7 th TERMIS World Congress Seattle,Washington June 25–28,2024

Biofabrication as a Method for Development and Implementation of Clinically Useful Products

Tuesday, June 25, 2024, 10:00 AM - 11:30 AM

5 - Matrix Stiffness Drives Piezo-1 Activation In Three-dimensional Head And Neck Cancer Spheroids And Derived Small Extracellular Vesicles

M. Sheth¹, M. Sharma¹, L. Esfandiari^1,2,3

¹ Biomedical Engineering, University of Cincinnati, Cincinnati, OH,

² Electrical Engineering, University of Cincinnati, Cincinnati, OH,

³ Cincinnati Cancer Center, Cincinnati, OH

*Purpose/Objectives: Extracellular biophysical cues such as matrix stiffness are key stimuli affecting tumor progression in vivo. However, it remains unclear how cells in a 3D tumor microenvironment perceive matrix mechanical stiffness stimuli and translate them into intracellular signals driving cancer. Mechanosensitive piezo1 ion channels, upregulated in many malignancies, mostly of epithelial origin, are major transducers of physical stimuli into biochemical responses. However, most piezo-1 mediated mechanotransduction studies are still limited to dynamic environments or two-dimensional culture conditions. Here, for the first time, we engineered a 3D spheroid culture environment with varying mechanobiological properties to study the effect of static matrix stiffness stimuli on piezo1 activation in oral squamous cell carcinoma (OSCC) tumors. Additionally, the role of small extracellular vesicles (sEVs) in matrix stiffness triggered OSCC invasiveness by means of piezo1 and CD44 signatures from parental spheroids was also studied.

*Methodology: Cal27 spheroids were generated using liquid overlay in sEV-depleted media. Matrix stiffness was defined as soft and stiff for 3 and 12 mg/mL Matrigel, respectively, and characterized by atomic force microscopy (AFM) and shear rheology. Day 14 spheroids were fixed and stained with DAPI, Piezo1-AF594, and CD44-APC and imaged using a Nikon A1R confocal microscope with multichannel z-stacks performed across the height of the spheroid. For flow cytometric analysis, day 14 spheroids were dissociated into single cells and stained with Piezo1 and AF594-secondary or CD44-APC. Flow data were acquired using Cytek Aurora and analyzed using FlowJo. sEVs were extracted from day 14 cell culture media using a lab-developed dielectrophoretic device. Nanoparticle tracking analysis (NTA) was performed using Nanosight NS300. sEVs were single stained with Piezo1-FITC, CD44-APC, and CD63-FITC. Imaging flow cytometry (iFCM) was performed using ImageStream^X Mark II and analyzed using IDEAS. Single sEVs immobilized on EV profiler chips and stained with a mix of CD63-Cy38 and Piezo1-FITC or CD44-APC were imaged using dSTORM with a Nanoimager S Mark II and analyzed using CODI.

*Results: Mechanical characterization of hydrogels indicated a 3-fold increase in stiffness at the intermolecular level and 8-fold increase at the bulk level using AFM and rheology, respectively. Morphological analysis using brightfield and confocal microscopy indicated significant increase in spheroid size with stiffness. Cellular flow cytometry demonstrated significant increase in piezo1 and CD44 expressions with increasing stiffness. NTA indicated a 5-fold increase in particle concentration between soft and stiff cultures. iFCM showed a 3.5- and 2-fold increase in Piezo1 and CD44 sEVs positivity, respectively, from soft to stiff conditions. dSTORM indicated presence of Piezo1 and CD44 in sEVs with increasing positivity with stiffness.

*Conclusion/Significance: Piezo1 is a mechanosensor of static extracellular matrix stiffness, and its activity drives cancer progression in OSCC spheroids. 3D extracellular matrix stiffness also alters sEVs production and composition, and thereby drives cancer progression by employing biomechanical Piezo1 and CD44 signatures from parental spheroids under distinct stiffness stimuli.

6 - 3D Collagen-based Scaffolds For The Investigation Of Extracellular Matrix Changes On Breast Cancer Progression, Chemotherapeutic Response And Gene Therapy In Breast Cancer

E. Sainsbury¹, F. O’Brien^1,2,3, C. Curtin^1,2,3

¹ Royal College of Surgeons Ireland, Dublin, IRELAND,

² Trinity Centre for Biomedical Engineering, Dublin, IRELAND,

³ Advanced Materials and Bioengineering Research Centre, Dublin, IRELAND

*Purpose/Objectives: Current 2D breast cancer (BC) models fail to recapitulate interactions between cancer cells and the extracellular matrix (ECM) as BC progresses, including the effect of an increase in glycosaminoglycan (GAG) deposition, stiffness and metastasis via epithelial-mesenchymal transition (EMT). Current 3D models for BC research are often composed of materials not found in native breast tissue and therefore limit our knowledge of BC behaviour. Additionally, the success of drug discovery and gene therapy targeting BC is hindered by the use of 2D monolayer and misrepresentative 3D cancer models, highlighting the need for the development of representative in vitro 3D models. This project aims to develop a scaffold model composed of breast ECM components, including collagen and GAGs; hyaluronic acid (HyA) and chondroitin sulfate (CS), known to be elevated in tumours, with stiffness comparable to cancerous breast tissue. These scaffolds will enable investigation of the role of ECM changes in BC progression and their potential use as a platform for gene therapy for manipulation of a specific miRNA of interest known to be overexpressed in patients with BC and associated with poor patient prognosis.

*Methodology: Collagen-based scaffolds comprised of varying concentrations of GAGs, HyA or CS, with stiffness comparable to healthy (0.4kPa) and cancerous (1 -5kPa) breast tissue were fabricated based on lab protocols. Scaffold characterisation and biocompatibility were performed using SEM, mechanical testing, metabolic activity, DNA and cytotoxicity assays using triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and MDA-MB-436. The effects of ECM changes on BC progression were investigated by cytokine array, qPCR, and histology. The use of scaffolds as platforms for drug discovery was assessed with chemotherapeutics, in addition to targeted gene therapy for a specific miRNA.

*Results: Collagen-based scaffolds composed of breast ECM components with physiological breast tissue stiffness, healthy-0.5kPa and cancerous-1kPa were successfully fabricated. Scaffolds were highly biocompatible, supported TNBC cell attachment and maintained characteristics associated with each cell line, such as the high migratory capacity of MDA-MB-231 cells and the highly proliferative nature of MDA-MB-436 cells. Changes in ECM composition did not affect cell metabolic activity/proliferation or migration but altered the expression of pro-inflammatory cytokines; an increase in HyA increased the cytokines while an increase in CS decreased them. ECM changes were also found to be involved in EMT, altering epithelial-mesenchymal gene markers with HyA-promoting metastasis-like features. ECM stiffness did not affect metabolic activity/proliferation but increased cell migration and the expression of an aggressive hybrid epithelial-mesenchymal phenotype. Scaffolds were suitable for the assessment of drug response to chemotherapeutics, with ECM stiffness found to alter chemoresistance. Delivery of targeted gene therapy was successful with the transfection of a miRNA inhibitor of interest, resulting in significantly decreased cell viability, migration and increased chemosensitivity as well as impeding the EMT process.

*Conclusion/Significance: This study demonstrates the development of the first physiological in vitro 3D BC models to investigate the role of HyA and CS deposition in TNBC progression. In addition, these scaffolds can act as platforms to investigate chemotherapeutic efficiency and as models for assessing targeted gene therapy.

10 - Matrix-bound Nanovesicles As Epigenetic Modulators Of The Myeloid Cell Phenotype

H. Capella-Monsonis, S. Badylak, G. Hussey

Surgery, University of Pittsburgh, PITTSBURGH, PA

*Purpose/Objectives: The role of extracellular vesicles (EVs) in regenerative medicine is gaining momentum as diagnostic and therapeutic tools development. Matrix-bound nanovesicles (MBV), a recently identified kind of EVs covalently attached to the extracellular matrix (ECM), have recently proven their potent immunomodulatory effect on myeloid cell populations. However, the effect that MBV have on myeloid cells role in adaptive immunity and on their innate memory have not been studied to date.

*Methodology: The capacity of fluorescently tagged porcine UBM-MBV to migrate to the bone marrow in mice was assessed through IVIS imaging after different doses regimes and routes of administration.

In vitro, flow cytometry analysis was performed to identify the different myeloid populations interacting with MBV. Then, the epigenetic changes elicited by MBV on monocyte progenitors and macrophages was investigated by carrying out an ATAC-seq assay on bone marrow-derived macrophages exposed to MBV at different stages of differentiation.

Finally, bone marrow macrophages response to inflammatory challenges was assessed after isolation from mice injected with MBV. Cytokines ELISA, RT-qPCR, total DNA methylation and histone methylation western blot analysis were performed to this end.

*Results: MBV were found to accumulate in the bone marrow of mice after local and systemic injection, where systemic injections showed a higher degree of accumulation. Flow cytometry analysis showed MBV to interact with neutrophils, natural killers and macrophages, all of which presented a high proportion of CD64+ cells, and myeloid progenitors (CD34+CD64+).

ATAC-seq revealed that MBV elicit stable epigenetic changes on myeloid progenitors and macrophages after exposure in vitro. These epigenetic changes modulated the chromatin accessibility to transcription factors such as BATF, JUN, FOS, REL and EHF. Interestingly, this accessibility modulation differed in function of the state of differentiation of macrophages (i.e. progenitors or differentiated macrophages).

In vivo, MBV effects on the response to LPS challenge in vitro (i.e. expression of inflammatory cytokines), were influenced by the route of administration, dose and number of doses, affecting specially the expression of IL-23 and IFNg. Several injections of a mild dose (10⁸ MBV/mouse) showed that MBV treatment increased DNA and histone H3K4 methylation.

*Conclusion/Significance: The present study demonstrates hat MBV interact with myeloid progenitors in the bone marrow and elicit epigenetic changes that affect the response to inflammatory stimuli. Given the source of MBV, the ECM, these findings could partially explain the regenerative potential triggered by decellularized ECM scaffolds. In addition, this study advances our understanding on the mechanisms by which MBV elicit their immunomodulatory effect and can pave the road to the development of future therapies.

12 - Electrospun Fiber Surface Roughness Modulates Macrophage Polarization: Implications For Fibrous Connective Tissue Injury Regeneration

A. Alemifar¹, J. Robinson¹, K. Burnette², H. Tse²

¹ University of Washington, Seattle, WA,

² University of Kansas Medical Center, Kansas City, KS

*Purpose/Objectives: Injuries to fibrous connective tissues suffer from poor healing. Even if the tissue does heal, the resultant tissue often has inferior mechanical properties. Treatment modalities often lead to pro-inflammatory activation of immune cells, precipitating the onset of osteoarthritis. Much research has focused on designing biomaterials for fibrous tissue engineering applications capable of modulating macrophage polarization to control the physiological transition from a pro-inflammatory (M1) state to a pro-regenerative (M2) state. Surface roughness (SR) plays an important role in controlling this response, as a narrow range of SR was seen to promote an M2 phenotype in 2D. Breast and dental implants with increasing SR led to increased inflammation in vivo. However, there have been no studies investigating the role that SR on the fibers of electrospun biomaterials used for tissue engineering plays in controlling macrophage phenotype. The objective of this study is to utilize a tunable, PCL-based electrospun fiber system to study the impact of SR on macrophage polarization. We hypothesize that a material with a SR on the same length-scale as the d-band of collagen (67 nm), the major source of SR on the collagen fiber, will minimize inflammation and promote regeneration.

*Methodology: Electrospinning was used to produce mesh with increasing SR (Ra = 9.6 ± 4.5, 32.9 ± 16.6, 58.9 ± 12.9 nm ). Primary macrophages were isolated from the blood from healthy volunteers (n =3, male, age 20-35). Cells were seeded at 60,000 cells/mesh and allowed to culture for 10 days. Cultured media was collected daily and analyzed using a Luminex assay for four pro- and four anti-inflammatory cytokines. Immunocytochemistry (ICC) was performed on mesh samples at 3 and 10 days, staining for pro- (CD 80) and anti-inflammatory (CD 206) cell surface markers as well as proteins associated with integrin signaling (paxillin and focal adhesion kinase (FAK)). Staining for actin (phalloidin) was used to assess cell morphology, and cell stretching was analyzed in ImageJ. Punches of the same mesh were implanted subcutaneously in MacGreen (GFP-labeled CSFR-1) mice for three days. Immunohistochemistry (IHC) was then utilized to stain for the same markers used for the in vitro studies.

*Results: Mesh with increasing SR were produced without significant differences in diameter or porosity (Figure 1A). Primary macrophages cultured on the high SR group showed a decrease in pro-inflammatory cytokines (Figure 1B) and cell surface markers at three days. Conversely, cells grown on the low SR group showed the opposite. These same results were seen in vivo (Figure 1C).

*Conclusion/Significance: Macrophage polarization can be controlled by changing the SR of implanted biomaterials both in vitro and in vivo, leading to changes in the expression of cytokines. A better understanding of this response and how to control it would allow for the design of biomaterials capable of controlling the polarization of macrophages to promote the regeneration of functional tissue after fibrous connective tissue injuries.

14 - Tailor-made Nanoswitches For Studying Piezo1-mediated Mechanotransduction And Modulation Of Stem Cell Fate

S. P. Teixeira¹, A. Pardo¹, R. L. Reis¹, J. G. Snedeker², M. E. Gomes¹, R. M. Domingues¹

¹ University of Minho, Guimarães, PORTUGAL,

² ETH Zurich, Zurich, SWITZERLAND

*Purpose/Objectives: Understanding tendon healing mechanisms is essential to improve therapeutic interventions, but despite recent discoveries, much of its molecular and cellular mechanisms remain unknown, particularly regarding cell and tissue responses to mechanical forces. PIEZO mechanoreceptors are being increasingly recognized to play critical roles in tendon mechanobiology, including regulation of cell mechanotransduction processes governing tissue biomechanics. However, their activation mechanism and downstream cell signaling are still not completely understood, mainly due to the current lack of effective tools to probe its multiple mechanotransduction effects. In this work, we explored molecular imprinting concepts combined with magnetic systems to develop new tailor-made nanoswitches enabling targeted wireless actuation on PIEZO1 in different cells relevant for tendon physiology, or with potential for regenerative applications.

*Methodology: Two different epitopes from functionally relevant domains of PIEZO1 topology (one from the Cap and one from the Blade) were rationally selected in silico by protein fingerprint analysis and used as templates for synthesis of molecularly imprinted nanoparticles (MINPs) by a solid phase imprinting method. Highly superparamagnetic zinc-doped iron oxide nanoparticles were incorporated into MINPs to grant them magnetic responsiveness. Affinity of MINPs for the target epitope peptide was characterized by surface plasmon resonance (SPR), while immunocytochemistry-like procedures were applied on endothelial cells (EC) to assess the recognition potential of MINPs for the full PIEZO1 protein. ECs and human adipose tissue-derived stem cells (hASCs) labeled with the deferent nanoswitches were cultured under (+Mag groups) or without the application of cyclical magnetomechanical stimulation. The downstream effects of PIEZO1 actuation on cell mechanotransduction signaling and stem cell fate were screened by analysis of their gene expression profiles.

*Results: The developed abiotic nanoswitches showed sub-nanomolar affinity for the respective epitope, being able to bind PIEZO1 expressed by ECs in a similar way to antibodies. Oscillating magnetic fields were applied to actuate PIEZO1 in EC membrane through the nanoswitches during in vitro culture time. The applied treatment led to significant changes in the expression pattern of genes downstream of PIEZO1 activity, demonstrating that these actuators can transduce magnetomechanical stimuli directly to PIEZO1 mechanoreceptors. Finally, this wireless actuation systems proved to be an effective tool to modulate the expression profiles of genes related to several musculoskeletal differentiation pathways in hASCs, including tenogenic markers. Importantly, the targeting of each epitope led to different signaling effects, implying distinct roles for each domain in the sophisticated physiological functions of these cell membrane channels.

*Conclusion/Significance: The wireless technology presented here is a promising new tool for studying fundamental mechanisms of PIEZO-mediated cell mechanobiology. Our results further suggest that by combining tailored molecular recognition with magnetomechanical actuation, these nanoswitches can be harnessed for therapeutic interventions having PIEZOs as targets, as well as in tissue engineering and regenerative medicine approaches of mechanosensitive tissues such as tendon.

Acknowledgements: European Union’s Horizon 2020 under grant No. 772817; FCT/MCTES for PD/BD/143039/2018 & COVID/BD/153025/2022, co-financed by POCH and NORTE 2020, under the Portugal 2020 partnership agreement through the European Social Fund, for 2020.03410.CEECIND and 2022.05526.PTDC; and Xunta de Galicia for ED481B2019/025.

15 - Adaptive Cyclic Load That Changes As Collagen Organization Increases, Does Not Improve Maturation In Engineered Ligaments

L. D. Troop, J. Puetzer

Biomedical Engineering, Virginia Commonwealth University, Richmond, VA

*Purpose/Objectives: Hierarchical collagen fibers are the primary source of strength in tendons and ligaments; however, these fibers don’t regenerate after injury or repair, resulting in limited treatment options. Previously, we developed a culture-system that guides ligament fibroblasts in collagen gels to produce native-sized hierarchical collagen fibers by 6 weeks, and we have demonstrated intermittent cyclic load at 5 or 10% strain further improves maturation. Interestingly, we found a shift in mechanotransduction with time, with 10% strain driving early improvements when cells were in unorganized gels, and 5% strain more beneficial later in culture once cells were anchored on aligned fibers. Therefore, our objective was to investigate whether adaptive cyclic load that changes as collagen fibers develop produces further maturation. We hypothesize decreasing cyclic strain as organization increases will drive cells to further improve maturation, producing significantly stronger replacements.

*Methodology: Bovine ACL fibroblasts were mixed with collagen to produce 20 mg/mL collagen and 5x106 cells/mL constructs. Constructs were loaded 3x/week with an established loading regime (Fig.1A). Controls were loaded at 0, 5, or 10% strain for 6 weeks. Adaptive load constructs were loaded with decreasing or increasing strain, changing as constructs formed aligned fibrils (2 weeks) and fibers (4 weeks) (Fig.1B). Post culture, organization, composition, and tensile properties were evaluated. Significance determined by 2-way ANOVA with Tukey’s post-hoc.

*Results: All constructs formed aligned collagen fibrils by 2 weeks, maturing into larger fibers and fascicles by 6 weeks (Fig.1C). However, adaptive load (increasing and decreasing) appeared to have less collagen crimp and reduced organization compared to steady load. Early in culture, when cells were in unorganized gels, 10% load increased GAGs and LOX activity (Fig.1D); and later in culture, once cells were on aligned fibers, steady 5% load significantly increased collagen (hypro). Interestingly, both adaptive loads did not similarly increase collagen. Instead, decreasing load resulted in a significant increase in GAGs and percent weight by 6 weeks compared to other groups (Fig.1D&E), suggesting that decreasing strain may cause an injurious response. Further, adaptive load did not improve LOX activity by 6 weeks, despite steady 5% and 10% load having increased LOX activity at 6 weeks. Mirroring organization, all constructs had improved elastic mechanics by 6 weeks (Fig1.E). However, steady 5% load had the highest elastic modulus and ultimate strength (UTS), mirroring collagen concentration, and steady 5 and 10% constructs had significantly improved toe moduli compared to other groups, mirroring increased crimp formation.

