overall slower movement. Fibrin beads create tunable microenvironments to sustain and optimize the performance of each cell type implanted into a wound-healing model. These microbeads can be implanted into a more realistic wound-healing model which will lead to greater understanding in the field as well as using cells for treatment by delivering cells in beads.
A Novel Dual Layered Biodegradable Scaffold System Through Electrospinning For The Simultaneous Delivery Of Hydrophilic And Hydrophobic Drugs For Application In Bone Tissue Engineering
Electrospinning has emerged as a versatile, cost effective and reliable technique for fabrication of micro/nano fibres and particles with a strict regulation of solution parameters. However, there is minimal evidence till date of any successful dual structured particle on fibre “Sandwich” system from electrospinning that provides controlled release of two or more different drugs. In this study we have successfully fabricated a dual structured electro-spun fibrous- micro particle system through electrospinning for delivery of a hydrophilic and a hydrophobic drug simultaneously. A blend of two FDA approved biodegradable polymers was used as the electrospinning solution. Preliminary work focused on optimization of the processing parameters to arrive at 5–8μm porous particles that were electro-sprayed on a fibrous mesh network of fibres. The particles were protected by another fibrous mesh on the top to prevent its loss during post fabrication, thereby the entire system resembling a sandwich. Characterization of the scaffold was carried out using Scanning Electron Microscopy, Rheometer and Fourier transform Infrared Spectroscopy. The hydrophobic Dexamethasone was loaded into the fibres while Epigallocatechin gallate - a green tea extract was the hydrophilic drug in the particles. The release profiles of drugs were quantified using High Performance Liquid Chromatography which enabled the detection of Dex at 246 nm and EGCG at 280 nm respectively. Future studies are directed towards understanding the influence of the carrier morphology, the efficacy of the proposed system and the synergistic effect of these drugs on the differentiation capabilities of mesenchymal stem cells via standard bone induction assays.
Novel Thermal-UV Responsive Hydrogel for Bioprinting and Tissue engineering
Characterization of a Gelatin-Hyaluronic Acid Scaffold for Dressing Wound Healing
1. O'Brien, 2011.
Improvement And Modulation Of Mechanical Properties Of Hydrogel From Ventricular Extracellular Matrix By Carbodiimide Crosslinker
Graduate School of Science and Engineering, Yamagata University, Yonezawa, JAPAN.
[1] AJ. Engler et al. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell 126, 677, 2006.
Biomedical Engineering, University of Michigan, Ann Arbor, MI.
Breast tumor cell-extracellular matrix (ECM) interactions contribute to malignant transformation, invasion, metastasis, and treatment resistance of breast cancer cells. Runt-related transcription factor 2 (RunX2) expression has been linked to metastatic behavior and poor clinical outcomes. As changes in matrix composition and stiffness are often associated with breast cancer aggression, there is an urgent need to better understand this interaction in physiologically relevant three-dimensional (3D) models in vitro. Therefore, our objective was to investigate how RUNX2 expression and activation are modulated by microenvironmental factors. We recently created a 3D, polyethylene glycol (PEG) hydrogel system containing type I collagen, Arg-Gly-Asp (RGD) and hyaluronic acid, whose stiffness can be tuned independent of the number of ECM components incorporated. RUNX2 activity was evaluated in mammary epithelial cells, MCF10A, and tumorigenic breast cell lines MDA-MB-231 (invasive) and MCF7 (non-invasive) in our biosynthetic hydrogel system compared to Matrigel controls. The synthetic system supported polarization of the cells and formation of acinar structures by MCF10A and MCF7s over a 23-day period in vitro, as verified by beta4 and beta-catenin staining. Quantification of RUNX2 phosphorylation within cell nuclei showed that this transcription factor is active during formation of acini and that activity levels were higher in MCF7 cells even though there was no difference in total RUNX2 expression compared to MCF10As. This novel system represents a reproducible framework where tumor cell behavior may be quantified as a function of a complex system in which combined microenvironmental parameters, including matrix stiffness, ECM composition, and their interactions, are systematically varied.
Adult stem cell research group, BioMediTech, University of Tampere, Tampere, FINLAND.
Polybutylene succinate (PBS) is an aliphatic biodegradable polyester with different processability and mechanical properties compared to polylactides (PLA)s, which are the most commonly used synthetic polymers in tissue engineering (TE). Since only few studies have evaluated PBS-containing materials for bone TE, we prepared PLA-PBS blends and analyzed material properties as well as cell attachment, proliferation and osteogenic differentiation of human adipose stem cells (hASCs) on scaffolds. In addition to PLA, PBS and PLA-PBS blends, PLA-polycaprolactone (PCL) and PLA-poly(trimethylene carbonate) (PTMC) blends were tested as reference materials. Polymer fibers were prepared using the melt spinning procedure. Knitted and rolled scaffolds (height 5mm, diameter 10mm) were manufactured, seeded with hASCs and cultured for up to 27 days in osteogenic medium. Cell spreading inside the scaffolds was evaluated by microcomputed tomography. Moreover, hASC viability, attachment and proliferation in the scaffolds were determined. The osteogenic differentiation was evaluated with alkaline phosphatase (ALP) activity and mineralization assays. All the scaffolds had open pores and porosity of 67.5 ± 6.0%, and cells were distributed evenly throughout the scaffolds. All the scaffolds supported hASC viability but cell spreading along the fibers was only detected in PBS-containing scaffolds. They also induced the strongest proliferative response and osteogenic differentiation. This was most evident with pure PBS and diminished with decreasing PBS content. On PLA, PLA-PCL and PLA-PTMC the osteogenesis was negligible. Based on these results, PBS is superior to PLA with respect to hASC attachment, proliferation and osteogenesis. This encourages to utilize PBS-based biomaterials more widely in bone TE applications.
Departamento de Biología Celular y Tisular, Universidad Nacional Autónoma de México, Mexico city, MEXICO.
Li G, Zhang X, Wang H, Wang X, Meng C, Chan C, Wai DT, Sze K, Li K, Tsai S, Ngai S, Chao Z, Lin MC, He M, Kung H. Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow, umbilical cord, and placenta: Implication in the migration. Proteomics 9, 20, 2009.
Biomimetic Surface Modification of PLLA Scaffolds for Bone Tissue Engineering
The ability to seed and culture in vitro adult stem cells on 3D scaffolds presents unique challenges due to the inert nature of commonly used polymeric or ceramic biomaterials. Mimicking the natural microenvironment of the target tissue can be of great benefit to improve the seeding efficiency and the differentiation of adult stem cells. The choice of scaffold used to support cells in culture plays a significant role in cell viability and Ex Vivo tissue development. Scaffold properties, such as rate of degradation, hardness, and biocompatibility must be manipulated to match desirable tissue properties and the rate of tissue growth to scaffold degradation. Surface modification primarily addresses the interface that the cells directly interact with, coating underlying material with undesirable properties (such as extreme hydrophilicity or hydrophobicity). Unmodified poly(L-lactic acid) (PLLA) scaffolds provide consistent cellular proliferation for both 2D and 3D MSC cultures, but low initial cell attachment rates result in excessive biomass loss. By attaching the peptide chain Arg-Gly-Asp (RGD), which is known to facilitate cell adhesion, to the surface of a 3D PLLA scaffold we are able to significantly increase MSC seeding efficiency and potentially improve cell physiology without compromising the mechanical and degradation properties of the underlying PLLA. The physical entrapment method of surface modification of PLLA scaffolds results in thorough coverage on both 2D films and 3D scaffolds. Furthermore, in vacuum conditions, this modification resists degradation up to four weeks, making this an ideal method for improving material surface expression for clinical use.
Enzymatically Crosslinked Silk-Hyaluronic Acid Hydrogels
Hydrogels are polymeric networks that can be utilized as scaffolds for a variety of biomedical applications. Previously, we developed a mechanically tunable elastomeric biomaterial through enzymatically crosslinking the tyrosines in silk fibroin. In the present work, we sought to extend these studies to address the need to reduce gel stiffening due to crystallization over time and to add cell-signaling features. Thus, composite hydrogels containing silk and hyaluronic acid (HA) were pursued. HA is hydrophilic and plays an important role in many biological functions. The main objective was to characterize the properties silk-HA hydrogel composites. Characterization techniques included determining the sol-gel transition, gelation kinetics, crosslinking efficiency, mechanics, secondary structure, and swelling behavior over time. The results showed that increasing HA concentration decreased gelation time and increased water-retention. Overall, this study demonstrated that hydrogel characteristics can be modulated by altering the two polymer concentrations. These composite hydrogels provide a tunable, versatile platform for a wide range of biomedical applications including cell encapsulation, tissue regeneration, and tissue augmentation.
Heparin-mimicking Sulfonated Hydrogel Promotes Osteogenesis Of Mesenchymal Stem Cells By Sequestering And Stabilizing Bone Morphogenetic Protein-2
Bioengineering, UCLA, Los Angeles, CA.
Heparin possesses a binding domain to a number of proteins and was often used to immobilize bone morphogenetic protein-2 (BMP-2) onto biomaterials for a controlled delivery. However, heparin suffers from natural variability in structure, difficult modification, and many non-targeting bioactivities and unknown physiological roles. In this work, we developed a hydrogel surface that can mimic heparin to stabilize BMP-2 and enhance osteogenesis by incorporating two heparin-mimicking polysulfonates, Poly-vinylsulfonic acid (PVSA) and Poly-4-styrenesulfonic acid (PSS) into photo-crosslinkable hydrogel already developed in our previously study using methacrylated glycol chitosan (MeGC) and riboflavin initiators. Bioactivity of BMP-2 was well retained under therapeutically relevant harsh environments in the presence of polysulfonates. The incorporation of polysulfonates into MeGC hydrogels did not significantly change the mechanical properties of hydrogels and supported proliferation of encapsulated cells with high cell viability. Sulfonated MeGC hydrogels significantly increased osteogenic differentiation of encapsulated bone marrow stromal cells (BMSCs) as observed by increased mineralization as well as upregulated osteogenic gene markers compared to unmodified hydrogel. This is mostly due to the sulfonated hydrogel surface that wequestered and augmented endogenous BMP-2 activity as validated by immunostaining for BMP-2. Moreover, sulfonated hydrogels supported sustained release of encapsulated BMP-2 with reduced initial burst compared to unmodified MeGC. In addition, the sulfonated hydrogels loaded with BMP-2 significantly increased osteogenesis of BMSCs compared with unmodified MeGC. These findings suggest promising hydrogel platform that can stabilize and augment BMP-2 activity to enhance osteogenesis for bone regenerative medicine.
Academician E.N. Meshalkin State Research Institute of Circulation Pathology, Novosibirsk, RUSSIAN FEDERATION.
Among all the suggested methods of tissue processing a biopolymer treatment is used widely. Chitosan is a biopolymer with variable linear molecular structure and physical properties. But the properties of non-linear chitosan isoforms studied very poorly. In this research we developed a method of bovine jugular veins conditioning using high-deacetylation degree (>93%) nanostructured chitosan (NC) as a way to prevent mineralization and improve biomechanical properties of material. NC is a chemically processed high-purity chitosan in form of nano-sized balls with a diameter of 20–200 nm. Samples treated by glutaraldehyde or epoxy solutions or decellularized followed by NC-treatment in ultrasound bath. For mineralization evaluation a tissue samples implanted subcutaneously in Wistar male rats for 1 month, then explanted and subjected to histological and atomic absorption analysis. Decellularized NC-treated samples showed same results as glutaraldehyde-fixed-only material on a tensile-strength-at-break test. But no mineral clusters were noted under histological analysis in all NC-treated samples (incl. GA-fixed). That was reaffirmed by atomic absorption spectroscopy showing calcium concentration under determining threshold (ω < 0,1%) in them. Although glutaraldehyde-treated tissues are widely used today, these materials have a number of practical limitations, due to mineralization and toxicity of glutaraldehyde. Chitosan is a natural biodegradable non-toxic biopolymer that has similar physical action on vein tissues properties. Nanostructured chitosan processing may be a perspective method of xenograft preparation preventing graft calcification and preserving biomechanical properties of an original tissue.
Fibrous Bundled Shaped Scaffold Fabricated By Phase-Separated Polymer Solution
In regenerative medicine, various methods to construct physiologically relevant tissues have been widely studied, and mimicking tissue microenvironment from both chemical and mechanical point of views have been considered to be very promising to alter cellular and tissue functions. For example, neuron and muscle tissue show fibrous hierarchical structures, therefore, appropriate tissue scaffold to promote regeneration and better guiding of cells have been anticipated. Here, we aimed to develop a method to fabricate biomimetic bundle-structured gel fibers and evaluate their potential to use as cell culture scaffold. To generate bundled gel fiber, we used phase-separated aqueous polymer blend solutions consisting of hydroxypropyl cellulose: HPC and sodium alginate: Na-Alg. The phase-separated polymer networks were stretched by applying shear stress within the microfluidic device, and were immediately fixed by cross-linking Alg with calcium ions in the co-flow region. We thus obtained bundled gel fibers and non-bundled gel fibers with long lengths and extremely high aspect ratios. The fiber bundles were 200∼400 μm in a diameter and consisted of approximately 102∼104 small fiber that were 1∼3 μm in a diameter. Fibroblast cells were aligned parallel to the microfibres, forming a cylinder-like structure along the fiber bundle while cells on the non-bundled gel fiber randomly spread. In addition, the gel fiber shows unique characteristics such as tunable microstructures, stiffness, and thermo-responsiveness which are useful for controlling microenvironment of cells and tissue. Therefore, we believe this bundled gel fiber should be a great candidate as a tissue scaffold.
A New Synthetic Matrix With Bimodal Pore-structure Provides Favorable Characteristics As A Transfer Vehicle For Adipose Derived Stem Cells And Fibroblasts
Dpt. of Surgery, University of Texas Medical Branch / Shriners Hospital for Children, Galveston, TX.
Biomedical Engineering Department, Northwestern University, Evanston, IL.
Thrombosis within small blood vessels is one of the major challenges when using decellularized tissues as biological scaffold materials for tissue or regenerative engineering. Here we describe an easy to implement approach to reduce the thrombogenicity without deleterious effects on the mechanical properties and ultrastructure of the extracellular matrix (ECM). Specifically, a collagen-binding peptide (CBP, CQDSETRTFY) is covalently linked to heparin using carbodiimide chemistry to synthesize collagen-binding heparin (CB-Hep). An inactive sequence of CBP (CBPi, CDEFQRSTTY) was used as a control. CBP (but not CBPi) binds specifically to collagen I and IV, with a higher binding affinity to collagen IV. The molar ratio and heparin bioactivity for both peptide-heparin conjugates were similar (1.60 ± 0.22 CBP/heparin vs. 1.77 ± 0.05 CBPi/heparin, p = 0.7929) (anti-Factor Xa activity 36.4 ± 5.7 U/mg in CBP-Heparin vs. 31.6 ± 3.1 U/mg in CBPi-heparin, p = 0.4056). ECM modified with CBP-heparin (but not CBPi-heparin) exhibited significantly reduced platelet adhesion (by over 70%) and whole blood clotting (by over 80%). Heparinization of the ECM via CBP also stabilizes endothelial cell adhesion on the lumen of vascular ECM, improving the long-term efficiency of re-endothelialization. Overall, CB-hep is a simple yet effective method to prevent thrombosis in decellularized tissue ECM, overcoming a significant challenge in tissue engineering of bioartificial organs.
Tuning the Properties of Injectable Hybrid Hyperbranched POEGMA hydrogels by Controlling the Degree of Branching in the Polymer Precursors
Fabrication Of 3d Aligned Gelatin Methacryloyl (gelma) Scaffold
School of Materials Science & Engineering, Nanyang Technological University, Singapore, SINGAPORE.