*Conclusion/Significance: Contrary to our hypothesis, adaptive load that changes as collagen organization matures, does not improve hierarchical organization, collagen crimp, or mechanics. Previously, it has been reported that cells respond to changes in load with catabolism, which only shifts to anabolism once cells modify their environment to sense the new load. This may indicate that adaptive cyclic load leads to repeated remodeling, and periods greater than 2 weeks is needed between load changes for improved maturation. This study provides new insight into how adaptive intermittent cyclic loading affects hierarchical fiber formation, which will help to inform rehabilitation protocols and engineered replacements.

16 - Metabolic Profiling Of Tendon And Cartilage Healing

I. Gerner¹, S. Meier-Menches², A. Bileck², S. Gueltekin¹, C. Gerner², F. Jenner¹

¹ Equine surgery, Vetmeduni Vienna, Vienna, AUSTRIA,

² Analytical chemistry, University of Vienna, Vienna, AUSTRIA

*Purpose/Objectives: Osteoarthritis and tendinopathies are prevalent, debilitating musculoskeletal disorders, characterized by a vicious self-perpetuating cycle of chronic inflammation and progressive degeneration. Considering the pivotal role of non-resolving inflammation in the pathogenesis of both conditions, along with the critical involvement of bioactive lipids like eicosanoids and endocannabinoids in inflammation initiation, maintenance, and resolution, this study investigates the role of metabolites in the in vivo injury response of cartilage and tendon. Employing targeted metabolomics analysis, we aim to explore temporal changes in metabolite levels during the healing process, contributing to a comprehensive understanding of the molecular mechanisms underlying the development and progression of osteoarthritis and tendinopathies.

*Methodology: Standardized cartilage and tendon lesions were created in musculoskeletally mature (2-5 years, body weight 95 ± 12 kg), female sheep without orthopaedic disease (n=3 per group). Tissue samples were harvested according to the three stages of healing on day 0 (uninjured control) and days 3, 21, and 150 post injury. Targeted metabolomics analysis were conducted using the MxP® Quant 500 Kit (Biocrates Life Sciences AG). Measurements were carried out using LC-MS and flow injection (FIA)-MS analyses on a Sciex 6500+ series mass spectrometer coupled to an ExionLC AD chromatography system (AB Sciex), using the Biocrates MxP Quant 500 kit column system and the Analyst 1.7.1 software with hotfix 1 (AB Sciex).

*Results: In both, tendon and cartilage tissue, increased expression levels of hypoxanthine indicating hypoxia, putrescine indicating lysosomal degradation of debris and increased lactate levels paralleled by decreased glucose levels indicating intensive glycolysis were detected. In tendons, a strong upregulation of cholesterol esters and glycosylceramides in response to injury indicates an overload of the lysosomal degradation capacity, potentially contributing to perpetuated inflammation. A strong decrease of triacylglycerols accompanied by an increase of diacylglycerols and corresponding acylcarnitines strongly suggests lipolysis. In cartilage in contrast, phosphatidylcholins were more abundant than triacylglyerols. However, lipolysis of both triacylglycerols (tendon) and phospholipids (cartilage) leads to formation of oxilipins/eicosanoids known to play an important role in inflammatory signaling and regulation.

*Conclusion/Significance: Cartilage and tendon injury resulted in increased levels of several metabolites of glycolysis and mitochondrial respiration, which are involved in adenine triphosphate (ATP) turnover and play an essential role in prechondrogenic condensation. Both musculoskeletal tissues had increased levels of hypoxanthine, an adenylate metabolite known to affect epithelial barrier function and wound healing. Similarly, both tissues had increased levels of the polyamine putrescine, which plays essential roles in promoting cell proliferation, migration and tissue healing. In contrast, glycosylceramides are involved in the regulation of expression of pro-inflammatory cytokines and have been reported to contribute to the establishment of chronic neuroinflammation. Our results offer a broad overview of the metabolic response to injury in cartilage and tendon tissues and contribute to the investigation of tissue-specific mechanistic interrelations between proteins and metabolites and their influence on resident tissue cells and immune cells recruited to the site of injury.

17 - Acoustic holographic bioassembly for tissue engineering

P. Fischer, M. Shi, K. Melde

Heidelberg University and Max Planck Institute, Heidelberg, GERMANY

*Purpose/Objectives: Acoustic holograms, which are phase plates designed to modulate wavefronts of sound waves, show potential as a tool for biological research, especially in the field of tissue engineering. Enabling the control of acoustic fields with exceptional intricacy, acoustic holograms can be employed to build tissue constructs of complex geometries from the bottom up by assembling biological components rapidly and in one shot.

*Methodology: Comparing with magnetic and electric fields that can also drive bioassemblies, acoustic fields confer superior biocompatibility, scalability of wavelength and intensity, as well as low attenuation in most media. Direct, contactless, and label-free acoustic manipulation of various biological building blocks such as cells, cell-encapsulated particles, spheroids, and organoids is therefore feasible under a range of experimental conditions.

*Results: Coupling single or multiple acoustic holograms to ultrasonic transducers, our group have showcased patterning of cells into complex shapes within a Petri dish [1] as well as three-dimensional assembly of hydrogel beads and cells in common labware [2]. Advancing from the two prior works, we have recently developed an acoustic holographic bioassembly platform that is able to assemble cells within a cell culture insert into centimeter-scale tissue constructs of arbitrary shapes and high cell densities (on the order of 107 cells/mL), without relying on the presence of an air-liquid interface. By demonstrating the viability, proliferation, and migration of the acoustically assembled cells over time, we show that this bioassembly platform has the potential to facilitate developing in vitro models for tissue-wide biological studies. Additional applications in acoustically-enhanced bioprinting and tissue graft fabrication can be envisaged as well.

*Conclusion/Significance: Offering a unique set of advantages, acoustic holographic bioassembly is expected to be applied more broadly in the field of tissue engineering in the future, both as a biofabrication method on its own and as a complement to other methods.

19 - Rapid Assembly Of Aligned Microvascular Networks For Cardiac Tissue Grafts

M. Jewett, F. Midekssa, E. Stanley, M. Hu, S. Bhirud, S. Xi, B. Baker

Biomedical Engineering, University of Michigan, Ann Arbor, MI

*Purpose/Objectives: Myocardial infarction leads to the formation of a matrix-dense, fibrotic scar that disrupts cardiac function. Many cardiovascular diseases are currently untreatable due to myocardium’s inherent inability to regenerate. As such, there is a pressing need to develop cell-based therapies to repair and restore injured myocardium. Current approaches being tested in patients do not possess highly aligned capillary-scale vasculature reflecting the vasculature of the native myocardium (Figure 1A), thus motivating the need for new technologies that can template organized capillary-scale vasculature in 3D tissues. Here, we developed a novel and versatile method to rapidly generate aligned capillary-scale microvasculature which can be readily integrated with hydrogels containing cardiomyocytes.

*Methodology: 1 wt% alginate was conjugated with RGD using sulfo-NHS/EDC activation, then dialyzed (Figure 1B). Alginate microfibers were crosslinked via injection of 1 wt% alginate through a 30 µm glass needle into a spinning bath of 100 mM CaCl₂. To precipitate super paramagnetic iron oxide nanoparticles (SPIONs) within alginate microfibers, microfibers were suspended in a 2:1 volumetric ratio solution of 100 mM iron (III) tetrahydrate and iron (II) tetrahydrate, respectively, with 2 mM CaCl₂ for 30 minutes before adding an equal volume of 0.5 M NaOH for another 30 minutes under argon. Microfibers were segmented manually and by sonication. Prior to seeding with endothelial cells (ECs), the microfibers were sterilized by soaking in 100% ethanol supplemented with 2 mM CaCl₂. Human umbilical vein endothelial cells (HUVECs) were resuspended in EGM2 supplemented with 2 mM CaCl₂ in a 0.6 mL tube containing microfibers and flipped 180 degrees every 5 minutes for 2 hours, then allowed to spread overnight. The following day, microfibers were encapsulated in a dextran vinyl sulfone (DexVS) or fibrin hydrogel. 3.2 wt% DexVS hydrogels were supplemented with 2 mM RGD, 4.8 wt% cysteine, and crosslinked with 11.07 mM VPMS. 10 mg/mL fibrin hydrogels were crosslinked with 1 U/mL thrombin. Following encapsulation, hydrogels were hydrated with cell culture medium supplemented with CaCl₂ until the day of microfiber degradation. To degrade alginate microfibers, the culture medium was supplemented with 1 U/mL alginate lyase for 48 hours.

*Results: SPION-modification of alginate microfibers (Figure 1C) and RGD functionalization did not affect microfiber diameter (Figure 1D). Further, SPION-modification rendered microfibers magnetic (Figure 1E) enabling alignment prior to immobilization upon hydrogel crosslinking (Figure 1F). Following encapsulation and alginate degradation, lumenization of the remaining network was visualized by 1 µm fluorescent bead perfusion (Figure 1G). Further, RGD-modified alginate microfibers were seeded with HUVECs prior to encapsulation in high-density fibrin (Figure 1H). When directly encapsulated in fibrin of equal density, HUVECs failed to form stable networks and commonly underwent apoptosis. In contrast, HUVEC-seeded microfibers and subsequent alginate microfiber degradation led to the formation of lumenized capillary-like networks.

*Conclusion/Significance: Here, we present a new method for fabricating aligned, capillary-scale vasculature using sacrificial, magnetic alginate microfibers that can be aligned during the gelation of natural or synthetic hydrogels. Future studies will explore the ability of organized capillary beds in supporting the viability and function of thick cardiac tissue grafts long term.

20 - Towards Clinically Relevant Cartilage Repair: Oxygen Control, Tissue Integration And Immunomodulation

G. Lindberg^1,2, A. Norberg², L. Veenendaal², M. Hofmann¹, N. Shchotkina¹, M. Gautreaux¹, K. Lim^3,2, S. South¹, N. Willett¹, P. Dalton¹, T. Woodfield²

¹ Bioengieering, University of Oregon, Eugene, OR,

² Orthopaedic Surgery, Centre for Bioengineering & Nanomedicine, University of Otago, Christchurch, NEW ZEALAND,

³ School of Medical Sciences, University of Sydney, Sydney, AUSTRALIA

*Purpose/Objectives: This presentation will address three key clinical hurdles in cartilage tissue engineering, focusing on articular conditions, particularly osteoarthritis. The first challenge involves fabricating larger cartilage constructs in the centimeter scale, where oxygenation constraints hinder cell survivability. The second challenge is insufficient lateral integration between implanted and host cartilage, limiting structural integrity and long-term success. To tackle these long-standing translational challenges, we use bioassembly techniques that employ hydrogel spheroids to create distinct tissue compartments, subsequently assembling them into larger, complex constructs.

*Methodology: This presentation will describe the development of our laboratory’s 1) controllable hydrogel platforms photo-polymerizable gelatin, heparin, hemoglobin and peroxides to augment hypoxia-induced cell death, and 2) ECM-hydrogels that enhance integrative cartilage repair strategies using Vitreous humor, Lysyl-oxidase-like-2, and copper. Across these biomaterial platform technologies, cellular health, tissue formation, multiplexed proteomics assays, and spatial transcriptomics were used to analyze the biological outcomes. The utilization of 10x spatial genomics represents an especially pivotal step in unraveling the intricacies of both the fused constructs developed, ensuring the integration region or specific tissue compartments can be analyzed separately from overall construct.

*Results: Firstly, our oxygen delivery investigations demonstrate that bioconjugation of Hep-SH provides a viable strategy for incorporation of oxygenated Hb within photo-crosslinkable microspheres, yielding a 7.5-fold increased equilibration time. Similarly, introducing peroxide-spheres to maintain higher O₂ levels significantly improved the viability of MSCs cultured under hypoxia for up to 5 days. Secondly, our implant integration models revealed that decellularized biomaterials highly supports lateral integration between mature tissues, while traditional biomaterials yield fibroblastic ECM and gene expression at the host-tissue interface. We furthermore discovered that LOXL2 enhances lateral integration across cartilage spheroids (6-fold increase in GAG/DNA). Thirdly, joint-organoids consisting of cartilage, synovial fluid, and T-cells were cultured successfully, yielding variable progression of OA disease stages between patient samples. We uncovered large variability in SF cytokine content across all demographics while age-related reduction in cartilage content was detected following growth factor stimulation. Addition of hydrogels to the inflammatory joint models resulted in modulation of cytokines expressed by inflammatory T-cells.

*Conclusion/Significance: This series of studies allowed us to develop hydrogels proficient to guide cartilage repair across a variety patient-centric condition. Ultimately, this talk will highlight some of the important advances in biofabrication and hydrogel design for more clinically-relevant cartilage repair and precision medicine, including 1) oxygen generating hydrogels, 2) integrative hydrogels, and 3) immunomodulatory hydrogels together with biofabricated 3D-models to inform regenerative needs in catabolic joint environments.

21 - Construction Of A Hydrogel Based, 3d Bioprinted Corneal Stroma

D. Basoz^1,2, A. Celebi^3,2, S. Buyuksungur⁴, A. Akalinli^5,2, D. Yucel^6,2, N. Hasirci^7,4, V. Hasirci^1,2,4

¹ Graduate Department of Biomaterials, Acibadem Mehmet Ali Aydinlar University, Istanbul, TURKEY,

² ACU BIOMATERIALS A&R Center, Acibadem Mehmet Ali Aydınlar University, Istanbul, TURKEY,

³ School of Medicine - Ophthalmology, Acibadem Mehmet Ali Aydinlar University, Istanbul, TURKEY,

⁴ BIOMATEN, CoE in Biomaterials and Tissue Engineering, Middle East Technical University, Ankara, TURKEY,

⁵ Biomedical Engineering, Acibadem Mehmet Ali Aydinlar University, Ankara, TURKEY,

⁶ School of Medicine - Histology and Embryology, Acibadem Mehmet Ali Aydinlar University, Ankara, TURKEY,

⁷ Department of Chemistry, Middle East Technical University, Ankara, TURKEY

*Purpose/Objectives: Cornea is the protective layer of the eye and transmits the light. In corneal damage, the loss is compensated by allografts of which there is limited availability. The purpose of the study was to produce hydrogel-based corneal stroma substitutes by 3D bioprinting.

*Methodology: Gelatin-based corneal stroma substitutes were produced by 3D-bioprinting and tested under in situ, in vitro and in vivo conditions. Methacrylated gelatin (GelMA, methacrylation degree 88.9%) was 3D printed (with and without human keratocytes, 1x10⁶ cells/mL) at 19°C. Discs (diameter 5 mm, thickness 500 µm) were punched out and characterized. In situ tests included measurements of equilibrium water content, compressive mechanical strength and light transmittance. Cell viability was determined with Live/Dead assay. In vivo studies involved implantation of corneal substitutes in New Zealand White Rabbit models by a lamellar cut in the cornea, a pocket creation in the stroma and placing the 3D printed discs. There were 4 groups: Group C: Control (untreated); Group S: Sham; Group HC-: Cell-free; Group HC+: Cell loaded. Rabbits were examined every two weeks for inflammation, edema, vascularization, ulcer formation, infection, condition of the incision, corneal integrity after NaF staining, implant rejection and implant color/status for up to 90 days.

*Results: 3D printed GelMA hydrogels exhibited an equilibrium water content of 89.2% ± 2. The phenotype of the cells was confirmed by ALDH1A and Lumican markers as keratocytes. Cell viability of the 3D-bioprinted samples was consistently high (>95%) for 21 days (Fig. 1 a). For both HC+ and HC- hydrogels the transparency was found to be around 70% in the visible light range (>400 nm), and presented very low transparency at the wavelengths lower than 400 nm, indicating low transparency against UV (Fig. 1 b). Compression elastic modulus was 174.2 ± 47 kPa on day 1 for HC- hydrogels which decreased substantially to 99.16 ± 12.14 by day 14. Conversely, for CH+ hydrogels, modulus increased from 171 ± 39 to 242.1 ± 33.5 by day 14. These indicate the hydrogel degrades when cell free but in the presence of cells the mechanical strength increases. In vivo tests showed that the stroma substitutes preserved their transparency and locations for 90 days, even when the cells were present. One major difference was vascularization observed in HC+ implants (Fig. 1 c-d). This might be interpreted as a disadvantage but it is known that vascularization is observed during regeneration, so it could be a sign of healing. Increase in mechanical strength of CH+ during test period might be a support for this interpretation. Finally, the transparency of the implants in vivo is another indication of suitability as a corneal substitute.

*Conclusion/Significance: 3D bioprinting of keratocyte loaded GelMA implants demonstrated comparable transparency, and higher and increasing mechanical properties compared to cell- free controls. In vivo studies showed that the implants did not cause inflammation, any maintained their positions in the stroma. In brief, GelMA stromal implants produced with 3D printing are promising, indicating a potential for advancement to the clinical stage. This study was supported by Health Institute of Turkey, (Project Number:11745).

22 - All-in-one Generation And Multiomic Profiling Of Human Whole Brain Organoid On A Microfluidic Plate

W. Zhao¹, Y. Wang², R. Hu², P. Chen¹

¹ Department of Biomedical Engineering, Wuhan University, Wuhan, CHINA,

² State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Innovation Academy for Precision Measurement Science and Technology, Wuhan, CHINA

*Purpose/Objectives: Human brain organoids (hBOs) have been increasingly recognized by neurobiologists and neurologists as transformative brain model in recent years. Particularly, hBOs have been used to probe mechanisms of neurological disorders, including microcephaly, schizophrenia, and Alzheimer’s disease, and provided important insights into the onset and progression of pathogenesis. Currently, the conventional standard culture (CSC) approach to generating hBOs starts from hPSCs and experiences complex parenchymal cell fate transitions from ectoderm, neuroectoderm, neuroepithelium, neural stem cells, and unmatured neurons to matured neurons. The procedure involves several laborious operations on multiple devices. Notably, the suspension culture is conducted in a bulk flow with a high Reynold number, which lacks precise microenvironment control over individual organoids, resulting in increased intra-batch morphological heterogeneity and decreased inter-batch reproducibility. This issue poses a great challenge for quantitative neural studies such as neuroprotectant screening and neurotoxicity assessment. Additionally, the suspension culture is inaccessible for in-situ and time-lapse observation of the same single organoids, which is essential to understanding neurodevelopment and morphogenesis. Therefore, there is a strong demand for innovative brain organoid culture methods that allow simple operation steps and reproducible organoid formation.

*Methodology: We developed an all-in-one microfluidic plate (AIOMP) to generate hBOs with a facile procedure on a single device without transferring. The AIOMP was conducted in a 12-well plate, with each well containing three hBOs in three separate microchambers. To optimize the biophysical microenvironment of the AIOMP culture, computational fluid dynamics (CFD) simulation was conducted using COMSOL Multiphysics. We applied comprehensive transcriptome, proteome analysis, RT-qPCR and immunofluorescence analysis to characterize hBOs.

*Results: The AIOMP with individually perfusable microchambers for hBOs offers significant advantages, including improved uniformity and reproducibility as well as the ability to observe morphogenesis in real time. Variability analysis revealed that the AIOMP had a low coefficient of variation for average diameter and a ratio of length to width than CSC group. Immunofluorescence and RT-qPCR analyses demonstrated that the markers of the forebrain (PAX6 and FOXG1) and hindbrain (ISL1 and PAX2) in the AIOMP were highly expressed, while the cortical plate of the hBOs had become thicker, suggesting that microfluidic technology can promote the differentiation and maturation of cortical structures. Furthermore, proteomics and transcriptomics results showed that the expression of neural-related proteins in hBOs in the AIOMP was upregulated, signifying the promotion of neurogenesis, indicating a higher accuracy to the human fetal brain compared to CSC group. Finally, Tau fibril-induced neuropathophysiology was studied in the hBOs using AIOMP, resulting in the identification of neurite loss and neuroinflammation, which are the main features of human neurodegenerative diseases.

*Conclusion/Significance: Overall, we provide an alternative bioengineered hBOs culture approach with simplified procedure, improved homogeneity and reproducibility, and accessibility to in situ imaging. More importantly, the hBOs are fully biologically evaluated at the transcriptome and proteome level. The hBOs demonstrate an increased correlation with early-stage human fetal brain with enhanced neurogenesis and corticogenesis. We expect this AIOMP to facilitate the widespread use of hBOs for investigating neurobiology and neurological diseases.

23 - The Pro-regenerative Extracellular Matrix Scaffold Microenvironment Enhances Cancer Vaccine Efficacy To Induce Anti-tumor Immune Memory

S. Pal¹, R. Chaudhari², I. Baurceanu¹, B. J. Hill³, B. A. Nagy¹, M. T. Wolf¹

¹ National Cancer Institute, Frederick, MD,

² Oregon Health & Science University, Portland, OR,

³ Frederick National Laboratory for Cancer Research, Frederick, MD

*Purpose/Objectives: Extracellular matrix (ECM) scaffolds derived from decellularized mammalian tissues are used during cancer care to surgically reconstruct tissues immediately following tumor resection. ECM scaffolds are immunomodulatory and initiate a mixed M1/M2 milieu of macrophage phenotypes and Type 2 immune signatures such as eosinophils and elevated IL-4 cytokine, essential to tissue regeneration and constructive scaffold remodeling. ECM scaffold implantation in the post-resection bed for tissue creates a unique opportunity for in situ cancer immunotherapy delivery (Figure 1A). While previous studies have explored the use of ECM scaffolds in cancer care, few have investigated their potential as delivery platforms for cancer vaccines, particularly in inducing cytotoxic cell-mediated immunity while also preserving the beneficial pro-regenerative hallmarks of the ECM scaffold response.

*Methodology: Porcine small intestine submucosa was decellularized (confirmed by the reduction in DNA content), lyophilized, and cryo-milled to form SIS particles. We first determined the optimal cancer vaccine adjuvant for combination with an ECM scaffold by characterizing the local, regional, and systemic in vivo immunological changes in C57Bl/6 mice after subcutaneously injecting SIS-ECM particulate with adjuvant via immunofluorescence, cytokine PCR, and plasma Luminex assay. The adjuvants CDA (cyclic diAMP), GM-CSF (granulocyte-macrophage colony-stimulating factor), MPLA (monophosphoryl lipid A), and vehicle were compared. We utilized an in vivo cytotoxic lymphocyte assay to elucidate an adjuvant formulation that induced antigen-specific cellular immunity when delivered with an ECM scaffold and characterized antigen release properties in vitro and in vivo. Finally, we implemented the resultant ECM scaffold-assisted cancer vaccine formulation to therapeutically treat established tumors and generate protective tumor antigen-specific immune memory, which we analyzed by FACS for memory T cells.

*Results: STING agonist CDA emerged as the most potent cytotoxic inducer when delivered with the SIS-ECM scaffold. Quantitative histology of the SIS-ECM implant showed that MPLA induced the greatest cellularity after 7-14 days, qualitatively mimicking infection. Multiplex immunofluorescent staining showed SIS-ECM alone attracted large numbers of leukocytes, primarily macrophages and antigen-presenting cells. (Figure 1B-E). SIS+CDA led to greater than 95% specific killing, indicating that CDA can be delivered locally with SIS to induce antigen-specific immunity (Figure 1F). Further, CDA did not diminish hallmark ECM immune responses needed in wound healing including IL4 signaling. Finally, SIS+CDA was delivered with OVA as a therapeutic vaccine to treat established OVA antigen-expressing E.G7 lymphoma tumors. SIS+CDA led to complete responses and protection on tumor rechallenge in curing 50-75% of animals. No tumor regression was observed in control SIS or CDA groups. SIS-ECM scaffold-assisted vaccination extended antigen exposure was mediated by CD8+ cytotoxic T cells and generated long-term antigen-specific memory for at least 10 months post-vaccination, producing 4-fold effector memory CD8+ T cells on tumor challenge than untreated controls.

*Conclusion/Significance: ECM scaffolds were an effective delivery system for a cancer vaccine, augmenting efficacy while preserving the distinctive characteristics of tissue regeneration immune responses. ECM scaffolds enhance cytotoxic T-cell priming while preserving tissue repair hallmarks with an appropriate immune adjuvant and may hold clinical promise for local immunotherapy post-tumor resection.

25 - Matrix Bound Nanovesicles Isolated From Immuno-Regulatory Tissue Have Distinct Biologic Functions

C. J. Patterson, V. Anto, J. Jones, G. Hussey, S. Badylak

University of Pittsburgh, Pittsburgh, PA

*Purpose/Objectives: Matrix-bound nanovesicles (MBV) are a recently discovered class of extracellular vesicles (EV) that are embedded within the extracellular matrix (ECM) of all soft tissues and organs until released by matrix turnover events. MBV have been identified as a key functional component responsible for the therapeutic immunomodulatory and tissue-reparative effects of ECM-based biologic scaffolds. Previous studies suggest that while MBV are ubiquitous throughout the ECM of all tissues, there are inter-tissue specific differences in cargo and function. However, whether and how MBV might differ within different parts of the same organ is unknown. Due to its distinct histological layers, the small intestine serves as an ideal organ for evaluating intra-organ differences in MBV composition and function. Each layer of the small intestine has a unique ECM composition, distinct cell population, and physiologic role. In particular, the Peyer’s Patch, the primary inductive site of mucosal immunity in the gut, provides a unique opportunity to explore hypothetical intra-organ differences in the composition and function of MBV isolated from a predominantly lymphoid tissue in comparison to non-lymphoid tissues within the same organ.

*Methodology: Small intestines were harvested from market weight pigs and manually separated into their individual histological layers (i.e. mucosa, submucosa, muscularis propria, serosa, and Peyer’s patch submucosa). MBV were liberated from the ECM by enzymatic digestion and isolated by ultracentrifugation. Following isolation, MBV were analyzed by 1) transmission electron microscopy and atomic force microscopy to evaluate differences in vesicle structure, mechanics, and morphology. 2) proteomic analysis to discern compositional differences, and 3) in-vitro functional assays to evaluate differences in MBV function on macrophage activation (an established phenotypic effect induced by MBV).

*Results: MBV from all layers displayed similar size and morphology by TEM imaging. While MBV from mucosa, submucosa, muscularis propria, and serosa layers showed similar immunomodulatory function (i.e. reduction of IL-6 and TNFα expression in recipient macrophages), MBV isolated from the lymphoid follicle-rich Peyer’s patch significantly increased expression of these pro-inflammatory cytokines. Proteomic analysis showed a differential enrichment of pro-inflammatory cytokines and growth factors in MBV isolated from Peyer’s Patch compared to MBV isolated from the surrounding non-lymphoid small intestine tissue.

*Conclusion/Significance: While MBV from most small intestinal layers were similar in size, morphology, and immunomodulatory function, Peyer’s patch MBV induced a more pro-inflammatory phenotype in recipient macrophages. These results suggest intra-organ differences in MBV composition and function, specifically when the tissue in question is composed of lymphoid follicles with an immune role. Further implying that differing components of various tissue types within an organ contribute to determining the biological output of the isolated vesicles. The resulting implication of this study is to uncover specific MBV characteristics that will ultimately be used for individualized treatments.

26 - Suspension Electrospinning Of Decellularized Extracellular Matrix: A New Approach To Retain Bioactivity

S. Jones¹, A. Luengo Martinez¹, S. VandenHeuvel², E. Burgeson³, S. Raghavan², S. Rogers⁴, E. Cosgriff-Hernandez¹

¹ The University of Texas at Austin, Austin, TX,

² Texas A&M University, College Station, TX,

³ University of Illinois Urbana-Champaign, Champaign, IL,

⁴ The University of Illinois Urbana-Champaign, Champaign, IL

*Purpose/Objectives: Decellularized extracellular matrices (dECM) have strong regenerative potential due to their complex composition that support tissue regeneration through angiogenesis, mitogenesis, chemoattraction, and immune regulation. However, current clinical options for dECM are limited to freeze-drying its native form into sheets. Electrospinning is a versatile scaffold fabrication technique that allows control of macro- and microarchitecture by tuning fiber size and orientation, as well as overall scaffold shape. It remains challenging to electrospin dECM while maintaining bioactivity. This has led researchers to either blend it with synthetic materials or use enzymatic digestion to fully solubilize the dECM. Both strategies reduce the innate bioactivity of dECM and limit its regenerative potential. Our lab has developed a new method of suspension spinning that does not require additives and preserves bioactivity. Systematic study of electrospinning parameters was used to identify key processing steps and rheological properties necessary to fabricate dECM fiber scaffolds with retained regenerative capacity.

*Methodology: Small intestinal submucosa (SIS) were decellularized using standardized protocols and electrospinning suspensions prepared using hexafluoroisopropanol. The effect of homogenization, concentration, and particle size on rheological behavior and electrospun fiber formation was then investigated. Brightfield imaging was used to characterize the electrospinning suspensions. The retention of bioactivity after electrospinning was assessed by comparing the angiogenic and immunomodulatory properties between a decellularized small intestinal submucosa (SIS) sheet and an electrospun SIS scaffold.

*Results: Homogenization was utilized to enhance particle interaction and increase elastic behavior of the suspension. Rheological analysis shows that the increase in particle interaction creates a viscoelastic solid that persists to larger strain amplitudes, facilitating electrospinning. A crossover strain amplitude of the loss and storage modulus greater than 100% was a strong predictor of dECM suspension spinnability. A direct correlation between concentration and viscosity was observed and impacted fiber morphology, similar to that observed in conventional electrospinning solutions. Dry particle size had minimal impact on suspension properties and fiber morphology, which was attributed to the lack of change in the suspension particle morphology. The dry particles of varying sizes broke down to a similar equilibrium state in suspension. The versatility of the dECM suspension electrospinning method was demonstrated by electrospinning SIS with three common decellularization techniques (Abraham, Badylak, and Luo) and dECM derived from three tissue origins (intestinal submucosa, heart, and skin). Bioactivity retention was indicated by a similar cell response between the SIS sheet and electrospun SIS for all assays. For angiogenic properties, an endothelial cell tube formation assay and a chorioallantoic membrane assay both showed the retention of angiogenic capacity. To examine immunomodulation, a macrophage-SIS contact and subsequent gene expression study was performed. Macrophage activation was similar in native and electrospun SIS, with a preference towards tolerogenic polarization (IL10, CHI3L3, VEGF). Interestingly, macrophages contacting electrospun SIS expressed 10-fold more TGFβ.

*Conclusion/Significance: Collectively, this work provides a user’s guide to electrospin dECM with bioactivity retention without the use of additives. This has broad utility in tissue engineering and regenerative medicine applications.

27 - The Effect Of Estrogen-mimetic Compounds In Cell Culture On Human Mesenchymal Stromal Cell (hmsc)

J. C. Bradford¹, S. Lynch¹, J. Robinson²

¹ Bioengineering, University of Washington, Seattle, WA,

² Orthopedics and Mechanical Engineering, University of Washington, Seattle, WA

*Purpose/Objectives: Women have a higher risk for several musculoskeletal injuries including anterior cruciate ligament (ACL) and meniscus tears. Human mesenchymal stromal cells (hMSCs) have the potential to promote repair and regeneration for these degenerative conditions and are a viable target for cell therapy. Male and female cells have varying response and exposure to estrogens transiently across the lifespan. Thus, when studying sex specific repair strategies there is a need to examine cellular response to estrogens. However, current cell culture medias contain exogenous estrogens in the form of phenol red and fetal bovine serum (FBS) and their effect on male and female human mesenchymal stem cells is unknown.

Herein, we investigate the effects of estrogen-mimetic media components and passage number on male and female hMSC metabolism, proliferation, senescence, and differentiation. We hypothesized that estrogen-like compounds in the form of phenol red and endogenous 17β-estradiol (E2) in FBS would increase metabolism and proliferation in a sex-based manner.

*Methodology: Bone marrow and adipose-derived human mesenchymal stem cells from 4 male donors ranging from 19 - 25 years of age, and 4 female donors ranging from 20 - 29 years of age were assessed. Male and female cells were grown in low glucose phenol red or phenol red free Dulbecco's Modified Eagle's Medium (DMEM) with either normal or charcoal dextran filtered FBS, abbreviated as phenol/free or normal/filtered for brevity. Cells were expanded in free/filtered media for at least 5 days prior to study. Cells at passages P2, P3, and P4 were cultured for 5 days in their respective media conditions and free/filtered + 20 pg/mL E2 was used as a positive control. Prestoblue and a Seahorse XF analyzer was utilized to assess metabolic activity. Picogreen was utilized to determine cellular proliferation. β-Galactosidase staining was used as a proxy for senescence and staining with alcian blue, oil red o, and alizarin red stains completed for trilineage differentiation potential.

*Results: Phenol red significantly increased the proliferation of male and female cells (Figure 1 A). Unfiltered FBS significantly increased the metabolism of all donors and more significantly increased the proliferation of female donors, similarly to exogenous estrogen (Figure 1 B-E). Basal metabolic rate from the Seahorse indicated filtered FBS caused a lower OCR/ECAR ratio for filtered FBS conditions, particularly in female cells, indicating a more stem like balance of metabolism (Figure 1 F-G). Beta galactosidase staining indicated that the phenol free/normal FBS group had the highest levels of senescence in female donors, while male donors showed little change in their senescence (Figure 1 H-I).

*Conclusion/Significance: Data demonstrates high variability of hMSCs response to common estrogenic media additives. For studies or trials utilizing hMSCs, particularly those involving sex differences or dimorphisms in disease, impact of phenol red and FBS composition on cell behavior must be considered.

29 - Single Nucleus Rnaseq And Spatial Transcriptomics Delineate The Sex Specific Pro-reparative Effects Of An Infusible Extracellular Matrix For Myocardial Infarction Treatment

J. Mesfin¹, M. Karkanitsa¹, J. Yu², A. Chen¹, V. Ninh², S. Paleti¹, E. Wong¹, C. Luo¹, Z. Fu², K. King², K. Christman¹

¹ Bioengineering, UC San Diego & Sanford Consortium for Regenerative Medicine, La Jolla, CA,

² School of Medicine, UC San Diego, La Jolla, CA

*Purpose/Objectives: Myocardial infarction (MI) treatments fail to target the pathological remodeling response. Recently, we developed an infusible decellularized ECM biomaterial (iECM), which can be administered through intracoronary infusion, localizes to the leaky vasculature caused by MI, and has shown pro-reparative effects, such as immunomodulation and cell survival. Given that therapeutic responses can vary due to sex, we investigated the iECM’s pro-repair mechanisms relative to sex using single nucleus RNA sequencing (snRNAseq) and spatial transcriptomics in both male and female rat MI models.

*Methodology: Either iECM or saline (n = 4 per sex) was administered through a simulated intracoronary injection after ischemia-reperfusion in both female and male rats, with harvest 3 days post-injection. Nuclei were isolated from left ventricular free wall tissue and 15,000 nuclei per sample was loaded onto a 10X Genomics Chromium Chip. The 10X v3.1 GEM kit was performed per manufacturer’s kit instructions. For spatial transcriptomics, harvested OCT-embedded short axis cardiac tissue slices (10 µm) were cryosectioned onto a 10X Visium slide, with Visium performed as per manufacturer instructions. Libraries for snRNAseq and spatial transcriptomics were sequenced by NovaSeq S4 technology, and data was analyzed using Seurat for R.

*Results: Spatial transcriptomic analysis between female and male iECM treated hearts demonstrates higher differentially expressed genes in female hearts. iECM elicited more immunomodulation (Angptl4, F13a1, Il18bp), fibroblast activation (Pi16, Tgfb1), and regulation of programmed cell death (Syk, Ripk3, Dapk3) in female hearts. In contrast, male hearts demonstrate interferon response genes (Mx2, Ddx3) alongside higher reactive oxygen species response (Reg3b, Eef1a1, Nid1). Given the major differences with spatial transcriptomics analysis, we then evaluated differences through snRNAseq within cardiomyocytes, macrophages, fibroblasts, and endothelial cells. Among cardiomyocytes, border zone genes (Cd44, Nppa) are more prevalent in females, while males have higher muscle development genes (Sox6, Gja1, Foxp2). Female iECM macrophages have high Negr1, Inhba, and Aldoa, which all play a role in M2 macrophage polarization, while male iECM macrophages exhibit a more inflammatory macrophage phenotype through interferon-related genes (Irf8, Enpp1). Female iECM fibroblasts at this timepoint exhibit an inflammatory phenotype (Mx2, Irf8), which is also conserved in males (Il6r, Il1r1). Finally, female iECM endothelial cells elicit a significant angiogenic response (Stab1, Adam12, Fbln5), which is also conserved in males (Vegfa, Actn2).

*Conclusion/Significance: Here, we establish sex-specific responses with iECM administration in a rat MI model. While the female iECM response elicits a greater number of differentially expressed genes, the male response also demonstrates immunomodulation, increased cell survival, and reactive oxygen species response. At the cell-specific level, we demonstrate sex-specific differences in eliciting pro-repair. Given that the sex-specific responses can vary chronologically, ongoing studies will evaluate both day 1 and day 7 post-infusion.