Cells cultured on 2-Dimensional (2D) and 3-Dimensional (3D) micro-environment platforms often display drastic differences in terms of cell shape, growth, morphogenesis, motility and differentiation. Henceforth, to better mimic the 3D in-vivo micro-environment, a novel electrospinning counter-electrode jig has been designed to enable the fabrication of 3D aligned fibrous scaffold. A rotating bath electrospinning jig was used to fabricate 3D aligned scaffold. The rotating shaft embedded in the bath aided the alignment of scaffold while the collection bath provided a stable platform for the formation of porous 3D scaffold. 20% w/v GelMA in 50:50 ratio of dimethylformamide and water was used as the electrospinning solution. 0.05 wt% of Pluronic® F-108 was added in bath solution and all scaffolds were photocrosslinked using (Irgacure 2959). 2D and 3D GelMA scaffolds were collected and characterized for imaging and mechanical properties. Briefly, the rotating bath electrospinning process enabled the fabrication of enhanced porous 3D GelMA scaffold as compared to the conventional electrospinning process which produced 2D GelMA scaffold. The tensile modulus was found to be higher for the 3D scaffold with better fibre diameter distribution and enhanced porosity as compared to the 2D counterpart. In summary, the rotating bath electrospinning jig has demonstrated as a good viability to produce 3D aligned scaffold. The aligned scaffold exhibited enhanced mechanical properties with better fibre diameter distribution as compared to the 2D counterpart. The novel 3D fibrous scaffold fabrication technique has demonstrated enhance porosity hence a promising alternative to any 2D fibrous scaffold for tissue engineering applications.
For decades, cancer biologists have relied on two-dimensional monolayer cell culture platforms and xenografts to investigate the mechanisms of cancer. Using these complementary systems, researchers have gained improved understanding of cancer biology and have developed many efficacious anti-cancer treatments. However, both monolayer cultures and xenografts have inherent limitations. Although studies based on animal models predict more pathologically relevant outcomes, the presence of many uncontrollable variables associated with these models makes it challenging to determine the impact of specific factors on tumour progression or to identify the therapeutic efficacies of novel personalized medicine. In this study we have used a hydrogel system that better mimics the topography and mechanical properties of the breast tissue. Further, we have incorporated specific components of the breast ECM, gelatin and fibronectin, thus recreating aspects of the native tissue microenvironment in-vitro to study breast cancer metastasis. Our data showed that these hydrogels have mechanical properties (Young's modulus, elasticity, pore-size) that are comparable to that of breast tissue. Culture of breast cancer cell lines MDA-MB-231 and MCF-7 resulted in spheroid formation. A comparative gene expression analysis revealed that cells grown in the hydrogels expressed increased levels of genes implicated in the events of metastatic progression. Hydrogel-cultured cells also showed increased invasiveness and spheroid formation efficiency in-vitro, and increased metastatic potential in-vivo. Thus, culturing breast cancer cells in 3D hydrogels that mimic the in-vivo tumour-like microenvironment enhances their metastatic potential. This system could serve as a comprehensive in-vitro model to investigate the manifold mechanisms of breast cancer metastasis.
Synthesis, Physicochemical and Mechanical Characterization of Small Intestinal Submucosa-Chitosan Tridimensional Scaffolds for Deep Wound Regeneration
Current deep wound healing solutions reduce injury size and promote scaring, but do not enhance regeneration. We propose Small Intestinal Submucosa(SIS)-Chitosan tridimensional scaffolds as a new material for tissue regeneration. Adding chitosan to SIS scaffolds could guide skin repair avoiding infections, and enhance their biostability and mechanical properties. We aimed to synthesize SIS-Chitosan scaffolds and evaluate Chitosan and crosslinking agent (CLA) concentrations effect on scaffolds properties. Scaffolds were fabricated using low and high levels of Chitosan and CLA: glutaraldehyde (GA) and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC) (n = 3). Functional groups were identified by Fourier Transformed Infrared Spectroscopy. Microstructure was assessed by Scanning Electron Microscopy (SEM) and gravimetric analysis. Influence of temperature on collagen and Chitosan denaturation was determined by thermogravimetry. In-vitro swelling and degradation tests were performed. Compression test was made to study compressive Modulus (CM). Collagen and Chitosan functional groups indicated successful scaffolds fabrication. SEM revealed porous microstructure, adequate for cell infiltration. Composite crosslinked scaffolds showed increased surface area and pore sizes. Porosity of all formulations was 90–96%. Thermogravimetry confirmed collagen and chitosan decomposition temperatures, varying with increasing CLA concentration. Chitosan addition led to decreased swelling; EDC scaffolds showed the higher water retention. Degradation of SIS-Chitosan scaffolds was lower than only SIS scaffolds; uncrosslinked scaffolds presented the higher weight loss. Incorporation of Chitosan into SIS scaffolds indicated a significant improvement in CM, as when varying EDC to GA. To conclude, physicochemical and mechanical properties of SIS scaffolds for deep wound healing could be increased by adding Chitosan and controlling CLA type and concentration.
Growth of Human Embryonic Stem Cells on Electrospun PLGA Scaffolds Coated with Vitronectin or Matrigel
Hematology and Stem Cell Laboratory, Faculty of Pharmacy, Federal University of Rio Grande do Sul, Porto Alegre, BRAZIL.
Human embryonic stem cells (hESC) can propagate indefinitely in vitro under appropriate conditions. The use of proteins, such as vitronectin or laminin, promote adhesion and maintain the pluripotency of ES in monolayer cultures. The growth of ES in tridimensional scaffolds could facilitate cell engraftment and potentially be used as a first step prior to the application of specific differentiation protocols for regenerative medicine. The aim of this study has been to evaluate the capacity of different poly(lactic-co-glycolic acid) (PLGA) scaffolds to support the growth of human ES in vitro. Scaffolds made of 30% PLGA in THF/DMF (7:3) were produced by electrospinning and treated with 0.25M NaOH. hESC (H9) were cultivated in vitronectin-coated plates. 5 × 104 cells were plated on NaOH-treated, NaOH-treated + vitronectin or NaOH-treated + matrigel scaffolds. After 1, 7 and 14 days, the viability was analyzed by the Wst-8 method. Viability was accessed using fluorescein diacetate and propidium iodide. Electrospun fibers with an average diameter of 2.9 ± 0.31μm were obtained. The viability increased in all the groups from day 1 to 14 except on the NaOH-treated scaffolds. The maximum increase was observed on the tissue culture plate. Microscopy analysis revealed that the cells formed round colonies in the NaOH-treated group and bigger and more spread colonies in the protein-coated scaffolds. No apparent differences in the number of dead cells were observed. In conclusion, H9 cells were able to grow in vitronectin and matrigel coated-PLGA scaffolds.
Biomedical Engineering, Universidad de los Andes, Bogotá D.C., COLOMBIA.
Hydrogels have been used in different medical applications due to their high hydrophilicity and soft structure. Nevertheless, body tissues have a wide range of physicochemical properties. We propose a Small Intestinal Submucosa and Chitosan (SIS/Ch) hydrogel as a new copolymer for tissue engineering (TE) applications. This hydrogel could promote hemostasis, angiogenesis and tissue regeneration. The objective of this study was to design a SIS/Ch material, using glutaraldehyde (GA) and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as crosslinking agents (CLA), with tunable physicochemical properties. SIS-Ch hydrogels (n = 3) were fabricated using high and low Ch and CLA concentrations. Functional groups were studied by Fourier Transformed Infrared Spectroscopy (FTIR). Characteristic decomposition patterns were assessed by thermogravimetry. Morphology was determined by Scanning Electron Microscopy (SEM). Swelling and degradation tests were performed. Rheology analysis included: flow, amplitude sweep and oscillation tests. Collagen structural stability and chitosan addition was confirmed by their FTIR and decomposition patterns, suggesting the production of a functional SIS/Ch hydrogel. SEM results showed that increasing CLA led to higher network density. Obtained porous and interconnected microstructure is optimal for cell infiltration. Although GA caused lower water uptake, hydrogels absorbed large quantities of water (30–120%), suggesting that all formulations could be used for bioactive molecules encapsulation maintaining a moisturizing environment that enhances regeneration. Degradation time was doubled by Ch and GA addition. Rheology evidenced hydrogels injectability and solid-like behavior; adding Ch and increasing CLA concentration improved network stability. In conclusion, we designed a new SIS-Ch copolymer hydrogel with tunable physicochemical properties for TE applications.