31 - Synergistic And Cell-specific Extracellular Vesicle Delivery Ofdevelopmental Transcription Factors To Human Intervertebral Disc Cells

K. Diop¹, S. N. Tang², M. Kordowski¹, A. Salazar-Puerta¹, Y. Liu¹, L. Bodine³, E. L. Yu⁴, S. Khan⁴, N. Higuita-Castro¹, D. Purmessur¹

¹ Department of Biomedical Engineering, The Ohio State University, Columbus, OH,

² Department of Orthopedic Research, Washington University St. Louis, St. Louis, MO,

³ Department of Mechanical Engineering, The Ohio State University, Columbus, OH,

⁴ Department of Orthopedics, The Ohio State University, Columbus, OH

*Purpose/Objectives: Intervertebral disc (IVD) degeneration is a major contributor to low back pain (LBP). However, current interventions fail to address the underlying disease pathology. Biological strategies such as viral gene delivery and cell-based therapies are hampered by critical limitations such as tumorgenicity/immunogenic responses and cell survivability. Engineered extracellular vesicles (eEVs) present a promising alternative which avoids these risks and carries the potential for tuning for cell-selective in-vivo delivery. [1,2]. This study aims to1)determine the therapeutic effects of synergistic EV delivery of developmental transcription factor Brachyury (T), FOXF1, and Brachyury+FOXF1 on catabolism, inflammation, pain predictors and matrix production in degenerate nucleus pulposus (NP) in 3D culture; and 2)in order to target LBP more comprehensively, eEVs were separately functionalized with NP and annulus fibrosis (AF) cell-specific ligands for targeting degenerate human NP or AF-cells and deliver cell-specific genetic cargo in-vitro.

*Methodology: Diseased AF and NP cells were isolated from human patient tissue (N=4, Male/Female, 19-70y.o) and AF or NP-cells from autopsy (Thompson grade <3) were used to generate eEVs as previously described [2, 3]. eEVs were generated via nanoelectroporation of human NP and AF cells with plasmids encoding for FOXF1, T, FOXF1+T, MKX, FOXF1+CD11a (NP-functionalized), MKX+Netrin-1 (AF-functionalized) or the empty vector pCMV6 (SHAM). eEVs were characterized via Nanoparticle tracking analysis and qRT-PCR for packing efficiency, concentration, and size distribution prior to use. For Synergistic studies, NP cells were expanded and seeded in 2% agarose gels (4x10⁶ cells/mL, 8mmØx4mm Height) and treated with FOXF1-eEVs, T-eEVs, or T+FOXF1-eEVs with respective SHAM-eEV controls (∼10⁸ particles/construct). Dependent variables were assessed at 2- and 8-weeks post treatment and included cell viability (Calcein/Ethidium), gene expression (qRT-PCR), Collagen, and Glycosaminoglycan (sGAG) content. For functionalization studies, NP and AF-cells were labeled with green and red cytotracker respectively, before being seeded in co-culture. eEVs loaded with FOXF1 or MKX were functionalized with CD11a or Netrin-1 to target NP or AF-cells, respectively.

*Results: For our Synergistic studies, significant increases in both T and FOXF1 expression was observed in FOXF1+T synergy groups compared to SHAM, FOXF1 or T alone. Interestingly, sex/age effects in catabolic and inflammatory genes were noted when assessing the individual patient response. Collagen content was significantly increased in all treatment groups compared to SHAM while T+FOXF1 combined resulted in significantly more sGAG accumulation at 8 weeks compared to other groups. For our Functionalization studies, eEVs were successfully derived after co-transfection of donor cells with FOXF1+CD11a or MKX+Netrin-1. eEVs loaded with FOXF1 or MKX and functionalized with CD11a or Netrin-1 were preferentially internalized by NP or AF cells, respectively, with increased cargo gene expression for FOXF1 or MKX, while non-functionalized eEVs were non-specifically captured by NP or AF cells, demonstrating no targeting.

*Conclusion/Significance: Our results demonstrate both successful delivery of desired genetic cargo via eEVs and enhanced synergistic effects of T+FOXF1 that improve cell phenotype with potential sex/age dependent responses to eEV treatment. In addition, we have demonstrated successful surface functionalization of human-derived eEVs with cell-specific ligands that preferentially target and deliver desired transcription factor cargo to NP and AF-cells respectively.

32 - A Novel PEG-Based Injectable Macroporous Hydrogel Scaffold

M. E. Dickenson, A. H. Morris

Biomedical Engineering, University of Michigan, Ann Arbor, MI

*Purpose/Objectives: Biomaterial scaffolds are a cornerstone of tissue engineering, used for tissue repair and regeneration, simulating tissue microenvironments in vitro, and controlled drug delivery. Poly(ethylene glycol) (PEG) hydrogels are broadly used due to their unique properties including biocompatibility and the ability to easily modify the polymer to fit a variety of needs. PEG hydrogels have submicron-sized pores, yet these are too small to enable the rapid cellular infiltration necessary for new tissue formation. Forming macropores within PEG gels enables more rapid cellular integration, but these formulations lose their injectability. Injectability of hydrogels is highly desirable, as these materials can be used in minimally invasive procedures and can easily form to and fill any void space. Thus, the proposed study aims to create a novel injectable macroporous PEG hydrogel, that capitalizes upon the known advantages of porous hydrogel materials as well as the ease of use of injectable biomaterials.

*Methodology: The novel injectable macroporous hydrogel scaffold is composed of two parts: an injectable non-degradable PEG bulk gel and rapidly degradable (plasmin-sensitive) PEG microspheres integrated throughout the bulk gel as porogens. Both standard rheometry techniques and Resonant Acoustic Rheometry were used to quantify bulk mechanical properties. Confocal imaging techniques and image analysis were used to quantify porosity and void volume within these materials. Cell survival, proliferation, invasion, and morphology were characterized via a variety of cellular assays after cells were cultured in vitro on our macroporous hydrogel scaffolds. In vivo cellular invasion into these novel scaffolds was evaluated post-injection in a healthy mouse model through utilization of histology and flow cytometry.

*Results: I have successfully created degradable microparticles and integrated them into an injectable bulk gel as porogens. These particles have been observed to degrade in a physiologically relevant timeframe in vivo, creating an interconnected pore network and allowing rapid cellular invasion into the hydrogel. The high level of tunability of this system has been confirmed through quantification of the mechanical properties and the interconnected porous networks for a wide array of gel compositions. This has confirmed that this system can be used to easily alter the bulk stiffness, pore size, and pore quantity of these scaffold materials. I have demonstrated in a mouse model injectability of the gel, that the interconnected pore network forms rapidly within the gel post-injection, and that cells invade into and can survive within the gel over the timespan of 2 days to 4 weeks. Both histology and flow cytometry have been leveraged to successfully profile the different types and quantities of cells being recruited into these scaffolds in vivo.

*Conclusion/Significance: I have demonstrated that I can create injectable microporous hydrogels that are mechanically robust and are highly tunable. I have additionally shown both in vitro and in vivo that these materials are biocompatible, and will rapidly recruit a wide variety of cell types post-injection in a mouse model. Ultimately, this novel injectable porous hydrogel scaffold design is a highly tunable system, being able to accurately recapitulate a wide range of natural biological environments.

33 - Fibroblast-selective Inhibition Of Mechanotransduction In A Biomechanically-relevant 3d Skin Model Of Fibrosis

A. Pappalardo, L. Garriga Cerda, H. Abaci

Columbia University Medical Center, New York, NY

*Purpose/Objectives: Human skin encloses the musculoskeletal system as a continuous organ in which there is a biomechanical balance between cells and their surrounding extracellular matrix (ECM). Skin wounds disrupt the biomechanical equilibrium and polarize fibroblasts to myofibroblasts (MFBs), which generate scars through ECM remodeling. Currently, corticosteroids injections are the only available treatment for skin scars and present underwhelming efficacy with the risk of skin atrophy and infections. Several animal studies demonstrated that the suppression of mechanotranduction in skin wounds results in scar-free healing and appendage regeneration. Though, a generalized suppression of mechanotransduction in the skin is not ideal as keratinocytes depend on YAP/TAZ signaling for proliferation, attachment, and differentiation. It was recently discovered that dopamine D1 receptor (D1R) agonists are able to suppress mechanotransduction through inhibition of YAP/TAZ signaling selectively in fibroblasts, which only express D1R, while basal keratinocytes express D2R.

*Methodology: We recently developed a novel 3D skin model biofabricated as a continuous tissue in desired anatomical shapes seamlessly conforming to the recipient site, e.g., skin gloves. Remarkably, this edgeless-skin technology reproduces the anisotropic properties of human skin, to our knowledge, for the first time in any 3D skin model, and therefore provides a unique tool to investigate fibrosis and screen antifibrotic therapies. Here, we first created a fibrotic environment in our edgeless 3D skin via TGFb and demonstrated that two D1R-selective agonists, dihydrexidine (DHX) and fenoldapam (FDP), can be repurposed for inhibiting MFBs polarization. We used verteporfin, a highly effective direct YAP/TAZ inhibitor, as a positive control.

*Results: Upon DHX, FDP and VTP treatment we observed through immunofluorescence an inhibition of YAP nuclear translocation and an enhancement of its inhibitory phosphorylation compared to the TGFβ control (up to 4-folds decrease). Similarly, the drugs were also able to prevent phosphorylation and nuclear localization of pFAK (up to 5-folds decrease), and pSMAD2 (up to 4-folds decrease), which are involved in mechanosignaling and TGFβR transduction respectively. DHX and VTP were also able to effectively supress αSMA expression, while FDP induced a partial suppression. DHX was able to contrast the suppressive action of TGFβ on cell proliferation, maintaining a percentage of Ki67+ cells similar to the untreated control (no TGFb). Finally, we assessed the secretion of pro-inflammatory cytokines in the culture medium of ex vivo foreskin cultures, and detected a 2-fold decrease in IL-6 for all drugs and in IL-8 for DHX and FDP. Importantly, the non-cell type specific mechano-inhibitor VTP showed an increase of IL-8, perhaps indicating epidermal toxicity. Finally, we demonstrated that DHX does not disrupt epidermal homeostasis unlike VTP. The untreated control and DHX-treated constructs displayed a healthy mature epidermis, with basal expression of Keratin14, apical expression of loricrin, and absence of Keratin17. In contrast, the VTP-treated sample shows an atrophic epidermis, with limited stratification, loss of basal keratinocytes and high expression of Keratin17, underscoring the toxicity of generalized YAP inhibition treatment.

*Conclusion/Significance: Overall, our data suggest the efficacy and advantages of fibroblast-selective mechanosignaling suppression as a topical antifibrotic therapy in the skin.

34 - Macrophages And T Cells Enhance Tissue Complexity And Function In 3D Skin-like Tissue Models Of Fibrosis

I. Singh, S. Shenk, J. Garlick

Tufts University, Boston, MA

*Purpose/Objectives: The influence of immune cells in the tissue microenvironment on the pathogenesis and progression of fibrotic diseases such as scleroderma is poorly understood. Most existing 3D skin-like human tissue models contain only 2 cell types - keratinocytes and fibroblasts, demonstrating an unmet need to incorporate immune cells to more fully recapitulate fibrotic diseases. The purpose of this research is to optimize tissue complexity of a 3D skin-like tissue 3D model of fibrosis that includes macrophages and T cells in the cellular microenvironment.

*Methodology: We tested the fabrication of 3D human skin equivalent (HSE) tissues with four cell types from scleroderma patients: fibroblasts and keratinocytes harvested from 3cm punch biopsies from forearm skin, and macrophages and T-cells from blood draws. T cells were expanded in 2D culture and characterized via flow cytometry using an optimized multicolor immunofluorescence panel, using antibodies including CD3, CD4, CD8, and CD44. Primary fibroblasts, T cells, and macrophages were then seeded in a bovine collagen matrix, keratinocytes were added 7 days later, and tissues were grown at the air-liquid interface. Tissue morphology was characterized by H and E staining and the presence of macrophages and T cells was determined by immunohistochemical (IHC) staining. T cell and macrophage function and cytokine cross-talk were determined by measuring IL-6 and IL-8 from 3D tissue supernatants. T cells were recovered from the collagen matrix after 14 days in 3D culture and T cell sub-populations were identified using the optimized panel. RNA was isolated from tissues and RT-PCR was performed to profile the production of fibrogenic markers in scleroderma fibroblasts (TGF-beta, Type I collagen, Type III collagen, and fibronectin).

*Results: We found that tissues demonstrated normal epithelial stratification and fibroblast morphology suggesting that immune cells supported keratinocyte growth and differentiation and did not alter skin tissue structure. IHC demonstrated the presence of T cells and macrophages throughout the connective tissue, confirming their presence in 3D tissues. Macrophage and T cell function were demonstrated by increased levels of inflammatory cytokines known to mediate crosstalk between stromal fibroblasts, macrophages, and T cells. When dermal cells were isolated from the 3D tissues and characterized via flow cytometry, we found about 60% of cells showed positive staining for the fibroblast marker CD90. 30% of dermal cells showed positive flow staining for the T cell marker CD3 and 50% of these were CD4+ T cells. Thus, functional T cell sub-populations were sustained in the 3D tissue microenvironment that augmented skin tissue morphology and function.

*Conclusion/Significance: Our findings represent an important step towards a more complex 3D human skin model that harbors scleroderma patient-specific macrophages and T cells. Increasing cellular complexity allows us to better reflect patient tissue and more accurately understand and study the role of macrophages and T cells in fibrotic diseases. We hope to broadly apply this tissue model to better understand the contribution of immune cells in a broad range of fibrotic diseases including scleroderma, liver cirrhosis, and interstitial lung disease.

35 - Biofabrication Of An Immunocompetent 3D Skin Tissue Equivalent Incorporating Primary Human Macrophages As A Pre-clinical Testing Platform For Inflammatory Skin Diseases

Y. Lim, H. Zarkoob, M. Song, M. Ferrer

National Institutes of Health, Rockville, MD

*Purpose/Objectives: The skin is the first line of defense against external insults by acting as a physical, chemical, and microbiological barrier. The skin harbors a highly specialized immunological niche crucial for tissue homeostasis, defense against harmful pathogens and modulating tissue repair. The skin immune system is tightly regulated for the proper functioning of the skin as a barrier interface, and its dysregulation leads to the manifestation of inflammatory skin diseases. Current in vitro skin models used for pre-clinical testing lack the cellular and physiological complexity of the skin immune components. Therefore, there is a critical need to develop immunocompetent three-dimensional (3D) skin tissue models to study the interplay between different immune cell subsets and the non-immune compartments in the skin and use as pre-clinical testing assays for the development of treatments for inflammatory skin diseases. We have previously created biofabricated 3D skin tissue equivalents that closely mimic human skin tissue and modeled skin diseases like atopic dermatitis (AD). We have now produced an immunocompetent 3D skin tissue models that incorporate primary human macrophages. Macrophages monitor the skin microenvironment for infection or cell stress signals to either promote or suppress inflammation. M1 macrophages are crucial to clear foreign debris, pathogens, and dead cells during an insult and initiate pro-inflammatory responses. Anti-inflammatory M2 macrophages support tissue repair by promoting angiogenesis and tissue remodeling. The numbers and phenotypes of the different macrophage subsets need to be properly regulated for skin tissue homeostasis. Excessive increase in M1 can lead to hyperinflammation, while increase in M2 can lead to skin fibrosis.

*Methodology: To generate an immunocompetent 3D skin model, M1 and M2 macrophages are polarized from CD14+ primary human monocytes and were introduced into biofabricated 3D full thickness skin equivalents with dermis and air-liquid interface (ALI) differentiated epidermis.

*Results: We first demonstrate that incorporated M1 or M2 macrophages survive and are functional until the end of the 3D skin culture (∼3 weeks). The addition of M1 and M2 macrophages into our biofabricated 3D skin tissues upregulates the production of chemokines such as CCL22 and CCL13, known to act as immune chemotaxis agent and modulate tissue remodeling. The macrophage-incorporated skin tissues also respond to bacterial and viral stimuli by producing pro- and anti-inflammatory cytokines. Furthermore, using a TGFβ-induced skin fibrosis model, we observed that M2 incorporation potentiates collagen deposition in the skin dermis.

*Conclusion/Significance: In conclusion, we have successfully generated biofabricated 3D full thickness skin tissues equivalent with functional macrophages in a 96-well microplate format that can serve as a platform to model immune skin diseases, including fibrosis, and can be used for drug screening.

36 - Tropoelastin Modulates Immune Responses To Accelerate Cutaneous Wound Healing

Z. Wang^1,2, H. Shi³, S. M. Mithieux^1,2, P. Silveira³, Y. Wang^3,4, A. S. Weiss^1,2,5

¹ University of Sydney, Sydney, Australia,

² Charles Perkins Centre, Sydney, Australia,

³ The ANZAC Research Institute, Concord, Australia,

⁴ Nanjing University of Chinese Medicine, Nanjing, China,

⁵ Sydney Nano Institute, Sydney, Australia

*Purpose/Objectives: The management of acute and chronic wounds remains a critical concern in clinical care due to their high occurrence rate. The synthetic dressing Integra is the leading treatment for wound repair but cost and repair limitations mean there are competitors and active research in the field. During dressing-assisted wound healing, the inflammation stage intuitively affects the healing rate and outcome. The use of novel dressings that aim to reduce inflammation or shorten the inflammation stage are promising approach to accelerate wound healing and repair. Here we show that a dressing made from a combination of tropoelastin (TE), a molecular promoter of wound repair, and polyglycerol sebacate (PGS), an elastic and biodegradable polymer, promoted wound healing through the regulation of local and systemic immune responses.

*Methodology: Using Integra and PGS dressings as controls, the ability of the electrospun TE-PGS dressing to promote wound healing was assessed in a full-thickness cutaneous wound model in mice. Wound size was measured on day 3, 7, 10 and 14. Wound morphologies and regeneration were assessed by H&E staining and Picrosirius red Staining. Immune cells in the wound sites were identified by immunohistochemistry. Immune cells in the spleen were assessed by flow cytometry. Cytokines in the blood serum were detected by multiplex cytokine assay.

*Results: We found the TE-PGS dressing promoted faster wound closure and collagen regeneration with improved tissue morphology. Locally at the wound site, iNOS⁺ M1 and CD206⁺ M2 macrophages differed spatially and quantitatively. In all groups, M2 (but not M1) macrophages populated the wound edge where tissue regeneration was active, while in the wound bed, both M1 and M2 macrophages were present. However, TE-PGS dressing promoted a significantly higher number of M2 macrophages at the wound edge with a higher M2/M1 ratio at the wound bed. The TE-PGS treated group also generated the highest CD4⁺ T cells and a rise in Foxp3⁺ regulatory T (T_reg) cells in the wound bed by day 7. The desirable outcome of increased M2 macrophage and T_reg levels is associated with an accelerated resolution of inflammation and improved tissue remodelling. Systemically in the spleen, we detected significant alteration in the immune cell population. TE-PGS dressing showed an early increase of F4/80⁺ splenic red pulp macrophage (RPM) on day 7 and an increased proportion of splenic CD4⁺ T cells. These were accompanied by an increase in serum IL-10 levels by days 7 and 14, pointing to an interaction between RPM and T^reg cells in the release of anti-inflammatory cytokines. In contrast, Integra triggered a late increase in splenic RPM on day 14, which we hypothesize is a consequence of a delayed interaction between RPM and T_reg cells in the spleen.

*Conclusion/Significance: Our results show that TE-PGS wound dressing leads to better healing outcomes by promoting a pro-healing profile in both local and systemic immune responses.