Department of Plastic Surgery, The Ohio State University, Columbus, OH.
The use of poly(propylene fumarate) (PPF) in tissue engineering dates back 20 years. However, only recently have we developed adequate process control of PPF synthesis and additive manufacturing (AM). In this work, a new synthesis method for PPF is combined with an additive manufacturing process to achieve fabrication of highly complex scaffolds possessing controlled chemical properties, porous architecture, and external geometry derived from patient data to optimize scaffold efficacy. A ring-opening polymerization of maleic anhydride and propylene oxide (coupled with a post-polymerization isomerization reaction) was used to produce large, highly-controlled batches of PPF. Low molecular mass oligomers, ranging from 700 to 3000 Da, were produced with narrow mass distributions (< 1.6). A resin suitable for 3D printing via photocrosslinking was formed by dissolving PPF into diethyl fumarate (DEF) in a 1:1 ratio. Photoinitiators, Irgacure 784 and Irgacure 819, and light absorber, oxybenzone, were are added to enable and control the crosslinking reaction. Biocompatibility of 3D printed PPF was confirmed by cytotoxicity testing with L929 mouse fibroblasts and human mesenchymal stem cells according to ISO Standard 10993-5. Scaffolds were manufactured with two polymer molecular weights (1500 Da, 2450 Da) and two architecture styles (200 μm, 400 μm struts). Degradation was assessed in an accelerated in vitro environment with a 0.1M NaOH solution at 37 deg C. Over 30 days, molecular weight (p < 0.001) and strut size (p < 0.05) were both shown to have significant effects on scaffold degradation rate.
Tissue Engineering Group, Pharmacy Department, Universidad Nacional de Colombia, Bogotá D.C, COLOMBIA.
Indolfi L. Biomaterials. 2012;33(29):7019–7027.
Cellular and Tissue Biology, Medicine School, Universidad Nacional Autónoma de México, Mexico, MEXICO.
1. Heris K., Rahmat Meysam, Mongeau Luc. Characterization of a Hierarchical Network of Hyaluronic Acid/Gelatin Composite for use as a smart Injectable Biomaterial. Macromol. Biosci. 12(2012):202–210.
Materials Science Program, University of Wisconsin-Madison, Madison, WI.
Le et al. Acta Biomaterialia 34, 93, 2015.
Molecular Biology, Instituto Potosino de Investigación Científica y Tecnológica, A.C. (IPICyT), San Luis Potosi, MEXICO.
Alginate and gelatin are two of the most common materials used as printable hydrogels. On the other hand, carbon nanotubes (CNTs) have unique physicochemical-mechanical properties with large surface area that introduces a surface roughness that is present in human extracellular matrix. Mixing CNTs with alginate/gelatin blend, or focal adhesion peptide modified alginate, can create bioprintable materials with tunable properties and broad applications ranging from tissue regeneration to cancer cell migration in 3D environments. Two types of composites were prepared: 1) alginate, gelatin and nitrogen-doped CNTs (CNx) hydrogel, and 2) oxidized alginate covalently crosslinked with CNx and mixed with gelatin solution. MDA-231 and IMR-90 cells were separately blended in the hydrogels before printed by an extrusion-based printer. The samples were analyzed mechanically, physically, and biologically. FT-IR spectrum confirmed the presence of carboxyl groups (∼1,700 cm−1) in both alginate and CNx, the presence of primary amines (at 3,295 cm−1) in CNx was found. SEM analysis showed a high porosity material, confocal images showed well-distributed cells inside of the hydrogel. Fibroblast IMR-90 showed a metabolism decrease while the concentration of CNx increased; meanwhile the proliferation of MDA cells maintained regardless of the presence of nanomaterials. The alginate-gelatin-CNTs composite hydrogel can enable the ability to tune properties such as the mechanical, electrical, or optical characteristics of the otherwise inert alginate gel.
Preliminar In-Vitro characterization of Small Intestinal Submucosa and Hyaluronic Acid scaffold as an open wound dressing material
Biomedical Engineering Department, Universidad de Los Andes, Bogotá, COLOMBIA.
Open wounds cause about 9.5% of annual deaths in the World and generate soft tissue separation. Typical treatments could reduce wound size and prevent further damage, but are insufficient to promote regeneration. Tissue engineering proposes new treatments based on natural scaffolds based on materials such as Small Intestinal Submucosa (SIS) and hyaluronic acid (HA), given their extracellular matrix-like environment. The aim of this study was to determine the influence of HA and crosslinking agent concentration on physicochemical and biological properties of SIS based scaffolds. SIS-HA scaffolds were prepared using with two concentrations of of SIS and HA. The scaffolds were crosslinked with glutaraldehyde (GA) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimida (EDC). Physicochemical tests included water uptake, bulk porosity, morphology and thermogravimetry. For biological evaluation kidney fibroblasts were cultured during 1, 3 and 6 days to study cell viability, adhesion and proliferation. Adhered cells and scaffolds structure were identified using hematoxylin-eosin staining (H&E) and scanning electron microscopy (SEM). Water uptake ranged from 3000–5000%, suitable for a healing environment. Furthermore, scaffolds presented bulk porosity over 90%. EDC crosslinked scaffolds had better water uptake and porosity than GA scaffolds. All samples showed a porous and interconnected microstructure (pore size: 30–60 um), optimal for cell infiltration. Thermogravimetric analysis confirmed collagen triple helix integrity. H&E test showed cell adhesion after 1, 3 and 6 days of culture. SIS-HA scaffolds revealed a higher cell viability than only SIS based. We conclude that SIS-HA scaffolds have improved physicochemical and biological properties and could be used as a wound dressing material.
In tissue engineering, hydrogels provide structural support and signaling molecules to adipose-derived stem cells for targeted tissue differentiation. With increased bioactivity observed in biologically-derived scaffolds, and mechanical tunability gained through synthetic reagents, a functionalized adipose-derived extracellular matrix (adECM) and a multifunctional acrylate were used to synthesize a hybrid hydrogel that was evaluated for its effect on morphology and maintenance of pluripotency in human adipose-derived stem cells (hASCs). Human adipose tissue was decellularized and enzymatically digested. It was then thiolated before dissolution in a 4-arm Poly(ethylene glycol) (4-arm PEGA) solution. The base-catalyzed hydrogel polymerization occurred upon the addition of Ethoxylated-Trimethylolpropane Tri(3-Mercaptopropionate) (ETTMP) and sodium hydroxide. Compressive testing, swelling and mass loss studies were performed to define the mechanical, chemical, and degradation properties of the hybrid scaffolds. Cells seeded on the hybrid scaffolds were cultured in stromal media (SM) and maintained viability for 14 days. Expression of hASCs' pluripotency markers (Nanog and Sox2) were quantified using qRT-PCR and morphology of hASCs was assessed via F-actin staining. Cell organization and the presence of spindle-shaped cells increased substantially with hydrogel adECM content at 7 and 14 days. In addition, significant Nanog and Sox2 expression was observed in hybrid hydrogels. With increasing adECM concentration, hydrogel crosslinking density and compressive strength decreased, while an increased rate of mass loss was noted. This study demonstrated the ability of adECM hybrid hydrogels to maintain pluripotency and regulate proliferation and morphology in hASCs. Future research will employ signaling molecules and controllable mechanical stability to guide a specific differentiation pathway.
Modification of Acellular Liver Scaffolds to Improve Maturation of iPS-hepatocytes in Co-culture
Enhanced Function Of Insulin Producing Cells Derived From Mouse Embryonic Stem Cells In 3D Engineered Tissues
Biomedical Engineering, New Jersey Institute of Technology, Newark, NJ.
Pluripotent stem cell-derived insulin producing cells (IPCs) have emerged as a promising cell source for treatment of type I diabetes. However, there are challenges in maintaining long-term viability and function of IPCs post-differentiation in in vitro culture. In this study, we investigated the effects of three-dimensional (3D) tissue culture of IPCs derived from mouse embryonic stem cells. 3D engineered tissues were successfully created by embedding IPCs in Type I collagen. While the expression of insulin and beta cell specific genes by IPCs were maintained in 3D engineered tissues, the viability decreased gradually with longer time in static culture. To determine whether perfusion flow allows long-term culture of IPC embedded 3D engineered tissues in vitro, a continuous flow was applied using a custom-built flow bioreactor system. The culture of IPCs in 3D engineered tissues under flow led to better survival of cells, especially under low flow rate of 0.02 ml/min. The culture of IPCs in 3D engineered tissues under flow also resulted in enhanced glucose responsiveness and upregulation of key beta cell marker genes compared to that of static culture revealing that flow can further enhance the function of IPCs post-differentiation. Overall, this study demonstrates the feasibility and benefits of culturing stem cell-derived IPCs in 3D tissue environment combined with perfusion flow in vitro.
Chemical, Electronics and Biomedical Engineering, UNIVERSIDAD DE GUANAJUATO, LEON, MEXICO.
The manufacture of tubular constructs for esophageal tissue engineering is focused on mimic the composition, structure and properties of the esophagus. Previous reports indicate that the tubular constructs derived from decellularized hollow conduit tissues provide chemical, physical and biological signals to support cell responses. This work reports the decellularization of the submucosal layers of small intestine (SIS) and esophagus (ES) by perfusion of anionic detergents; sodium dodecyl sulphate (SDS) or sodium deoxycholate (SD), and the relationship between the tissue source (piglets and adult pigs) and the residual composition (genomic DNA, laminin, fibronectin, and sulphated glycosaminoglycans/sGAG) in the decellularized tubular constructs. It reports also a strategy to recoat decellularized constructs derived from piglet esophagus submucosal tissue with collagen gels containing epoxyeicosatrienoic acids (EET) and its performance in a rabbit esophageal substitution. The results indicated a higher decellularization efficacy by using SDS in comparison with SD. The retention of total protein, fibronectin, laminin and sGAG in piglet tissues decellularized with SD is higher in comparison with the ones decellularized with SDS. The residual composition in decellularized ES obtained from piglets and adult pigs is not significantly different. The recoating of decellularized ES with collagen gel + EET appears to induce the neovascularization around of the implant after 3 days of implantation in the cervical esophageal portion. Results suggest that the modification of decellularized tubular constructs with EET is adequate to enhance the scaffold bioactivity, which deserve further studies to explore the advantages of the present modification in the esophageal tissue engineering.
Cartilage injury represents a leading cause for disability. Previous strategies for cartilage repair employed hydrogels as 3D niche for cell delivery due to its injectable nature to fill defects of any shape. However, conventional hydrogels are inherently mechanically weak and often lacks macroporosity that is desirable for cell proliferation and new matrix deposition. Our research group has recently developed a method to fabricate hydrogels into microribbon(μRB)-like structures, which support direct cell encapsulation into a macroporous scaffold with cartilage-mimicking shock-absorbing capacity. The goal of this study is develop methods for fabricating of various extracellular matrix components into μRB-based hydrogels, and evaluate their potential for supporting mesenchymal stem cell-based chondrogenesis. Using a wet-spinning technique, μRBs were fabricated from methacrylated gelatin, chondroitin sulfate (CS), hyaluronic acid (HA), and polyethylene glycol (PEG). Human mesenchymal stem cells were encapsulated in μRB-based hydrogels with above compositions, and cultured in chondrogenic medium for three weeks. Outcomes were evaluated using live/dead staining, biochemical assays, mechanical testing and histology. All μRB compositions supported high MSC viability and enabled neocartilage formation with increased compressive moduli over time. Among all four compositions tested, gelatin-based μRBs led to the highest amount of cell proliferation, collagen deposition, and glycosaminglycan production. Such ECM-based μRBs provide a novel biomaterials platform that enables direct encapsulation of cells in 3D macroporous cell niche with tunable biochemical and mechanical cues. While this study focuses on stem cell chondrogenesis, the biomaterials platform may be broadly applicable for engineering artificial niche to modulate different cell fates or tissue types.
Improving Degradable Biomaterials for Orthopedic Fixation Devices
Bioengineering, Temple University, Philadelphia, PA.
Advanced Convergence Technology, Korea Polytechnic University, Si Heung, KOREA, REPUBLIC OF.
Extracellular matrix (ECM) plays a key role in terms of providing topological cue and biochemical support to the mammalian cells. For this reason, numerous researchers have exploited natural/synthetic hydrogels for providing to cells in the 3D culture environment that mimic in vivo-like tissue/organ instead of human natural ECM due to their extremely complexes characteristics and a variety of components of ECM. To overcome this problem, in this study, we fabricated extracellular molecule-based matrix (EMM) by using proteins which is synthesized by Normal Human dermal fibroblast (NHDF) on nanofibrous membrane and has a similarity of natural ECM in terms of providing topological structure and extracellular components. We cultured NHDF to obtain EMM for 7 days, and then cellular components were completely eliminated from nanofibrous membrane expect the ECM proteins. To assess the characteristics of manipulated EMM, we measured ECM proteins such as collagen, fibronectin, and GAGs and repopulated Normal Human Epidermal Keratinocyte (NHEK) on the EMM for verifying difference of cell functions such as cell attachment/proliferation compared with collagen hydrogel. Our results shown that EMM could serve cell adhesion sites and mechanically strong supports for tissue-engineered human tissue. Thus, our engineered niche has a potential in terms of mimicking in vivo-like ECM by exploiting human cells.
Biomedical Engineering, North Carolina State University, Raleigh, NC.
1. ISO 23317: Implants for surgery - In vitro evaluation for apatite-forming ability of implant materials. 01-10-2012.
2. Oyane et al. J Biomed Mater Res A. 65(2): 188–95, 2003.
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, JAPAN.
For tissue engineering and cell therapy, purification of stem cells is one of important technologies. In many cases, stem cells were labeled by fluorescent and magnetic modified antibody and purified by FACS and MACS system, respectively. In the present study, for label-free capture and collection of target cell, we developed a surface immobilizing an antibody and investigated the capture and release of target cells on it. Polyethylene films were modified by grafting of poly(acrylic acid) (PAAc) and biotin was introduced into the PAAc graft. The desthiobiotin-antibody (anti-mouse CD45 antibody) was immobilized on the film through desthiobiotin-avidin interaction. Mouse bone marrow cells (CD45 positive) and HL60 cells (CD45 negative) were seeded on the films with and without immobilizing CD45 antibody, the selective adhesion of cells was observed. When biotin conjugated water-soluble polymers were used as dissociation agents, the captured cells were completely released, indicating that the dissociation of desthiobiotin-avidin associates occurs due to the different association constant of desthiobiotin and biotin to avidin. From these results, it was suggested that target cells could be selectively captured and collected by using a device having surface immobilizing antibody via desthiobiotin-avidin interaction.
Biomedical Engineering, Drexel University, Philadelphia, PA.