53 - Biosensor-integrated Multiorgan-on-a-chip Platform For Real-time Monitoring Of Organoid Function

S. Jeon, J. Cheng, A. Mehta, K. You, A. Atala, S. Lee, S. Soker, J. Yoo

Wake Forest Institute for Regenerative Medicine, Winston Salem, NC

*Purpose/Objectives: The emergence of an organ-on-a-chip system aims to replicate the complex physiological functions of the human body. To assess responses to drugs or other insults, biological analytes are typically analyzed using basic laboratory techniques such as spectrophotometry and immunoassays. However, the manual sampling and offline analysis are limited in capturing the dynamic cellular changes in real-time and are prone to user-dependent errors.

*Methodology: In this study, we developed a novel integrated platform that combines a multiorgan-on-a-chip with environmental and biomarker sensors to monitor the organoid function in real-time. The multiorgan-on-a-chip (liver, pancreas, and ovary) was fabricated using a precision organoid bioprinting technique directly onto a polydimethylsiloxane (PDMS)-based chip. Electrochemical aptamer-based biosensors such as albumin, insulin, and estradiol were established to measure the levels of secretory proteins from the organoids. Environmental sensors measuring pH, oxygen, glucose, lactate, and temperature enabled real-time monitoring of culture conditions. The multi-organ-on-a-chip and sensor components were integrated into a single inline flow loop with automated control and measurement capabilities.

*Results: Three types of organoids showed high cell viability and expressed cell-specific markers during 8 days of flow culture. Using aptamer-based protein sensors, standard curves were drawn according to incensement of each protein concentration. The accuracy of the aptamer-based biosensor measurements was validated with conventional enzyme-linked immunosorbent assay (ELISA). As a proof of concept, automatic media exchange was demonstrated in a feedback loop with measured environmental parameters. Finally, all dynamic changes of organoid function and cultural environment from multiorgan-on-a-chip were recorded over 48 hours.

*Conclusion/Significance: Our approach represents an innovative multiorgan-on-a-chip platform that allows for continuous monitoring of organoid function with automated maintenance of physiological environmental conditions.

56 - Tissue Engineering A Humanized Rat Model For Osteosarcoma Research

J. Gospos^1,2,3, S. Saifzadeh^1,4,5, M. Laubach^1,2,5, F. Savi^1,2,3, D. W. Hutmacher^1,3,6, J. A. McGovern^1,3,6

¹ Queensland University of Technology, Brisbane, Australia,

² Centre for Biomedical Technologies, Brisbane, Australia,

³ Max Planck Queensland Centre (MPQC) for the Materials Science of Extracellular Matrices, Brisbane, Australia,

⁴ Medical Engineering Research Facility, Brisbane, Australia,

⁵ Australian Research Council (ARC) Training Centre for Multiscale 3D Imaging, Modelling, and Manufacturing (M3D Innovation), Brisbane, Australia,

⁶ ARC Training Centre for Cell and Tissue Engineering Technologies, Brisbane, Australia

*Purpose/Objectives: Osteosarcoma (OS) is the most frequent malignant bone tumour; commonly affecting children, adolescents and young adults. The survival rate of metastatic OS is less than 25%. OS is treated with a combination of surgical resection and systemic chemotherapy with doxorubicin (DOX). However, surgical resection and high dose DOX treatment cause serious long-term adverse effects. To overcome the limitations of current OS treatments, we have developed an advanced rat OS model that converges tissue engineering and regenerative medicine principles to improve treatment outcomes for OS patients. This model provides a platform to study OS primary tumour development and metastasis in an appropriately sized animal model that will concurrently allow for studying complex surgical interventions.

*Methodology: A humanized bone microenvironment was created by implanting an orthotopic tissue engineered bone construct (ohTEBC) biofabricated using melt electrowritten medical grade polycaprolactone (mPCL) scaffolds (measuring 6x5mm in dimension) coated with calcium phosphate (CaP). The TEBC was then seeded with human osteoblasts that were explanted from hip arthroplasty surgery and expanded and then cultured onto the scaffold for 6-weeks prior to orthotopically implanting the ohTEBC around the femur of an immunocompromised rat. Once the humanized bone microenvironment was established, the SAOS-2 OS cell line was injected orthotopically into the ohTEBC and key diseases processes were monitored via dynamic bioluminescent imaging (BLI) and µCT. Further characterisation was carried out via histological and immunohistochemical analysis.

*Results: The implanted ohTEBC formed a chimeric human bone that contained a human-derived extracellular matrix fraction that was infiltrated with bone marrow as determined by histology analysis. The injected SaOS-2 OS cell line formed a primary orthotopic tumour within the human bone microenvironment in 100% of the rats. BLI and µCT demonstrated the formation of a primary tumour and the development of lung metastases over a 15-week period. Furthermore, all rats developed lung metastasis which accurately mimics human disease progression. µCT characterization revealed the destructive nature of OS with evidence of bone remodelling at the niche and primary tumour and degradation of the endogenous femoral cortical bone. Histology and immunohistochemistry analysis demonstrated extensive OS-derived hsCol-1 networks infiltrating the entire tumour microenvironment with evidence of osteoclast-mediated bone remodelling at the niche and primary tumour. Furthermore, the OS tumour was characterized by a dense aggregation of M2-polarised tumour-associated macrophages at the tumour periphery.

*Conclusion/Significance: We have tissue engineered an orthotopic OS tumour model that recapitulates the hallmarks of human disease within an immunocompromised rat. This model will allow to study complex surgical interventions and regenerative medicine techniques never possible. The outcomes of this study will improve the chemotherapeutic and limb sparing surgery options for children and adolescents with OS.

57 - RCAN1.4 Regulates Tumor Cell Engraftment And Invasion In A Thyroid Cancer To Lung Metastasis-on-a-chip Microphysiological System

S. R. Anderson, K. G. Nairon, A. Nigam, T. Khanal, N. Rajan, M. D. Ringel, A. Skardal

The Ohio State University, Columbus, OH

*Purpose/Objectives: Progressive metastasis is the primary cause of cancer-related deaths. It has been recognized that many cancers are characterized by long periods of stability followed by subsequent progression. Genes termed metastasis progression suppressors (MPS) are functional gatekeepers of this process, and their loss leads to late-stage progression. Previously, we identified regulator of calcineurin 1, isoform 4 (RCAN1.4) as a functional MPS for several cancers, including thyroid cancer, a tumor type prone to metastatic dormancy. RCAN1.4 knockdown increases expression of the cancer-promoting transcription factor NFE2L3, and through this mechanism increases cancer cell proliferation and invasion in in vitro and in vivo environments and promotes metastatic potential to lungs in tail vein models. However, the mechanisms by which RCAN 1.4 regulates specific metastatic steps is incompletely characterized. Studies of the metastatic cascade are limited in mouse systems due to high cost and long duration. Additionally, immune cell contributions to the tumor microenvironment and subsequent T cell exhaustion are difficult to recapitulate as some of them are not expressed in mice at all (e.g., Interleukin-8). Bioengineered human cell-based models such as organoids and tumor-on-a-chip systems can serve as effective alternative model systems.

*Methodology: Here, we have shown the creation of a thyroid-to-lung metastasis-on-a-chip (MOC) model to address these limitations, allowing invasion analysis and quantification on a single cell level. We then deployed the platform to investigate RCAN1.4 knockdown in fluorescently tagged hTh74 and FTC236 thyroid cancer cell lines. Cells were circulated through microfluidic channels, running parallel to lung hydrogel constructs allowing tumor cell-lung tissue interactions. Similar to studies in mouse models, RCAN1.4 knockdown increased NFE2L3 expression, globally increased invasion distance into lung hydrogels and had cell line and clonally-dependent variations on bulk metastatic burden. In line with previous in vivo observations, RCAN1.4 knockdown had a greater impact on hTh74 metastatic propensity than FTC236. Following validation of this “tail vein” analogous MOC, we increased the complexity of the thyroid-to-lung MOC by incorporating a primary thyroid tumor construct comprised of FTC236 cells encapsulated in 3D hydrogels. In addition to increased physiological accuracy the previous cell line-based lung constructs were replaced with lung constructs comprised of primary human lung epithelial cells and fibroblasts.

*Results: After initiation of fluid flow, RCAN1.4 knockdown resulted in rapid metastasis. Fluorescent cells were observed to travel to and invade the lung constructs in 24 hours. In contrast, the control cells failed to invade the lung constructs, even after 2 weeks of MOC operation. In summary, we have developed and validated a novel MOC system for thyroid cancer research, in which RCAN 1.4 MPS knockdown of primary tumor increases metastasis to lung organoids, both in the form of initial engraftment and invasion.

*Conclusion/Significance: This system creates opportunities for more detailed and rapid mechanistic studies of the metastatic cascade and creates opportunities for translational assay development. Next steps will include assessment of immune cell populations and secreted cytokine impacts on the TME and tumor progression.

58 - Engineered Human Bone Marrow Captures Breast Cancer-induced Changes In Hematopoiesis

P. L. Graney, D. N. Tavakol, G. Vunjak-Novakovic

Columbia University, New York, NY

*Purpose/Objectives: The bone marrow (BM) is a major regulator of homeostasis and disease, as the residing site of hematopoietic stem and progenitor cells (HSPCs). The functional role of the BM is to produce downstream blood and immune cells, acting as a renewable source of myeloid and lymphoid progenitors that can respond to stimuli during injury, regeneration and disease. In the presence of a primary tumor, circulating tumor-secreted factors migrate to the BM and redirect the lineage commitment of HSPCs, causing accumulation of myeloid-derived suppressor cells (MDSCs) that promote tumor progression. Currently, it is poorly understood how the systemic perturbations to the BM, which are distinct from those occurring at the primary tumor site, alter the BM niche and how these changes bias hematopoiesis toward myeloid-skewing. The objective of this work was to characterize the immune cell responses to tumor-released circulating signals, which have been shown to reshape the immune cell landscape and promote metastasis.

*Methodology: To this end, we used an engineered model of healthy human bone marrow (eBM) that is capable of producing downstream blood/immune cells. eBM microtissues were prepared from decellularized bovine-derived bone scaffolds, seeded with human mesenchymal stem/stromal cells, osteoblasts, endothelial cells, and CD34+ HSPCs encapsulated in fibrin gel. As a preliminary model of tumor-released circulating signals, culture media were collected from cultures of the parent MDA-MB-231 triple negative breast cancer line, lung-targeting and bone-targeting subsets derived from this line by the Joan Massagué lab at MSKCC, an MCF10a control breast epithelial line, and healthy SFEM II basal media control. The eBM was cultured in conditioned media for 10 days and changes to HSPCs were characterized by flow cytometry of cells produced by the eBM.

*Results: Notably, metastatic culture supernatants induced significant skewing toward CD45+CD11b+ myeloid cells and generation of monocytic and granulocytic MDSCs that were distinct from non-cancerous epithelial and healthy controls. These changes were observed as early as after 4 days and maintained over 10 days in culture. These findings were recently recapitulated using serum derived from patients with varying stages of breast cancer and healthy patient serum controls. In ongoing studies, we are using serum from treatment-naïve patients to delineate the contributions of breast cancer stage and subtype to myeloid skewing, and identify changes to the eBM stromal microenvironment.

*Conclusion/Significance: Overall, these data suggest that human eBM tissue can be used to model tumor-specific changes to hematopoiesis in vitro, enabling the identification of unique populations of altered myeloid and stromal cells that evolve during breast cancer metastasis. Improved understanding of the tumor-specific changes in the immune landscape has potential to lead to the development of new tools for patient-centric therapeutic intervention.

59 - Nuclear Deformation And Rupture During Intravascular Migration Through A Mineralized Matrix Are Key Mediators Of Prostate Cancer Metastasis To Bone

A. Athirasala¹, C. Franca¹, A. Mansoorifar¹, R. Subbiah¹, M. Q. Lima Verde¹, M. Fraga¹, D. Keene², L. Bertassoni¹

¹ Oregon Health & Science University, Portland, OR,

² Shriners Childrens Hospital, Portland, OR

*Purpose/Objectives: Several neoplastic diseases preferentially target bone tissue, which presents a substantially different microenvironment from that of the primary tumor. Both the physical properties of the bone microenvironment as well as the biological function of its diverse population of resident cells are derived from the unique nanoscale structure and chemical composition of its highly calcified matrix, which is likely to also play a significant role in the survival and growth of metastatic tumor cells. However, these key interactions are far from being understood, given that current in-vitro bone models fail to replicate the structural, cellular and extracellular complexity of native bone. Here, we developed and characterized a novel bone-on-a-chip platform that replicates the critical hallmarks of bone tissue.

*Methodology: The platform constitutes a microfluidic model of nanoscale mineralized cell-laden collagen hydrogel, incorporating embedded osteoblasts, osteocytes, mesenchymal stem cells and a perfusable pericyte-supported vasculature interacting with circulating prostate cancer cells. The on-chip format of the platform enables live tracking of early metastatic events such as transendothelial migration and extravasation as well as spatially resolved analysis of cellular events through immunofluorescence imaging.

*Results: Prostate cancer cells were observed to have a clear predilection for dissemination through the engineered microvasculature into the bone like matrix when compared to non-mineralized scaffolds. Moreover, it was evident that the process of intravascular migration and extravasation through the more constrictive and calcified matrix induced significant nuclear deformation, rupture and DNA damage pointing to possible mechanisms through which the bone microenvironment could contribute to genomic instability and acquisition of more aggressive phenotypes following metastasis.

*Conclusion/Significance: In conclusion, the engineered bone on a chip platform elucidated novel mechanisms driving metastatic cancer progression in bone tissues.

63 - Bioprinting And Microvascular Assembly Within PEGNB Granular Materials

I. W. Zhang, A. J. Putnam

University of Michigan, Ann Arbor, MI

*Purpose/Objectives: The development of functional, multiscale, and perfusable vascular networks remains one of the largest challenges in tissue engineering. We previously demonstrated that PEG hydrogel microbeads containing endothelial cells (ECs) and stromal cells support the formation of primitive vascular networks within the beads and nucleate extensive interconnected microvascular networks in vitro. Leveraging the use of colloidal bead suspensions as supportive media for bioprinting, the purpose of this study was to evaluate if clickable PEG microbeads could support bioprinting of mesoscale structures, and whether the beads could then be clicked together into a granular hydrogel scaffold capable of supporting microvascular self-assembly.

*Methodology: PEG-norbornene (PEGNB) hydrogel microbeads were formed via microfluidic droplet generation with 0.5% PFPE in NOVEC-7500 and UV photopolymerized. Beads were comprised of 10 wt% 8-arm PEGNB, 2 mM LAP, PEG-dithiol (PEGDT), and 2 mM RGD with a molar ratio of thiol:ene of 0.375:1. Beads were jammed into a slurry via vacuum filtration and then could be secondarily photopolymerized into an annealed granular construct. Rheological characterizations of both the PEGNB bead slurry and the clicked constructs were performed using a 20 mm diameter parallel plate head. For bioprinting, jammed bead suspensions with additional LAP and PEGDT were packed into a PDMS mold, and then used to support a 2.5% (w/v) gelatin bioink extruded with an Allevi-3 bioprinter using 30G, 0.5-inch blunt needle tips. After printing, the PEG beads were photocrosslinked together to form a granular construct with the embedded print. To instigate vascular formation, a 1:1 mixture of ECs and lung fibroblasts (LFs) at 2 M/mL was suspended within a 500 μL volume bead/LAP/PEGDT slurry and UV crosslinked to form cell-laden constructs. These were then cultured for 7 days in EGM2 and then fixed and stained with UEA (an EC-specific lectin), phalloidin, and DAPI to visualize microvascular networks.

*Results: PEGNB hydrogel microbeads were reliably formed with an average diameter of 372.8 ± 39.6 μm (Fig 1A). The colloidal microbead slurry exhibited shear-thinning mechanical behavior suitable for suspended bioprinting (Fig 1B), supported suspended printing of sacrificial bioinks, and could be UV crosslinked into a free-standing granular construct post-print (Fig 1C). Cell-laden PEGNB constructs yielded microvessel-like development in the inter-bead void space, with cells spread along the surface of the nondegradable RGD-modified microbeads (Fig 1D, E). UEA-positive tubule formation was observed, with F-actin staining showing LFs supporting branching morphogenesis in a pericyte-like manner (Fig 1D). Characterization of the shear storage modulus (G’) of clicked acellular and cell-laden PEGNB granular constructs after 1 and 7 days of culture in media confirmed that all constructs maintained mechanical stability over culture (Fig 1F).

*Conclusion/Significance: Our results show that PEGNB microbeads support suspended bioprinting, secondary photocrosslinking into granular constructs, and self-assembly of microvasculature within the inter-bead voids. Ongoing studies are focused on optimizing sacrificial ink evacuation of the bioprinted mesoscale structures and bioprinting into cell-laden PEGNB bead baths. Future work will be focused on evaluating vascular inosculation across length scales in the granular constructs towards the goal of functional perfusion of a hierarchical, engineered vascular tree.

66 - Role Of Macrophage In Sensory Innervation And Cutaneous Wound Healing

J. Xu, W. Qiao, K. W. Yeung

The University of Hong Kong, Hong Kong, China

*Purpose/Objectives: Cutaneous wound healing is a complex physiological process that relies on the orchestrated interactions of various cell types. Among these, macrophages and sensory neurons emerge as critical players involved in maintaining skin regeneration following injury. However, the precise roles and interplay of these cell types during cutaneous wound healing remain incompletely understood. Therefore, this study aims to investigate the relationship between these cell types in the context of cutaneous wound repair and its impact on healing dynamics.

*Methodology: The dynamic polarization of macrophages and its influence on wound healing were studied by single-cell RNA sequencing and validated with LysMCre-iDTR mouse, a transgenic mouse model for inducible depletion of macrophages. Moreover, two-photon in vivo imaging and immunofluorescent staining were employed to investigate the crosstalk of macrophages and sensory neurons during the wound healing process. Additionally, the levels of inflammatory cytokines and neuropeptides related to neuroimmune crosstalk were measured by ELISA.

*Results: Single-cell RNA sequencing data revealed the changes in the phenotypes and cytokine release profiles of macrophages at different time points of wound healing. Macrophage depletion at the early stage of wound healing led to a significant decrease in sensory nerve density, as well as alterations in key cytokines and growth factors essential for sensory neuron activation and wound healing. As a result, the closure of the wound was significantly impaired.

*Conclusion/Significance: This study uncovered the critical involvement of the neuroimmune axis in cutaneous wound healing, as macrophages orchestrate the reinnervation and regeneration of the injured skin. Our findings provide valuable insights into the regulation of the wound repair process and inspire the development of novel therapeutic strategies for cutaneous wound healing.

67 - Exploring The Dynamic Interplay Of Neuroinflammatory Signals In The Intestine: Insights Into Pro- And Anti-inflammatory Cytokines And Neuropeptides

E. Escarda-Castro, T. Coimbra, L. Moroni, P. Wieringa

Maastricht University - MERLN Institute, Maastricht, Netherlands

*Purpose/Objectives: The intestine wall is able to self-regulate multiple aspects such as cell differentiation and barrier integrity. However, external cues from the nervous system and the immune system also impacts intestinal homeostasis. Currently, the precise contributions of these regulatory elements, as well as their interplay, remain unclear and are only partially understood. In this study, we begin to unravel the complex neuroinflammatory interplay between pro- and anti-inflammatory cytokines and neuropeptides within the intestinal microenvironment, deciphering the role of the innervation in inflammation and immune regulation in the gut.

*Methodology: Using differentiated Caco-2 monolayers as a well-established intestinal epithelium model, a panel of pro- and anti-inflammatory cytokines were applied to mimick the role of the immune system. As a starting point, we studied the effects of the cytokines in the intestinal epithelium, and we selected working concentrations and combinations for the subsequent experiments. We chose four distinct inflammatory scenarios: basal, in which there were no cytokines present, representing a physiological state; pro-inflammatory, with the presence of TNF-α, IL-1β, and IFN-γ; anti-inflammatory, with just the presence of IL-10; and resolving, in which both pro- and anti-inflammatory cytokines were present. We then concurrently applied extrinsic nociceptive and sympathetic neural signals, which we emulated via neuropeptides known to be secreted by these neural subtypes: Substance P, VIP, CGRPα, CGRPβ, and NPY. By studying the effects of neuropeptides in different inflammatory states induced by cytokines, we seek to reveal their specific contributions to inflammation, immune responses, and gut homeostasis. Epithelial cell function was by assessed via cell viability, metabolic activity, ROS presence, and the secretion of inflammatory mediators (IL-6 and IL-8). We also performed gene expression analysis and we investigated the barrier function response of the tissue. As final validation, we co-cultured the epithelial monolayers with iPSC-derived neurons within a 3D in vitro gut model to validate findings from the neuropeptide investigation.

*Results: This study sheds light on the multifaceted roles of nervous system in various inflammatory states of the intestinal epithelium. Our findings revealed distinct responses of the epithelium to CGRPα and CGRPβ, highlighting their unique characteristics despite their similar nature as peptides. Even though they have been treated as equivalent in the literature, CGRPα responses seem to stimulate inflammation, and CGRPβ seems to mitigate it. Additionally, while VIP is generally considered an anti-inflammatory factor in the intestine, our research demonstrated its contribution to the inflammatory environment under pro-inflammatory scenarios. On the other hand, Substance P appeared to have a minor role in intestinal inflammation. Interestingly, NPY exhibited pleiotropic effects, with its impact on inflammation being dependent on the specific conditions, demonstrating its complexity in modulating inflammatory responses.

*Conclusion/Significance: By unveiling the distinctive behaviors of neuropeptides in response to different inflammatory conditions, our findings underscore the complexity of neuroinflammatory interactions in the gut and emphasize how we can harness neural signaling to steer the pathological resolution and regenerative state of intestinal tissue.

68 - In Vitro Vascularization Predicts Allograft Healing Of Hydrogel-based Tissue Engineered Periosteum

A. March¹, D. S. Benoit², R. Choe¹

¹ University of Rochester, Rochester, NY,

² University of Oregon, Eugene, OR

*Purpose/Objectives: Bone graft procedures cost over $2.5 billion in the US annually, with decellularized cadaveric bone grafts (allografts) used in approximately one-third of these procedures. Although allografts remain the gold standard for treating critical-size bone defects, approximately 60% fail within ten years post-implantation. Allograft failure is directly linked to the absence of periosteum, a highly vascularized tissue surrounding bone that promotes bone healing and host tissue recruitment through periosteal paracrine signaling. We have developed a tissue-engineered periosteum (TEP) to improve allograft healing, based on cell degradable poly(ethylene glycol) (PEG) hydrogels encapsulating mouse mesenchymal stem cells (MSCs) and osteoprogenitor cells that mimic the periosteal cell population and post-injury paracrine signaling (Fig 1A). A positive correlation between host tissue infiltration and integration and bone biomechanical strength for TEP-modified allografts was observed. Despite this promise, evaluating TEP efficacy in vivo is low throughput and costly. Therefore, to further hasten the development of hydrogel-based periosteum, we investigated the efficacy of in vitro screening methods. We hypothesize that in vitro 3D spheroid sprouting metrics, including early sprouting and network formation over time, have predictive power for in vivo host tissue integration and healing, measured by vascularization, for the engineered periosteum (Fig 1A).

*Methodology: Human umbilical vein endothelial cells and human MSCs were used to form vascular spheroids. Hydrogels entrapping spheroids were formed via enzymatically degradable (D) or non-degradable (ND) linkers and functionalized with the cell adhesive peptide, RGD, or a non-adhesive control, RGE. Sprouting parameters (total network length, number of sprouts, and branches per sprout) and cell-secreted angiogenesis factors (angiocrines) were quantified over 7 days. To evaluate hydrogel-mediated bone allograft healing in vivo, allografts were modified with TEP and implanted into 10-12 week-old female C57Bl6/J mice. Bone healing was evaluated at 3-, 6-, and 9-weeks post-implantation with microcomputed tomography, histology, and biomechanical analysis.

*Results: RGD-functionalized degradable hydrogels best-supported sprouting parameters. Spheroids within enzymatically degradable RGD hydrogels developed a total network length 26-fold and 390-fold higher than degradable RGE and non-degradable hydrogels, respectively, at day 7. Angiocrine secretion supported the importance of hydrogel degradation, as degradable RGD or RGE hydrogel groups demonstrated significantly 5.6-fold and 2.8-fold higher vascular endothelial growth factor-A secretion at day 7 than non-degradable hydrogels. In vivo, hydrogel degradation was a dominant factor for allograft healing, with enzymatically degradable RGD TEP-modified allografts resulting in a 1.6-fold and 4.2-fold greater bone callus volume at 6 weeks post-implantation over non-degradable RGD TEP-modified and unmodified allografts, respectively. Several correlations were made to discern the predictive power of sprouting parameters for measures of in vivo healing. Early (day 1) number of sprouts positively correlates to 6-week graft vascularization (R² = 0.62), in addition to bone callus volume (R² = 0.32) (Fig 1Bi). Furthermore, network formation in vitro at day 7 positively correlates to 6-week graft vascularization and bone callus volume (R² = 0.57 and 0.32, respectively) (Fig 1Bii).

*Conclusion/Significance: This study demonstrates a significant positive correlation between in vitro angiogenesis and in vivo healing, suggesting the promising predicive value of vascular spheroid sprouting for developing tissue-engineered periosteum.

69 - 3d-printed Polycaprolactone-allograft Constructs With Controlled Osteoinductive Releasing Nanogels For Bone Regeneration

E. Mirdamadi^1,2, N. Tang³, H. Tian³, S. A. Meyr¹, M. A. Reynolds², M. O. Wang¹, M. Iwamoto³, M. J. Hartman^2,4, T. L. Lowe^1,2

¹ University of Maryland College Park, College Park, MD,

² University of Maryland School of Dentistry, Baltimore, MD,

³ University of Maryland School of Medicine, Baltimore, MD,

⁴ Hartman Oral and Maxillofacial Surgery, Mechanicsburg, PA

*Purpose/Objectives: Globally, bone defects are projected to cost 17 billion USD by 2030. A class of large bone defects which cannot heal on their own and remain challenging to treat are known as critical-sized bone defects (CSBDs). To improve CSBD treatment outcomes, bioactive materials are implanted along defect voids to facilitate natural bone regeneration. Autografts are the gold standard bioactive material for CSBD healing due to their strong bone regenerative properties and low immunogenicity. However, the process of autografting only provides scarce amounts of bone material while causing donor site morbidity. Alternatively, bone allograft is a relatively more abundant autograft counterpart but with weaker bone forming activity. In addition, bone grafts rapid in vivo resorption and weak mechanical strength limit the materials utilization for patient-and defect-site specific CSBD regeneration. The purpose of this work is to manufacture 3 dimensionally (3D) printed slow biodegrading polycaprolactone-allograft (PCL-ALLO) constructs, with bone-mimicking physical and mechanical properties for bone regeneration. To enhance bone forming activity, bone morphogenetic protein 2 (BMP-2) loaded nanogels are coated on the 3D printed constructs to slow release osteoinductive cues necessary for bone repair which are assessed in vivo.

*Methodology: PCL pellets, combined with 0, 10, or 30 wt% ALLO, were combined in a single screw extruder to make filaments of PCL-ALLO. The PCL-ALLO filaments were then fed on a fused deposition modeling (FDM) 3D printer to manufacture 6 mm discs containing either 250, 500, or 750 µm interconnected pores. BMP-2 loaded nanogels were synthesized by combining poly(N-isopropylacrylamide) and biodegradable dextran-poly(lactate-2-hydroxyethyl-methacrylate) macromer through UV emulsion polymerization. BMP-2 was loaded in nanogels up to a 3 µg growth factor per 1 mg nanogel concentration.

*Results: The compressive moduli of constructs with varied ALLO content and interconnected pore sizes ranged from 0.45 to 6 MPa. Monolithic PCL-ALLO structures, containing either 10 or 30 wt% ALLO, have 160 and 120 MPa compressive moduli as well as 182 and 122 MPa tensile moduli which were all in range of native bone stiffness. Dental pulp stem cells (DPSCs) and fibroblast cells (L929), in the presence of 1 month degraded PCL-30 wt% ALLO extract products, exhibited no cytotoxicity when evaluated by an MTT assay. In addition, nanogel vehicles were not cytotoxic to DPSCs at concentrations up to 5 mg·mL^-1. Constructs with greater allograft content increased construct-nanogel binding affinity and adhered dental pulp stem cell proliferation by 50% after a week in culture. Nanogels loaded with BMP-2 released BMP-2 with near zero-order release kinetics for 35 days. A BRE-luc assay revealed that the BMP-2 in the nanogels had nearly equal bioactivity as biologically active naked BMP-2. Histology and micro-CT showed that 3D printed constructs, containing allograft and BMP-2-loaded nanogels, played important roles in increasing construct cellular penetration and mineralization in rodent heterotopic as well as critical sized calvarial defect models as early as 3 weeks.

*Conclusion/Significance: 3D-printed BMP-2-nanogel-containing PCL-ALLO constructs have great potential to provide CSBD implants for patient-specific and defect-site-specific bone regeneration.

70 - Biomimetic Electrical/photothermal Coordinated Nanocomposite Membrane For Modulating Inflammatory Microenvironment And Improving Bone Regeneration

Y. Guo

Peking University School of Stomatology, Beijing, CHINA

*Purpose/Objectives: Bone defects under complex conditions often heal with unsatisfactory outcomes. Therefore, there is an urgent need to develop biomimetic materials with multi-physical properties to treat complex bone defects.

*Methodology: Herein, an electrical/photothermal coordinated nanocomposite membrane based on the bionic bone tissue microenvironment was designed. The piezoelectric polymer polyvinylidene-trifluoroethylene P(VDF-TrFE) was used as the substrate, and the Polydopamine (PDA) coated BTO as the filler.

*Results: This nanocomposite membrane realized the accurate remote control of the physiological optimal temperature and potential of bone defect repair through near-infrared irradiation. Biological efficacy evaluation showed that this biomimetic electric/photothermal material has a regulatory effect on macrophage phenotype and significantly promotes bone regeneration in vivo and in vitro. In addition, HSP70 and PI3K-AKT signaling pathway were recognized as mechanistic targets for the electrical/photothermal synergies that promote phenotypic transition of macrophages.

*Conclusion/Significance: These findings provide innovative strategies and theoretical basis for the design and development of biomaterials with precise remote regulation of multiple physical properties for bone regeneration.

71 - Spatio-temporal Neuroangiogenic Scaffold For Tissue Regeneration In Medication-related Osteonecrosis Of The Jaw Using Bioactive Elastin-like Recombinamers (ELRs)

I. Gonzalez de Torre¹, C. García Sierra², F. Gonzalez Perez¹, L. M. REdondo²

¹ University of Valladolid, Valladolid, Spain,

² Hospital Universitario Río Hortega, Valladolid, Spain

*Purpose/Objectives: Bone defects, a challenge for orthopedic experts, arise from tumor removal, medication-related bone necrosis, and nonunion post-fractures. The objective of the work is construction of a multi-bioactive scaffold based on that allows a space/time control over the regeneration of damaged bones due to Medication-Related Osteonecrosis of the Jaw (MRONJ) by the combination of the two main investigation lines in bone regeneration as angiogenesis and new innervation of the damaged area, using a minimal invasive approach based on the injection of the fast-degrading pro neuro and angiogenic ELR based hydrogels

*Methodology: Chemical crosslinking facilitated the creation of multi-bioactive scaffolds using ELRs with reactive groups. Fine-tuning physical and mechanical properties involved adjusting reaction conditions, incorporating techniques like rheology, stretching-compression measurements, SEM, and porosity assessments. Cell-loaded multi-bioactive scaffolds, prepared and incubated, underwent evaluation for adhesion, proliferation, angiogenic, and neurogenic potential. In vitro assessments utilized immunofluorescence staining and ELISA assays, while live-recorded monitoring and live-dead analysis ensured cytocompatibility. In rat and rabbit models, preformed scaffolds were subcutaneously implanted, and the regenerative process was evaluated over time. Rabbit models with MRONJ underwent traditional or percutaneous implantation, with histological evaluation following established bone histological techniques.

*Results: The combination of protease-sensitive sequences with different degradation rates, the VEGF-mimetic peptide (QK) and the IK-VAV pro-neurogenesis peptide covalently tethered in a 3D model ELR scaffolds demonstrates, within the same scaffold, the preferential guidance of angiogenesis and neurogenesis in a spatiotemporal manner. The synergistic effect of protease-sensitive factors and biochemical signaling in cell infiltration, and subsequent preferential vascularization or innervation, was confirmed. Interestingly, the non-protease-sensitive or slow-protease-sensitive ELR hydrogel containing the pair of ELR cylinders allowed a highly defined one-directional pattern of vascularization and innervation in each cylinder, promoting a faster integration within the host tissue in the presence of the slow one. As such, we have shown that vascularization and innervation can be spatiotemporally controlled by selecting the bioactivities and degradation rates in a single ELR scaffold. The 3D model ELR scaffolds designed open up the possibility of novel strategies in tissue-engineering applications in which the spatiotemporal control of angiogenesis and neurogenesis, as well as the resorbable and nondegradable long-lasting nature of the bioinspired scaffold, play a paramount role. The multi-bioactive scaffold was successfully implanted in a MRONJ model from a living rabbit and evaluated the performance of the scaffold as well as its regenerative capacity in a necrotic environment. A high degree of bone regeneration was founded as histologic and NMR images demonstrated.

*Conclusion/Significance: A groundbreaking dual approach, simultaneously targeting angiogenesis and innervation, addresses the necrotic bone in MRONJ syndrome. Vascularization and nerve formation play pivotal roles in driving reparative elements for bone regeneration. The scaffold, enriched with biofunctionalities like RGD, QK peptides, VEGF mimetic, IKVAV peptide, and protease-sensitive domains, achieves effective time/space control over necrotic bone regeneration. These results pave the way for regenerating significant defects in the maxillofacial area, offering promising prospects in tissue engineering.

72 - Life On Hold - Cryopreservation Of Kidneys And Pancreatic Islets By Vitrification For Transplantation

J. Rao, Z. Han, J. C. Bischof, E. B. Finger

University of Minnesota, Minneapolis, MN

*Purpose/Objectives: Effective protocols to indefinitely extend the lifespan of organs/ cell clusters for transplantation would effectively abolish time from organ donation and distribution, facilitating therapeutic protocols of immune tolerance and recipient pre-conditioning, thereby improve long-term outcomes. Due to an imbalance between ice nucleation and cryoprotective agent (CPA) toxicity, cryopreservation of organs for transplantation has failed to clinically translate. We describe kidney and islet cryopreservation by vitrification and demonstrate post-rewarming cellular/ organ level health and transplant outcomes.

*Methodology: Rat kidneys were vitrified to cryogenic temperatures of -192°C using VMP (CPA) and rewarmed using silica-coated iron oxide nanoparticles generating heat from alternating electromagnetic fields of a radiofrequency coil. Kidneys were assessed for morphology (histology and TEM) and vascular integrity (confocal staining and vascular resistance), in vitro function using normothermic oxygenated machine perfusion (NMP), and in vivo by transplantation.

Pancreatic islet cryopreservation of mouse, porcine, non-human primate, human stem cell derived beta clusters and human deceased donor islet isolation was performed using a cryomesh designed at the University of Minnesota. Viability was assessed by live/dead staining. Cell health by Tetra Methyl Rhodamine Ester stain for mitochondrial membrane potential, SeaHorse oxygen consumption rate, ATP measurement, TEM for mitochondrial morphology, Annexin translocation for apoptosis, and TUNEL stain for DNA integrity. Architectural integrity by histology and TEM. Function by glucose-stimulated insulin release and transplantation.

*Results: Vitrified-nanowared kidneys on non-hematocrit normothermic machine perfusion made urine, consumed oxygen, glucose, and demonstrated venous and urine electrolyte compositions similar to controls. In syngeneic transplants (nephrectomized recipients), all nanowarmed kidneys (n=7) reperfused immediately and uniformly and made urine. Outcomes of cryopreservation time periods of 1 day to 100 days demonstrated no significant differences. Although there was a period of early graft dysfunction lasting 2-3 weeks (peak Cr 12.6 mg/dl), renal function fully normalized after that (Cr 0.4-0.8). Metabolic dysfunction (hyperkalemia, hypervolemia, metabolic acidosis) resolved by POD14. At POD30, serum and urine analysis demonstrated complete normalization of renal function. Microscopy showed focal ATN, hyaline change with intact basement membrane, near normal architecture, and vascular endothelium.

Vitrified islets demonstrated >89% cellular viability with restored ATP content, normal mitochondria and respiration, and regulated insulin secretion in vitro. Vitrified islets durably restored normoglycemia in 9/10 syngeneic transplants. Islets were cryopreserved for the longest period of 9 months and demonstrated insignificant differences in viability and in vitro function and transplant outcomes to that of short periods of cryogenic storage. 9/10 transplants demonstrated reversal of diabetes and normoglycemia within 24-72 hours post transplantation. Engraftment of islets was achieved between 20-30 days with excellent glycemic control for 150 days (study-end point).

*Conclusion/Significance: Vitrification and rewarming of islets and kidneys maintained biological integrity and function for successful transplantation. Biological characterization for immunological response and long-term graft survival will be critical for clinical translation. Although the first series of successful cryopreservation of rat kidneys demonstrates biological feasibility, scaling up to human kidneys involves substantial modifications to cryoprotective agents and cooling-rewarming protocols. High throughput vitrification of pancreatic islets is achievable within acceptable limits of viability and recovery for clinical translation.