Immediately following the implantation of biomaterials in the body, like sutures, a foreign body response (FBR) commences, which is characterized by recruitment of inflammatory cells, fusion of macrophages into multinucleated giant cells, and formation of a collagenous fibrous capsule surrounding the biomaterial. Analysis of detailed kinetic mechanisms of the FBR has been limited due to the lack of live in vivo analysis tools. Zebrafish (Danio rerio) are an attractive model organism for the study of FBR because they have been used for live exploration of other inflammatory pathologies, including cancer and fibrosis. To confirm and characterize the FBR, polypropylene sutures were implanted into adult wild-type zebrafish, with samples taken for histological analysis (Masson's Trichrome for collagen deposition and L-Plastin immunostaining for leukocytes) at time points ranging from one hour to 21 days post-implantation. Our data showed significantly increasing numbers of L-Plastin+ cells and increased collagen deposition around the suture over time, which are characteristic of a classic FBR. To explore the FBR progression in real-time and potential FBR-modulation, polystyrene beads adsorbed with interleukin-10 (IL10), an anti-inflammatory cytokine, were injected into the dorsal region of Tg(mpx:GFP)(mpeg:mCherry) larval zebrafish. Live confocal microscopy showed a significantly reduced number of neutrophils, with no change in macrophage count, surrounding IL10-adsorbed beads compared to controls after 24 hours, supporting reports from murine models that IL10 inhibits neutrophil recruitment (Penaloza HF et al., Immunology 146(1), 2015). Together, these results suggest that zebrafish exhibit the FBR and may be used to screen novel immunomodulatory biomaterials to reduce FBR.
Investigating Cellular Response of Mesenchymal Stromal Cells to Therapeutic Ultrasound in 3D Scaffold
Radiology and Imaging Sciences, NIH, Bethesda, MD.
Mesenchymal stromal cells (MSC) have been shown to downregulate inflammation or stimulate endogenous stem cell proliferation and thereby provide a promising cell therapy for regenerative medicine. In our study, application of therapeutic ultrasound (TUS) was shown to retain more MSC in the TUS-treated murine muscle after direct transplantation of MSC. However, responses of MSC to TUS in the tissue microenvironment have not yet been investigated. Therefore, we investigated cellular and molecular responses of MSC in 3D alginate scaffold after TUS treatment. MSC-alginate scaffolds were prepared using 1.2% alginate and formed into 6mm diameter discs of 25ul volume consisting of 2.5x105 cells. TUS treatment group showed more initial cell death compared to the control, however, the surviving cells in TUS group showed higher proliferation. After 1–2 weeks of culture in alginate scaffolds, MSC lost their characteristic surface markers, but there was no difference in the ability to differentiate. Cytokine secretion and RT-PCR of cells harvested from alginate scaffolds showed remarkably elevated anti-inflammatory and immunosuppressive markers as well as higher migration ability compared to 2D cultured MSC. TUS treatment in combination with 3D alginate culture seems to enhance those properties further. Our results suggest that 3D culture alone and TUS exposures may enhance potency, migration and immunosuppression ability of MSC, therefore it can be used as a preconditioning method for MSC in cell therapy.
Effect Of TNF-α, LPS And IFN-γ On The Phenotype Of Mesenchymal Stem Cells From Human Placenta
This research was supported by project DGAPA PAPIITIN218315
The Effect of Natural Extract on Hair Loss Improvement
WINNOVA, SEOUL, KOREA, REPUBLIC OF.
Natural substances are extracted by a variety of extraction methods such as hot water extraction, distillation and extraction, organic solvent extraction, and ultrasonic extraction method. Among them, BH-ORGA41 was used the vapor extraction to extract the effective complex such as Eosengcho, licorice, green tea and etc. Extracted solution (BH-ORGA41) showed various functions. Especially, this research is accomplished to study the effect of natural extract on hair lost. The suitability of a cytotoxicity assay based on the MTT method has been evaluated for BH-ORGA41. It showed non-toxic in dermal papilla cells. Dihydrotestosterone(DHT), the primary mediator of prostate growth, is synthesized in target tissues from the circulation androgen testosterone through the action of steroid 5α-reductase. To determine whether this control is mediated by DHT, we investigated the effect of BH-ORGA41 on the expression of 5α-reductase and Bax. Experiments were conducted using a DHT-induced in dermal papilla cells in vitro. The expression of 5α-reductase was lower than in finasteride, positive control and the expression of Bas was lower than in the control group. These results support the hypothesis that BH-ORGA41 prevents DHT-induced hair cell apoptiosis. The effect of BH-ORGA41 on hair growth in 6 week-old C57BL/6 mice was assessed. Hair growth compared to the minoxidil group was faster in the BH-ORGA41. BH-ORGA41 group had 73% hair coverage on the 21st day, while the minoxidil had 38% hair coverage on the 21st day. Hair thickness compared to thecontrol group was greater in the BH-ORGA41. This result suggest that BH-ORGA41 might have prevent hair loss.
Biomedical Engineering, University of Michigan, Ann Arbor, MI.
Over the last decades, the number of young women surviving cancer has increased due to efficient chemo- and/or radio-therapeutic treatments, impairing the patient's reproductive function and causing premature ovarian failure. Therefore, developing a system to provide ovarian endocrine support is of utmost importance. We hypothesize that the use of immunoisolators will support ovarian endocrine function of the implanted tissue while protecting it from immune rejection and eliminating the need for immune suppression. We used PEG-VS hydrogels and commercially available immunoisolating device, TheraCyte, to verify our hypothesis. We encapsulated ovarian pieces from 6–8 days old Balb/c mice in PEG hydrogels and TheraCyte and subcutaneously implanted them on the dorsal side of ovariectomized adult B6CBAF1 mice. Blood was collected at regular time intervals in addition to daily analysis of vaginal cytology. Flow cytometry analysis of antibodies, IgG and IgM produced over time in the serum was analyzed. Following ovariectomy, confirmation of ovarian endocrine cessation was verified by vaginal cytology corresponding to the increase in follicle stimulating hormone (FSH) levels. After implantation of encapsulated ovarian tissue, FSH levels decreased indicating restoration of the Hypothalamus-Pituitary-Gonadal axis. We observed antibodies in sera by day 21 in mice with non-encapsulated ovarian tissue, whereas no antibodies were detected in the sera of encapsulated ovarian tissue even at day 60 of the implantation period. In summary, our results demonstrate that synthetic PEG hydrogels and TheraCyte are able to restore ovarian endocrine function as well as immunoisolate the donor ovarian tissue from the host.
Biomedical Engineering, University of Michigan, Ann Arbor, MI.
Due to increasingly effective chemotherapeutic treatments, the number of children and young women surviving cancer in recent years has risen. Unfortunately the toxicity of these treatments leads to premature ovarian failure (POF) in almost half of survivors. POF results in irregular ovarian endocrine function which impacts pubertal development and overall health. Implantation of ovarian tissue from a donor could restore ovarian function and the physiological balance of gonadotropins and sex hormones. We hypothesized that encapsulation of ovarian tissue in an immunoisolating hydrogel, such as poly(ethylene glycol) vinyl sulfone (PEG-VS) will support ovarian tissue and prevent rejection. For this purpose we tested three different conditions: (1) non-degradable photo-polymerized PEG-VS, (2) degradable PEG-VS crosslinked via Michael-type addition and (3) a dual construct with a degradable core and non-degradable shell; all having similar bulk properties. To determine the survival and the duration of function of the encapsulated graft we implanted constructs subcutaneously on the dorsal side of ovariectomized mice for 7 and 30 days. Blood was collected weekly via the tail vein and daily vaginal cytology was carried out. The non-degradable hydrogel poorly supported graft survival, while the degradable construct was good for survival but would not work for allogeneic applications due to its degradable network. The dual construct was optimal as it supported graft survival and longevity while providing an immunoisolating barrier. This study sets the foundation for applying PEG-based immunoisolating devices to promote endocrine function restoration in ovariectomized mice.
Biomedical Engineering, University of Florida, Gainesville, FL.
The local delivery of immunosuppressive agents could significantly impact the success of islet transplantation for treatment of diabetes. Fingolimod, a clinically-approved sphingosine-1-phosphate receptor agonist, has been found to delay islet rejection in rodent models when delivered systemically [1]. Herein, we engineered a platform for the local delivery of fingolimod by incorporation within a macroporous polydimethylsiloxane (PDMS) scaffold (0.1%-1% w/w) [2]. In vitro release studies quantifying release rates and duration observed sustained release within targeted dosages over >7 d. Fingolimod-PDMS scaffolds containing syngeneic islets (600 IEQ) were subsequently transplanted into diabetic mice. Surprisingly, either delayed or abrogated efficacy was observed when fingolimod doses exceed 0.5%. Histological assessments indicate reduced host cell engraftment and a trend of elevated T cell migration. Mechanistic in vivo and in vitro studies of scaffold implants and islets are on-going, but current results support systemic impacts of FTY, with reduced white blood cell counts, and potential impairment of islet viability and function when locally delivered, despite drug release rates up to 80-fold less than published reports [3–4]. Overall, this study confirmed the ability to modulate local delivery of fingolimod in a sustained-release manner using a 3-D scaffold; however detrimental impacts at the site of islet transplantation was observed in mouse models.
1. Liu L, et al. Microsurgery. 27(4), 300–4, 2007.
2. Weaver, J. D. et al. Tissue Eng. Part A. 21, 2250–2261, 2015.
3. Yang Z, et al. Clinical Immunology. 107, 30–35, 2003.
4. Truong, W. et al. Am. J. Transplant. 7, 2031–2038, 2007.
Institute for Frontier Materials (IFM), Geelong, AUSTRALIA.
Electrically excitable tissues like nerve and muscle have shown promising results in regeneration on conductive scaffolds. In this study a solution of 14% PCL was electrospun on a rotating collector forming aligned nanofibres with the average diameter of 0.69 μm. The fibre mats were dip coated by the conductive polymer PPy (polypyrrole) to form a substrate capable of stimulation of nerve cells. The fibre mats were then made hydrophilic by 1 minute continuous oxygen plasma treatment (50 watt power). The hydrophilicity of the samples were tested in one month after the treatment and the water contact angle remained 0 degree, the same as immediately after the treatment. The conductive scaffolds with 85% porosity delivered 15.7 MPa Young's modulus which is far higher than the native nerve tissue with less than 5 MPa modulus. Fibroblast cells were cultured on the scaffolds and results show that there are significantly more cells attached to the plasma treated fibre mats compared to samples without plasma treatment. PC12 cells along with nerve growth factor, were cultured on the aligned nanofibers for 7 days to evaluate to nerve cell growth on these scaffolds. Formation of neurites in the direction of fibres suggests that the electroactive PCL-PPy scaffold can support the differentiation of PC12 cells into nerve cells.
In Vivo Therapeutics Corporation, Cambridge, MA.
A promising strategy for the treatment of spinal cord injury is human neural stem cell (hNSC) implantation. Typical approaches to deliver hNSCs involve multiple bolus injections of cells caudal and rostral to the injury. This may result in cellular reflux, tissue damage at each injection site, and a lack of immediate cellular connectivity across the area of injury. These limitations may be overcome by using a single injection site to deliver a continuous rostrocaudal trail of hNSCs across the injury. Here, we describe a proof-of-concept device that produces longitudinal cellular transplants in the spinal cord. A stepper-motor driven nitinol needle was introduced from the dorsal surface at a shallow angle for distances up to 4cm, a relevant length for bridging human spinal cord lesions. Trails were deposited by retracting the needle during injection of hNSCs suspended in a viscous carrier. Injections into collagen spinal cord mimics demonstrated reduced cell reflux compared to bolus injections and improved homogeneity when cells where injected in viscous carrier compared to PBS. The device was used to deliver 12 mm long trails in nude rat spinal cords. Immunofluorescence labeling for human cytoplasmic marker STEM121 identified hNSCs with extended processes at 4 weeks post-injection. Finally, the device was used to deliver 4 cm long trails in Gottingen minipigs. A plexus of STEM121-positive cells was detected at one week post-injection. These results demonstrate the preliminary advantages and feasibility of generating bioengineered hNSC trails in spinal cord. Future work will examine the long-term viability, differentiation, and function of the trails.
Tufts University, Medford, MA.
The human brain is a massive network comprised of billions of neurons interconnected through trillions of synapses. In addition to the complexity of the brain, its fragile nature also presents a significant hurdle in our pursuit for understanding the organ. While much of our knowledge surrounding the human brain is the result of animal based in vivo models, there are disadvantages in drawing conclusions between such disparate organisms. With advances in tissue engineering, research has expanded to the use of human stem cells to show neuronal development and organization. The long-term growth and differentiation of neurons derived from human induced pluripotent stem cells (hiPSC) in a 3D framework was demonstrated in porous silk fibroin 3D scaffolds. The hypothesis was that by differentiating the hiPSCs into neurons within a neural-supportive 3D scaffold, a tissue system that more accurately represents the connectivity found in a brain-like environment would be achieved. This model has shown prolonged growth of neurons and astrocytes as well as synaptic formation based on immunohistochemistry and electrophysiological local field recordings. This 3D neuro model can be utilized for the study of network development and plasticity, as well as the progression of disease states by utilizing patient derived hiPSCs.
Institute of Child Health, University College London, London, UNITED KINGDOM.
There is much need for developing 3D (3-dimensional) -systems that can closely mimic human neural tissue behavior and provide reproducible models for studying central nervous system normal and abnormal development and function, responses to putative therapeutic agents, and possibly aid repair in injured or diseased brains. Our aim is to establish a novel 3D-model that can be precisely patterned using human neural stem cells (hNSCs) and a brain-derived matrix. To build this 3D-model, we are using the bio-electrospraying technique which consists of a needle-based delivery system driven by an electric field that can be used to generate cell-laden scaffolds into specific architectures using a variety of matrices. To achieve this we: 1) assessed hNSC survival and their ability to grow and differentiate along neural and glial lineages after bio-electrospraying, and 2) established conditions for the preparation of a suitable brain-derived matrix to generate the scaffold to be used in 3D-systems. To this purpose, we used porcine brain and compared two different protocols for their ability to provide decellularized extracellular matrix (ECM) that maintains biological activity. We show here that bio-electrospraying does not negatively affect either viability or differentiation of hNSCs. We also show that following both decellularization protocols cells were fully removed from the ECM and DNA content significantly reduced. However, one of the protocols used provided a higher yield of ECM than the other and was superior in preserving glycosaminoglycan content. Hence, ECM obtained using this protocol is undergoing further characterization and the feasibility of bio-spraying is being tested.
Repair of the nerves is one of the major challenges in field of regenerative medicine. Development of bioactive scaffolds that provides physical support along with local release of biomolecules required for cell proliferation and differentiation could be an alternative for development of nerve guides for peripheral nerve regeneration. In that effort, biocompatible and biodegradable coaxial Cellulose Acetate (CA) with growth factor core are electrospun to obtain aligned nanofibers. PC12 cells cultured on characterized CA nanofibers showed similar levels of viability and proliferation as that of control substrate (glass). Moreover, the viability of cells cultured on the substrates increased with time up to 7 days indicating active proliferation. Following this, NGF released from the core of the nanofibers aid in differentiation of PC12 cells to check the compatibility of the substrate for neurite growth. Confocal microscopy of differentiated PC12 cells show that CA substrates promote neurite growth. Following this we present the results of the effect of sustained release of NGF from CA based coaxial nanofibers on promoting neurite extension in comparison to burst release of NGF.
Bioengineering Islet Organoids From Expanded Porcine Pancreatic Cells
Microfabricated Immune-isolating Devices For Transplanting Therapeutic Cells In Vivo
MIT, Cambridge, MA.