74 - Vascular Microphysiological Systems As An Organ Preservation Testbed

Y. Kim, E. Gill, S. R. Mahmoodi, A. Consiglio, J. Velazquez, K. E. Healy

University of California, Berkeley, Berkeley, CA

*Purpose/Objectives: Endothelial cell (EC) damage during cold storage and reperfusion injury after organ transplantation causes deterioration of EC barrier function (BF) and ultimately edema, leading to failure of organ transplants. Here, we introduce a vascular microphysiological system (MPS) as a testbed to investigate the combinational effect of thermal and fluid perturbations (i.e., fluid shear stress (FSS)) on human endothelial BF with the real-time monitoring and quantification of BF using electrical impedance spectroscopy (EIS).

*Methodology: Based on computational modeling (COMSOL Multiphysics) microfabricated polydimethylsiloxane-based microfluidics chips were designed to provide variable FSS ranging from 0 to 20 dynes/cm². The MPS integrated with gold electrode arrays enabled the EIS measurement and quantification of BF through the equivalent circuit model (Fig. 1A). A 3D lumen structure was formed within the vascular MPS using human coronary artery ECs, and was exposed to 40 µL/min of flow for 6 hours before preservation (Fig. 1B). Static cold storage (SCS) at 4^oC was used as clinical control and isochoric supercooling (ISC) at -3^oC was used to preserve vascular MPS in organ transplantation media (i.e., University of Wisconsin solution). After 24-96 hours of preservation, vascular MPS were exposed to variable FSS to simulate the reperfusion injury after organ transplantation and measured with EIS to monitor BF simultaneously.

*Results: FSS > 8 dynes/cm² induced artery-like morphology and VEGFR-3 expression, a permeability-related marker, in the ECs compared to lower FSS and no flow. Tight junction resistances (an indicator of BF) declined to 20-50 % compared to before storage (p<0.0001) (Fig. 1C); however, resistances recovered after rewarming and reperfusion in 24 hours of preservations (p<0.0001). For ISC, 72-96 hours of preservation showed significantly improved recovery of BF compared to SCS (72h: p=0.0077 and 96h: p<0.0001). (Fig. 1D).

*Conclusion/Significance: These results demonstrate that the vascular MPS platform can provide a physiologically relevant in vitro model with variable FSS as a testbed recapitulating ischemia-reperfusion injury in ECs and adding in the optimization of organ preservation protocols.

Figure 1. (A) Photograph of vascular MPS instrumented with gold electrode arrays at 4 regions with different wall shear stresses. The outline of the cell chamber is marked with fiduciary black lines. Scale bar: 1 mm. (B) Confocal immunofluorescence images of human coronary artery ECs inside the channel of the vascular MPS. Inset right demonstrates EC covering the geometrical surface area of the cell channel, clearly developing a 3D open lumen. Scale bar: 100 µm. Green: VE-Cadherin, Blue: DAPI. (C) Tight junction resistances (R_TJ) of EC monolayers on electrodes in the vascular MPS before, immediately post ISC for 24 hours, and after rewarming and reperfusion for 4 hours. (D) R_TJ measured in rewarming and reperfusion for 4 hours after 24-96 hours of SCS and ISC. R_TJ were normalized by before preservation.

75 - Electrospun Magnetic Membranes For Cryopreservation

S. Leal Marin^1,2, A. Lopera Sepulveda^3,4, L. Gangwar⁵, O. Onyinyechukwu⁵, C. Garcia⁴, J. Bischof⁵, B. Glasmacher^1,2

¹ Leibniz University Hannover, Garbsen, Germany,

² Lower Saxony Center for Biomedical Engineering, Implant Research and Development, Hannover, Germany,

³ Universidad Nacional de Colombia sede La Paz, La Paz, Colombia,

⁴ Universidad Nacional de Colombia sede Medellin, Medellin, Colombia,

⁵ University of Minnesota, Minneapolis, MN

*Purpose/Objectives: Cryopreservation is a key technique for the long-term storage of biological samples, but its effectiveness is limited by the harmful effects of ice formation and recrystallization on tissue viability upon freezing and thawing. In response to this challenge, researchers have used magnetic nanoparticles (MNPs) to perfuse organs such as kidneys and liver and apply alternating magnetic fields for thawing to facilitate a uniform rewarming mechanism, mitigating the adverse consequences of prolonged and uneven thawing. This study aimed to synthesize magnetic nanoparticles via the sol-gel combustion method and include them in electrospun membranes to analyze their effect on homogenous and controlled thawing in order to use these membranes for tissue engineering as a helping tool for cryopreservation.

*Methodology: Using the sol-gel combustion method, MNPs were synthesized employing iron nitrate as a precursor and 6-aminohexanoic acid as a combustion agent. The magnetic membranes were fabricated by electrospinning from polycaprolactone solution (140 mg/ml in TFE) and 5% (w/v) of MNPs. The magnetic membranes were frozen by direct immersion in liquid nitrogen in cryovials containing 1ml of VMP (16.8 wt% ethylene glycol, 22.3% DMSO, 12.9% formamide, 1% X-1000, 1% Z-1000) and with a fiberoptic temperature probe inside to measure the freezing-thawing temperatures. After freezing, the cryovials were thawed under an alternating magnetic field using a 120 kW radiofrequency (RF) coil (AMF Life Systems, Auburn Hills, Michigan) at 98% power (provides an RF field at 35 kA/m and 360 kHz). Reference samples of 5% (w/v) MNPs in solution with VMP were used for comparison and commercial MNPs. The morphologic properties of the membranes were analyzed by SEM, the magnetic properties by vibrating sample magnetometry, and the thawing rate and specific absorption rate were calculated. The biocompatibility was assessed by metabolic activity and live staining with human dermal fibroblast.

*Results: The obtained membranes exhibited a fiber size of approximately 400 nm without agglomerations of magnetic nanoparticles (MNPs). Since the size of the MNPs is around 50-100 nm, the hypothesis is that the majority of particles are located within the fibers. Magnetic characterization revealed superparamagnetic properties for the synthesized MNPs and ferromagnetic properties for the obtained membranes. Regarding the specific absorption rate values, the synthesized MNPs in solution showed higher values (586.3 W/gFe), than commercial particles (393.1 W/gFe). However, the values obtained for the magnetic membranes were only 6% (35.2 W/gFe) compared to particles in solution. These results correlated with faster thawing rates of 50 K/min for the synthesized MNPs in solution in comparison to 36 K/min for the commercial MNPs. The biocompatibility test results showed the growth of the cells on the magnetic membranes and metabolic activity comparable with membranes without MNPs.

*Conclusion/Significance: Although the magnetic membranes' specific absorption values were very low, the synthesized MNPs have great potential for improved thawing without adverse effects on biocompatibility. Future studies should be done on increasing the concentration of the particles in the membranes for application in cryopreservation.

79 - Controlled Delivery Of UPI Peptide Via PLGA Microparticles To Enhance Endothelization

S. Changizi¹, N. Muthu Vijayan¹, H. Chen², C. A. Bashur^1,3

¹ Florida institute of technology, Melbourne, FL,

² Boston Children’s Hospital, Boston, MA,

³ Florida institute of technology, Melboure, FL

*Purpose/Objectives: Tissue-engineered vascular grafts (TEVGs) are an alternative graft, particularly for vessels with diameters ≤6 mm. Our prior research showed that epsin-mimetic UPI peptides can promote endothelial cell (EC) proliferation by preventing vascular endothelial growth factor receptor 2 (VEGFR2) internalization, and incorporation within TEVGs allowed rapid endothelialization and 100% patency at 1 week. However, we hypothesize that local, more sustained release from microparticle carriers is important for longer-term grafting outcomes. Thus, the goals of this study are to develop microparticle-incorporated gelatin coatings for more extended delivery of UPI peptides and assess their effects on graft endothelialization and remodeling.

*Methodology: The UPI peptide was entrapped within poly (DL-lactide-co-glycolide) (PLGA) microparticles using a water/oil/water double emulsion technique with 2mg/g peptide/PLGA. After lyophilization, peptide release in saline was evaluated via a BCA assay. These microparticles were integrated with a luminal gelatin coating within electrospun polycaprolactone (PCL) conduits using a custom mold. SEM imaging characterized the individual microparticles and final graft structure. Human umbilical vein ECs (HUVECS) were used to assess the effects of exogenous UPI peptide on cell migration (scratch test, 0-18 h intervals) and on cell attachment to PCL spincoated films 1 day after seeding. Scrambled peptides (control group) were also tested to determine the specificity of the UPI response. Further, the specificity of the iRGD sequence of the peptide for ECs was tested by comparing the response of smooth muscle cells (SMC) and normal human dermal cells (NHDF). Immunofluorescence and real time-PCR was performed to characterize cell number and phenotype in vitro. Finally, end-to-end rat aortal grafting is being performed for 4 weeks prior to graft characterization with histology and immunofluorescence. One-way ANOVA and Tukey comparisons were performed.

*Results: HUVECs migrated significantly further with exogenous 100 µg/mL UPI peptide compared to scrambled peptide and culture media only (n=3), demonstrating specificity. Preliminary results also appear to show that incorporating UPI peptide improves EC organization at day 1 and 3 of culture compared to scrambled peptide (n=3), with dense areas of circular organization observed. For scrambled peptides, the cells appeared more rounded at this dose. Further, the 3 day culture appeared to show higher cell confluency for the UPI peptide condition. Spherical microparticles with/without peptide loading were prepared with an average diameter of 6.62±4.07 µm. UPI peptide exhibited an extended release from PLGA microparticles and continued to release over the 28 days tested so far (42% released) and is ongoing. Microparticles were incorporated within the gelatin coating, and SEM of the lumen showed a mostly consistent layer of gelatin with protein loaded microparticles. We are currently analyzing the impact of the gelatin/microparticle coated grafts in vivo both with and without UPI peptides.

*Conclusion/Significance: This study showed that UPI peptides exhibit specific benefits for EC migration and organization not seen with scrambled peptides, which may explain how they improve in vivo TEVG endothelialization. Further, extended and controlled release of UPI peptides can be achieved for PLGA microparticles embedded conduits. This may be important clinically where graft endothelialization takes longer than in rodent models.

81 - Single Cell Analysis Reveals Diverse Senescent Cell Phenotypes in the Foreign Body Response to Synthetic Implants

A. N. Rindone¹, S. Nagaraj¹, K. Krishnan¹, J. C. Mejías¹, F. Haoning Yu¹, C. Christopher², L. Davenport Huyer³, E. J. Fertig¹, J. H. Elisseeff¹

¹ Johns Hopkins University School of Medicine, Baltimore, MD,

² C M Cherry Consulting, Baltimore, MD,

³ Dalhousie University, Halifax,, NS, Canada

*Purpose/Objectives: The foreign body response to biomaterials (FBR) is a significant barrier to the success of regenerative medicine therapies. We recently showed that senescent stromal cells (SnCs)—non-proliferative cells with a secretory phenotype—accumulate and drive excess fibrosis during the FBR. However, SnCs are known to play diverse roles in wound healing and fibrosis, and our lack of understanding of in vivo SnC phenotypes hinders our ability to target pathological SnCs in the FBR. Here, we conduct single cell RNA-sequencing (scRNA-seq) on human breast implant capsules, and apply a transfer learning approach, ProjectR, to an in vivo-derived senescent signature (SenSig) to define the SnC phenotypes in the FBR and identify SnC-specific therapeutic targets.

*Methodology: To conduct scRNA-seq of the FBR, we obtained de-identified fibrotic capsules from patients undergoing breast implant replacement surgeries under an approved JHU IRB exemption. Cells from the capsules were isolated, enriched 75:25 for CD45+ immune cells, and sequenced using 10x Chromium. Data were processed and analyzed using a standardized pipeline in Seurat. We applied transfer learning of SenSig to the capsule dataset via ProjectR (SenSig-ProjectR) to identify cells that were concordant with the signature. Cells with a positive projection weight and adjusted p-value less than 0.01 were considered senescent. To characterize SnC phenotypes, we conducted differential gene expression analysis between SnCs and non-SnCs on non-cycling clusters with greater than 5% SnCs and 500 total cells.

*Results: Using SenSig-ProjectR, we identified three cell clusters enriched in SnCs (Figure 1A): Myofibroblasts/cartilage-like fibroblasts (Fib_1), stromal/progenitor-like fibroblasts (Fib_2), and pericytes/smooth muscle cells (Pericyte_SMC). These SnC subtypes shared common gene expression patterns. Notably, all SnC subtypes upregulated genes associated with ECM deposition and remodeling, including COL1A1/2, POSTN, and TIMP1 (Figure 1B). Both fibroblast SnC subtypes also upregulated cartilage ECM genes such as COMP, COL11A1, and LUM, suggesting both subtypes adopt a fibrocartilage-like phenotype.

However, SnC subtypes exhibited distinct gene expression patterns, suggesting differences in their phenotype and function. Fib_1 SnCs upregulated translation and cytoskeletal contraction genes, which are associated with a pro-fibrotic secretory and contractile phenotype, while Fib_2 SnCs expressed a greater diversity of collagens and inflammation genes (Figure 1B-C). Pericyte_SMC SnCs uniquely expressed lipid metabolism genes (e.g. CD36, PPARG, LPL) linked to homeostatic and pathological vascular function.

Furthermore, SnC subtypes expressed distinct cell surface protein genes that may serve as therapeutic targets (Figure 1D-F). Both fibroblast SnC subtypes upregulated LRRC15, a marker associated with pathological fibrosis. However, Fib_1 SnCs specifically upregulated TSPAN2, LAMP5, and SDC1; while Fib_2 SnCs specifically upregulated NGFR. There were no markers exclusive to Pericyte_SMC SnCs, but SEMA5B, NOTCH3, and STEAP4 were highly expressed in Pericyte_SnCs relative to other cell types.

*Conclusion/Significance: Using scRNA-seq and SenSig-ProjectR, we identified one perivascular and two fibroblastic SnC subtypes in the FBR. While all SnCs upregulated ECM-associated genes, Fib_1 SnCs exhibited the most distinct pro-fibrotic phenotype, while Fib_2 and Pericyte_SMC SnCs expressed genes associated with diverse processes including fibrosis, inflammation, and vascular function. These data can inform SnC-targeted therapeutic development to mitigate pathological fibrosis and improve outcomes of biomaterial-based regenerative therapies.

82 - Morphology-based Deep Learning For Prediction Of Osteogenic And Adipogenic Differentiation

S. Wang

University of New Haven, West Haven, CT

*Purpose/Objectives: Human mesenchymal stem cells (hMSCs) are multipotent progenitor cells with the potential to differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes. These cells have been extensively employed in the field of cell-based therapies and regenerative medicine due to their inherent attributes of self-renewal and multipotency. Traditional approaches for assessing hMSCs differentiation capacity have relied heavily on labor-intensive techniques, such as RT-PCR, immunostaining, and Western blot, to identify specific biomarkers. However, these methods are not only time-consuming and economically demanding, but also require the fixation of cells, resulting in the loss of temporal data. Consequently, there is an emerging need for a more efficient and precise approach to predict hMSCs differentiation in live cells, particularly for osteogenic and adipogenic differentiation. In response to this need, we developed innovative approaches that combine live-cell imaging with cutting-edge deep learning techniques, specifically employing a convolutional neural network (CNN) to meticulously classify osteogenic and adipogenic differentiation.

*Methodology: We developed four notable pre-trained CNN models, VGG 19, Inception V3, ResNet 18, and ResNet 50, were developed and tested for identifying adipogenic and osteogenic differentiated cells based on cell morphology changes. We rigorously evaluated the performance of these four models concerning binary and multi-class classification of differentiated cells at various time intervals, focusing on pivotal metrics such as accuracy, the area under the receiver operating characteristic curve (AUC), sensitivity, precision, and F1-score.

*Results: Among these four models, both VGG 19 and ResNet 50 showed excellent performance with high accuracy for both binary (0.9572) and multi-class classification (0.9474). ResNet 50 showed consistent performance with high AUC (0.9936) for multi-class classification. Importantly, all these four models exhibited exceptional performance with the overall accuracy of more than 0.93, and overall AUC score of more than 0.94. By analyzing the daily images of differentiated cells, all these models can accurately detect subtle morphological changes within 1 day of differentiation.

*Conclusion/Significance: In summary, our study underscores the immense potential of using a CNN approach to predict stem cell fate based on cellular morphological changes of differentiated cells. This approach holds promise for enhancing the application of cell-based therapy and expanding our knowledge of regenerative medicine. Additionally, this non-invasive method, relying solely on basic bright-field microscope images, has the potential to facilitate biomanufacturing and the translation of these advancements into practical cell-based therapies.

83 - Functional Guide-rna Cell Barcoding For Tracking Hipsc Clonal Lineages During Directed Differentiation Into Cardiomyocytes

S. Sohn, J. Zoldan, D. Morgan, A. Brock

The University of Texas at Austin, Austin, TX

*Purpose/Objectives: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have great potential in tissue engineering applications, but their clinical relevance is hindered by the heterogeneity of cardiac differentiation, which yields varying abundances of CMs and CM subtypes, even within a single hiPSC line. A potential source of this heterogeneity is differential responses to cardiac differentiation cues that arise from epigenetic differences between subpopulations of hiPSC clonal lineages. This may make lineages “primed” to differentiate into different cell fates in the cardiac niche. A potential solution to address this challenge is to employ cell barcoding to analyze lineage dynamics during cardiac differentiation. Utilizing the functional cell barcoding platform ClonMapper from Dr. Amy Brock's lab, it is possible to live-isolate specific lineages throughout differentiation. This approach can facilitate evaluation of differential expression in lineages that are “primed” for different cell fates within the cardiac niche. Characterizing the cardiac differentiation properties of different hiPSC lineages may elucidate methods into improving homogeneity of cardiac differentiation. This will be accomplished with the following objectives: i) establish a ClonMapper barcoded hiPSCs to assess if barcoding negatively impacts differentiation capacity, and ii) determine if hiPSC clonal dynamics are influenced by cardiac differentiation.

*Methodology: ClonMapper guide-RNA barcodes were integrated into hiPSC genomes via lentivirus transduction. Successfully transduced hiPSCs were sorted via fluorescence activated cell sorting, as the barcodes are conjugated to a gene encoding blue fluorescent protein. Barcoded (BC), sorted hiPSCs were seeded on Geltrex coated plates for cardiac differentiation via Wnt modulation. Pluripotency was assessed via immunostaining for pluripotent markers in BC hiPSCs, and via immunostaining and qPCR for cardiac markers in BC hiPSC-CMs. qPCR was performed on mRNA extracted from hiPSC-CMs on days 7-28 for cardiac genes, using gene expression on day 0 (undifferentiated hiPSCs) to determine ΔCt, and using ΔCt values of BC and non-barcoded (NBC) samples to determine ΔΔCt and calculate fold-change.