Cell based therapies has the potential to treat a variety of chronic disease including type 1 diabetes, anemia, liver failure and Parkinson disease. Transplanting engineered or stem cell derived cells that secrete therapeutic factors for long periods of time have been an enduring goal. Implanted cells are often immunogenic, and in absence of immunosuppression are rapidly rejected by the host immune system. Cell encapsulation provides a safer alternative to immunosuppression for implanting foreign cells in vivo. In this work we present a microfabricated cell-encapsulating device having optimized geometry and pore-size, which protects the graft from immune cells while providing an optimal environment for their survival and growth. The devices are flexible allowing them to be well integrated into surrounding tissue. We also developed a chemistry to graft antifibrotic polymers on the device surface resulting in dramatic reduction in foreign body reaction and fibrotic over growth on these devices. Lastly we demonstrate the therapeutic potential for these devices in cure of diabetic mouse using primary rat islets, and using human EPO secreting cells to increase mouse hematocrit.
The cell-cycle inhibitor flavopiridol has been shown to improve recovery from spinal cord injury in animal models. However, the systemic dose of flavopiridol has side-effects and the mechanism of action is not clear. This study aimed to develop a strategy for the local delivery of flavopiridol and investigate its mechanisms of action. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) were used for the sustained delivery of flavopiridol. The spinal cord was right-hemisectioned and NPs were delivered into the injury site. Transparent spinal cord technology was used for the three-dimensional observation of anterograde tracing. The results showed that flavopiridol NPs had a sustained release of up to 3 days in vitro. Flavopiridol NPs significantly decreased inflammatory factor synthesis in astrocytes. The inflammatory factor multiplex immunoassay revealed the systemic cytokine profile. And flavopiridol NPs alleviated the reduction of GM-CSF and the increase of IP-10. In-vivo study also demonstrated that flavopiridol NPs decreased cell-cycle activation, inflammatory expression and glial scarring, and facilitated neuronal survival and regeneration. The cavitation volume was decreased by ∼90%. Administration of flavopiridol NPs also improved the motor recovery of injured animals. These findings demonstrated that local delivery of flavopiridol in PLGA NPs improves recovery from spinal cord injury by systemic regulation of inflammatory cytokines, and inhibition of astrocyte proliferation, migration and inflammatory factor synthesis.
A Microfluidic Platform to Study the Effects of GDNF on Neuronal Axon Entrapment
Department of Biomedical Engineering, Washington University in Saint Louis, Saint Louis, MO.
Peripheral nerve injury (PNI) affects 360,000 Americans annually1. One potential strategy to treat these patients is to enhance axon regeneration by transplanting glial cell line-derived neurotrophic factor (GDNF) overexpressing Schwann Cells (SCs). Unfortunately, we and others have found that constitutive GDNF overexpression results in failure of regenerating axons to extend beyond the GDNF source, a phenomenon termed the “candy-store” effect. Little is known about the mechanism of this axon entrapment in vivo. Here we developed a reproducible in vitro culture platform using a microfluidic device to model axon entrapment and to investigate the mechanism by which GDNF causes axon entrapment. The device is comprised of three culture chambers connected by two sets of microchannels, which prevent cell soma from moving between chambers, but allow neurites to grow between chambers. Different levels of GDNF were applied to each chamber and the resulting neurite extension was measured. We found that, while physiological levels of GDNF promote neurite extension, supra-physiological level of GDNF induced axon entrapment. This indicates that high levels of GDNF can directly impact neurite extension resulting in axon entrapment. This also demonstrates that we have developed a method to model axon entrapment in vitro. This in vitro system can also be used to explore the specific signaling mechanisms by which entrapment occurs.
1. J. Kelsey, Upper extremity disorders (Churchill Livingstone, New York, 1997).
Spinal cord injury (SCI) is a devastating and irreversible event with no effective therapy in clinical practice. However, studies have shown that the administration of curcumin, with potent anti-inflammatory effect, play a role in the repair of SCI. In the present study, we investigate whether combining curcumin, as a polyacetal (PA) nanoconjugate to improve hydrosolubility, with another promising therapy as transplantation of ependymal/ progenitor cells (epSPCs), would produce synergistic effects on locomotor recovery and which mechanisms would be involved. We found that curcumin significantly reduced glial scar volume as it showed lower volume of negative area for glial fibrillary acidic protein (GFAP) and improved axon growth which resulted in an increase of β-tubulin positive fibers, maybe due not only to the antioxidant ability of curcumin but also to the inhibition of Rho/Rock pathway as it is seen in culture neurons by lower phosphorylation of its target protein LIMK1. We also observed a decrease of ED1 expression that could indicate a lesser amount of macrophages in the area and therefore a decrease in the inflammatory component. Caspase 9 was also decreased by curcumin treatment showing reduced apoptosis. All these effects were accompanied by an improved locomotor activity as shown BBB rating. Same results were obtained in the chronic treatment of SCI where the combination therapy showed a synergistic effect between epSPCs and curcumin nanoconjugate with the best results on motor recovery. PA-Curcumin conjugate and epSPCs improves functional recovery following SCI through the enhancement of anti-apoptotic, anti-inflammatory and axonal growth stimulation effects.
Nanoparticle-assisted Stem Cell Delivery in the Eye
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA.
[1] Manuguerra-Gagne R et al., Stem Cells 2013;31(6):1136–48.
[2] Gottanka J, et al., IOVS 2004, 45(1):153–8.
Regeneration Permissive Cues in Lower Vertebrate Retina to Inform Retinal Regenerative Medicine
Chemical Engineering, Northeastern University, Boston, MA.
Evaluation Of Human Epithelial Cells and Keratocytes Growing On A Swine Cornea Scaffold
Argüelles M, 2014 Integracion de queraocitos humanos en estroma corneal desvitalizado de cerdo.
Toward the Defined Xeno-Free Differentiation of Human Protein-Induced Pluripotent Stem Cell-Derived Retinal Progenitors
In-vivo Recellularization of Decellularized Pig Aortic Valves in Sheep
Comparison of Different Ages of Cell Donors for the Production of Tissue Engineered Intestine (TEI)
Center for Perinatal Research, Research Institute at Nationwide Children's Hospital, Columbus, OH.
This study was to develop a new hybrid graft composed of chondrocytes and osteoblasts with similar characteristics to the osteocartilaginous tissue and to probe its potential application as a new strategy for the treatment of OA in humans. The hybrid graft joint was manufactered with autologous stem cells differentiated to cartilage in bioabsorbable polymers made by three-dimensional printing technology for recreating cartilaginous articular surface. The designed construct had similar shape, size and properties to native tissue, which guaranteed to restore the loss of a damaged joint segment in OA. Our group offered a new biological solution to restore joint in young patients doomed to suffer pain and disability function.
Optimizing Animal Models to Examine Cardiovascular Biomaterial Hemocompatibility
Biomedical Engineering, Oregon Health and Science University, Portland, OR.
Bioengineering, Rice University, Houston, TX.
Integra LifeSciences, Plainsboro, NJ.
Chronic wounds affect over 6 million people in the US due to slower wound healing associated with aged skin, impaired blood circulation, diabetes and other comorbidities [1]. Rodent or porcine diabetic models are frequently used to model chronic wounds, such as diabetic ulcers, but these models are unable to fully replicate the clinical complications, especially chronicity of the wound. In this study, we developed a model to address healing impairment that is caused by glycosylation of collagen resulting from extended exposure to hyperglycemia [2]. To rapidly simulate glycosylation of collagen, wounds were treated with glutaraldehyde to crosslink collagen. Eight circular full-thickness wounds (2 cm diameter) were created on the dorsal skin of six Yorkshire pigs (25–40 kg). Wounds were either treated with a weak glutaraldehyde solution for 5 minutes on days 0 and 1 post-wounding or left untreated. By day 14, 93 ± 4% of the untreated wounds had completely reepithelialized. In contrast, the glutaraldehyde treated wounds took until Day 42 to reach the same threshold. Healing was significantly delayed in glutaraldehyde treated wounds at Days 7, 14, 21, 28 (t-test vs. untreated, p < 0.0001) and 35 (p < 0.05). To our knowledge, this study presents the first in vivo evidence that glutaraldehyde crosslinking of dermal tissue can significantly impair wound healing.
1. Wound Repair Regen. 2009;17(6):763–71.
2. J Pediatr Surg. 1990;25(1):75–8.
None.