*Results: Immunostaining showed BC hiPSCs retain expression of Oct4 and Sox2 and can differentiate into cTnT-expressing hiPSC-CMs, suggesting barcoding does not negatively impact pluripotency. Both NBC and BC populations had similar levels of TNNI3 and TNNI1 expression until day 28, where NBC hiPSC-CMs had significantly greater expression of TNNI3 and significantly lower expression of TNNI1 relative to BC hiPSC-CMs, suggesting BC hiPSC-CMs are less mature, as a higher TNNI3:TNNI1 ratio is associated with CM maturity. Conversely, relative expression of calcium handling genes RYR2, CACNA1C, and CASQ2 was significantly greater in BC than NBC hiPSC-CMs at several timepoints. Increased expression of these genes indicates longer action potential duration and increased calcium storage and cycling, suggesting BC hiPSC-CMs are more mature than NBC controls.

*Conclusion/Significance: We have shown BC hiPSC-CMs maintain pluripotency and cardiac differentiation capacity, though they differ from NBC hiPSC-CMs in expression of several genes linked to cardiomyocyte maturity. While additional analyses are needed to determine whether ClonMapper barcoding affects function of hiPSC-CMs in a meaningful way, our data indicate that barcoded hiPSCs are pluripotent and ClonMapper is a viable method for examining clonal dynamics of hiPSC cardiac differentiation.

84 - Determining Optimal Scaffold Geometries For Rotator Cuff Repair: A Parametric Finite Element Approach

S. E. Winston, K. McGilvray

Colorado State University, Fort Collins, CO

*Purpose/Objectives: There are >250,000 rotator cuff repair surgeries in the United States annually. Unfortunately, these repair strategies fail at rates upwards of 90%. Examining these repaired tissues shows that the native biomechanical gradient between bone and tendon, the enthesis, is not regenerated post-op.Most tissue engineering approaches to repair the rotator cuff enthesis have utilized scaffolds with graded material properties to cue the local cell milieu to deposit tissue-specific (e.g., bone to tendon) extracellular matrix and aligned collagen at the repair site. However, advanced additive manufacturing techniques, specifically melt electrowiriting (MEW), allow the creation of complex, cellularly instructive architectures with a single material to attune local mechanics. Using MEW our group has developed a novel tissue engineering scaffold with an elegant, arced geometry that demonstrates a gradient in localized stiffness using a single material across a length scale translational to that observed at the human enthesis. Our group aims to investigate how the arced radius of curvature, fiber spacing, and fiber diameter of our biomechanically instructive scaffold affect the stiffness gradient properties of these scaffolds using a parametric finite element (FEA) approach. We hypothesize that our novel architecture will recapitulate the biomimetic gradient of stiffness seen across the rotator cuff enthesis, resulting in biomechanically robust enthesis formation at the repair site.

*Methodology: A single arced scaffold architecture was printed using MEW using polycaprolactone (Fig 1a). Samples (N=4) underwent uniaxial tensile testing to 4% global strain. Regional strain across the length of the scaffold was measured using digital image correlation (MATLAB 2022b). These data served to validate a 2D beam FEA model of the arced geometry (Abaqus 2021) (Fig 1a). This model was then subject to a parametric study where the radius of curvature, spacing, and fiber diameter, were varied across the printability of our MEW system. 540 simulations have been analyzed. For each simulation, the ‘strain gradient factor’ (SGF - unitless) was calculated as the ratio of strain at the bony end of the scaffold to that at the tendon side (Fig 1a).

*Results: Variations in the SGF were correlated with alterations in the scaffold's radius. Spearman correlation coefficients were determined as -0.84 for the radius of curvature, -0.24 for fiber diameter, and -0.08 for fiber spacing, suggesting a strong inverse relationship with radius, a weak inverse relationship with fiber diameter, and a negligible inverse relationship with spacing in terms of their impact on the SGF. The SGF exhibited a range from 63.5 to 1.7 (Fig. 1b) when the radius was varied from 38.5 mm to 150 mm. At the smallest assessed radius, the SGF was modulated from 42.4 to 7.9, correlating with fiber diameters ranging from 10 µm to 70 µm.

*Conclusion/Significance: The introduction of this novel, arced geometry facilitated the creation of a tunable gradient structure, resembling the gradient observed in the enthesis (Fig. 1c). Future investigations will focus on examining the cellular responses elicited by the scaffold, with the goal of generating a gradient transition of tissue from bone to tendon for use in rotator cuff repair.

86 - Vascularized Stem Cell-based Constructs For Bone Tissue Engineering

J. Musilkova¹, A. Sedlar¹, L. Svobodova¹, M. Beran², L. Bačáková¹

¹ Biomaterials and Tissue Engineering, Institute of Physiology CAS, Prague, CZECH REPUBLIC,

² Food Research Institute in Prague, Prague, CZECH REPUBLIC

*Purpose/Objectives: Tissue engineering aims to create tissue prostheses as a combination of biodegradable polymeric scaffold and cellular component that enables tissue self-regeneration. The biodegradable scaffold serves as a temporary mechanical support for cells, offers a suitable environment for the adhesion, proliferation, and differentiation of the stem cells, and stimulates the production of extracellular matrix proteins. The self-regeneration proces could be supported by the vascularization of the prostheses and prolonged their lifetime.

*Methodology: In our work we tested 10 different types of 3D printed scaffolds with fully interconnected porous system, prepared from bioresorbable polymer PLA, functionalized by mineralization with simulated body fluid solution. The scaffolds differ from the inner architectures of polymer fibers. As a cellular component, human adipose tissue-derived stem cells (ASCs) and human umbilical vein endothelial cells (HUVECs) were used. The vascularization process of the prostheses was supported by stimulation in the dynamic system STUART. The cell adhesion, number, morphology, and cell penetration inside the scaffolds were followed by fluorescence staining with TRITC-conjugated phalloidin and DAPI, using a Dragonfly 503 confocal microscope. Possible vascularization of the scaffolds was tested in experiments with co-cultivation of ASCs with HUVECs. Later immunostaining of specific endothelial markers E-cadherin for specification of endothelial cells was used.

*Results: All the 3D printed scaffolds are very stiff and comply with demands for mechanical properties of bone implants. So the basic criterion for the scaffold type selection was the density of the ASCs colonization of the scaffolds. 4 types of 3D-printed scaffolds were found as appropriate cell carriers. The cells were spread along the fibers, proliferated well, and subsequently colonized the inside of the scaffolds to the depth of approx. 400 μm. The cell penetration inside the scaffolds is an important prerequisite for the successful creation of functional 3D tissue replacements. We found, that the honeycomb-like inner architecture of scaffold fibers is more suitable for cell colonization than the rectangular. We estimated, that ASCs promoted the the self-assembling of endothelial cells in a ratio of 1:1 (ASCs : HUVECs). For the cell-cocultivation and probing the optimal conditions for vascular base self-assembling, we selected four scaffold types. On three of them, we detected noticeable endothelial structures that could be the base for graft vascularization, after both static and dynamic cultivation. The basic vascularization of the constructs was stronger under dynamic conditions.

*Conclusion/Significance: PLA 3D-printed scaffolds are therefore a suitable material for creating stem cell-based vascularized bone grafts. They enabled a sufficient penetration of cells inside the construct and simultaneous co-cultivation of ASCs with endothelial cells to promote the construct's vascularization.

Supported by Ministry of Health of the Czech Republic (grant No. NU20-08-00208), and by the Praemium Academiae grant No. AP 2202 provided by the Czech Academy of Sciences.

87 - Preclinical Evaluation Of Polymer-based Composites For Patient-individual Additive Manufacturing Of Biodegradable Jawbone Implants - A Minipig Study

C. Vater¹, E. Jarmer², A. Lode¹, T. Fecht³, S. Grom³, A. Placht¹, T. Ahlfeld¹, M. Scharffenberg⁴, J. Wittenstein⁴, R. Jung⁵, S. Heinemann⁶, A. Höß⁶, G. Lauer², F. Reinauer³, T. Wolfram³, M. Gelinsky¹, C. Bräuer²

¹ Centre for Translational Bone, Joint and Soft Tissue Research, University Hospital Carl Gustav Carus and Medical Faculty of Technische Universität Dresden, Dresden, GERMANY,

² Department of Oral and Maxillofacial Surgery, University Hospital Carl Gustav Carus and Medical Faculty of Technische Universität Dresden, Dresden, GERMANY,

³ KLS Martin SE & Co. KG, Mühlheim, GERMANY,

⁴ Department of Anesthesiology and Intensive Care Medicine, Pulmonary Engineering Group, University Hospital Carl Gustav Carus and Medical Faculty of Technische Universität Dresden, Dresden, GERMANY,

⁵ Experimental Center of the Medical Faculty Carl Gustav Carus, Technische Universität Dresden, University Hospital Carl Gustav Carus and Medical Faculty of Technische Universität Dresden, Dresden, GERMANY,

⁶ INNOTERE GmbH, Radebeul, GERMANY

*Purpose/Objectives: A promising therapeutic option for the treatment of critical-size jawbone defects is the implantation of biodegradable, porous structures which are produced patient-specifically by using additive manufacturing techniques. In this work, the degradable poly(DL-lactide) polymer (PDLLA) was combined with different mineral phases (calcium carbonate - CaCO₃, strontium-modified hydroxyapatite - SrHAp,) with the aim to buffer its acidic degradation products, which can cause inflammation, and to stimulate bone regeneration in a jawbone defect model.

*Methodology: A segmental lower jawbone defect of approximately 3 cm was created in 14 adult minipigs (56,0 ± 6,5 kg) and filled with individually manufactured implants consisting of: (1) control PDLLA (n = 4), (2) composite PDLLA:CaCO₃ (n = 2, PDLLA blended with CaCO₃ particles), (3) composite PDLLA:SrHAp (n = 4, PDLLA blended with SrHAp particles) and (4) hybrid PDLLA-SrHAp (n = 4, PDLLA encasing a SrHAp core). The defect was stabilized by an individual titanium reconstruction plate. During the 6 months-lasting observation period, the progress of bone regeneration was monitored by repetitive digital volume tomography scans. Additionally, 3 different fluorescent dyes were administered to gain information about the way of bone formation. In order to better address the clinical situation, one animal of group (1), (3) and (4) was observed for 14 month following removal of the reconstruction plate.

*Results: All 14 animals survived the 6 months observation period, but n = 12/14 animals showed intra-oral suture dehiscences and/or extra-oral fistulas independently of the implant. Due to strong swelling, composite implants made of PDLLA:CaCO₃ (n = 2/2) and PDLLA:SrHAp (n = 1/4) had to be removed prematurely by 6, 8 and 14 weeks postoperatively. For the hybrid implant PDLLA-SrHAp, dislocation and fracturing of the SrHAp core occurred in n = 1/4 animals. Generally, after 6 months defect bridging could be observed in all animals with formation of new bone starting within the first 5 weeks and originating from the defect borders and the periosteum along the implant/reconstruction plate and around the implant, respectively. Bone ingrowth into the implant could be detected only for PDLLA-SrHAp. After 14 months, the remaining 3 animals demonstrated an ongoing bony consolidation of the defect area with increasing bone volume. Probably due to material degradation for PDLLA and PDLLA:SrHAp, new bone formation occurred around the implant, whilst bone ingrowth into the implant could only be seen for PDLLA-SrHAp.

*Conclusion/Significance: In this experimental model, due to their swelling and inflammation-inducing degradation products, the designed scaffolds in this study based on PDLLA and PDLLA blended with CaCO₃ or SrHAp seem not suitable for the treatment of large bone defects in this minipig model. In contrast, PDLLA-SrHAp induced bony ingrowth and might therefore be the most promising option for the regeneration of such defects. However, the amount of PDLLA within these implants should be kept as low as possible.

89 - Development Of A Scaffold-based Platform For Simultaneousdelivery Of Microrna Mimics And Microrna Inhibitor For The Treatment Of Large-volume Bone Defects

J. Sadowska, A. Matheson, M. Ziminska, A. Balouch, S. Wojda, C. Ferreira, H. McCarthy, S. Donahue, F. O’Brien

Royal College of Surgeons in Ireland, Dublin, IRELAND, Queen’s University Belfast, Belfast, UNITED KINGDOM, University of Massachusetts Amherst, Amherst, MA

*Purpose/Objectives: The treatment of large-volume bone defects remains one of the most significant challenges in modern tissue engineering. The delivery of microRNAs from scaffolds emerges as a promising strategy to tackle this issue, as they enhance the expression of beneficial genes while suppressing detrimental effects. Scaffold provides structural support to bone tissue and controlled delivery of microRNAs (miRs) to induce endogenous cells to produce relevant therapeutic proteins with a physiological release profile and minimal side effects. In this study, collagen-nanohydroxyapatite (coll-nHA) scaffold were combined with the cell-penetrating RALA peptide as a vector. This created a miR-activated scaffold system for the simultaneous delivery of both miR-26a mimics and miR-133a inhibitor for bone repair.

*Methodology: Development and physicochemical characterisation of dual-miR scaffolds: The coll-nHA scaffolds were prepared using freeze-drying techniques followed by the incorporation of a mixture of miR-26a-RALA and antagomiR-133a-RALA nanoparticles at 1 μg and 3μg concentrations. The dual-miR scaffolds were assessed in terms of: the miR and antagomiR NPs distribution and release, morphology, weight loss, and calcium release. In vitro evaluation with human mesenchymal stem cells: The biological validation included assessment of: metabolic activity, DNA content, cell distribution (H&E staining), transfection efficiency, expression of osteogenic targets and mineralisation (Ca²⁺ quantification and Alizarin Red staining). In vivo evaluation in a calvarial defect in rats: The scaffolds were implanted into 7 mm defects; bone volume and density were assessed by μCT at 4- and 8 weeks post-surgery. The samples were harvested at 8 weeks and assessed histomorphometrically using H&E staining.

*Results: The scaffolds facilitated a controlled and prolonged release of the therapeutic cargo over 24 hours, with a retention of approximately 60% for the 1μg dosage and about 80% for the 3 μg dosage of NPs spanning the 28 days of the study. The dual-miR scaffold effectively transfected human mesenchymal stem cells (hMSCs) with both therapeutic agents, resulting in a 20-fold increase for miR-26a mimics and a 10-fold decrease for antagomiR-133a (Fig.1A). The hMSCs cultured on dual-miR scaffolds demonstrated increased mineralisation and calcium production (Fig.1B). The dual-miR scaffolds significantly accelerated bone repair in critical-sized calvarial defects of male rats (Fig.2) leading to over a 50% higher bone volume compared to the miR-free group (Fig.2A). The scaffolds yielded a higher bone mineral density (Fig.2C), underscoring their potential to foster the growth of tissue with superior quality.

*Conclusion/Significance: This study describes the development of a 3D dual-miR scaffold system utilising a novel cell-penetrating peptide for combined and controlled delivery of osteogenic miR and antagomiR for orthopaedic applications. The dual-miR scaffold was capable of transfection of hMSCs with both miR and antagomiR, which translated into an enhanced expression of targeted, osteogenic genes (ALP, BMP2). The increased level of ALP and enhanced calcium deposition shows that the miR-26a mimics and miR-133a inhibitor positively regulate osteogenesis. This in vitro success faithfully mirrored in vivo outcomes, indicating potent healing of critical-sized defects, yielding a highly mineralised bone matrix. This demonstrates that the scaffold developed herein produces a new tissue of superior quality, indicating an exciting therapeutic outcome.

90 - Photocrosslinked Silk Fibroin Microgels And Microgel Scaffolds For Biomedical Applications

F. Karimi¹, N. Farbehi¹, M. Haghighattalab¹, K. Lau², M. Kordanovski¹, K. Lim², J. Rnjak-Kovacina¹

¹ Graduate School of Biomedical Engineering, University of New South Wales, Sydney, AUSTRALIA,

² University of Sydney, Sydney, AUSTRALIA

*Purpose/Objectives: Silk fibroin hydrogels, widely investigated for tissue engineering, have limited utility due to their nanometre pore size, restricting adequate cell, tissue, and vascular infiltration and remodelling post-implantation. Microgel scaffolds are an emerging class of microporous biomaterials formed by annealing small microscale hydrogels (microgels) into a 3D construct.This study aims to address the limitation of nano-porosity in silk fibroin hydrogels by developing photocrosslinked silk fibroin microgels and microgel scaffolds with micro-porosity (Figure 1).

*Methodology: Silk microgels were generated using a microfluidic device that allowed tuning of the microgel diameter (100-400 µm) and were stabilized via visible light-initiated photo-crosslinking of native tyrosine residues in silk (using the photoinitiator ruthenium). Microgels were then covalently annealed using silk solution as glue and the same cytocompatible visible light-initiated crosslinking to form microgel scaffolds.

*Results: Small (∼100 µm diameter) and large (∼400 µm diameter) spherical silk microgels were generated by a water-in-oil emulsion approach within a flow-focusing microfluidic device. The photocrosslinking reaction allowed the formation of microgels in the absence of silk modification, as well as covalent encapsulation of biological molecules in the microgels. Microgels were then covalently annealed using silk solution as a glue, making this into a cytocompatible cell encapsulation platform. Unlike the nano-porosity of bulk photo-crosslinked silk hydrogels, the microgel scaffolds had an average pore diameter of 29±17 µm or 192±81 µm depending on the microgel size, with enhanced mechanical properties compared to bulk hydrogels. The tuneable nature of this platform allows the formation of heterogeneous materials biofunctionalized with different molecules at different ratios and with spatial control. The microporosity supported enhanced cell spreading and proliferation in vitro and increased scaffold remodeling in vivo, encouraging improved tissue infiltration and matrix deposition. Analysis of the immune reaction to these implants suggested that large microgels support pro-healing macrophages (CD206+) and material remodeling relative to small microgels. Finally annealing of the microgels into microgels scaffolds supported improved immune response relative to loose microgels with fewer macrophages and fewer pro-inflammatory (MHCII+) macrophages.

*Conclusion/Significance: This work establishes silk microgels and silk microgel scaffolds as promising therapeutic candidates for tissue engineering and regenerative medicine applications, addressing the limitations of traditional silk hydrogels.

91 - Impact Of Microgel Size And Interstitial Voids On Cartilage Tissue Formation By Encapsulated Mesenchymal Stromal Cells

T. Nguyen¹, F. Li², V. Truong³, N. Medhekar¹, H. Thissen², J. Forsythe¹, J. Frith⁴

¹ Materials Science and Engineering, Monash University, Clayton, AUSTRALIA,

² CSIRO Manufacturing, CSIRO, Clayton, AUSTRALIA,

³ Institute of Sustainability for Chemicals, Agency for Science, Technology and Research, Singapore, SINGAPORE,

⁴ Materials Science and Engineering, Monash University, Melbourne, AUSTRALIA

*Purpose/Objectives: Microgels are attracting increasing attention for tissue regeneration applications, including cartilage tissue-engineering where assembled microgels laden with mesenchymal stromal cells (MSCs) have been shown to outperform bulk hydrogels. Whilst the reasons for this are not fully understood, it is clear that cells migrate to the microgel surface and deposit new tissue in the voids between microgels. Microgel size is therefore likely to be an important contributor to the success of microgel-based cartilage tissue engineering, but the impact of this on MSC chondrogenesis and cartilage tissue formation is not yet known. In this study we developed a system to produce microgels of varying diameter and used these to investigate the impact of microgel size on MSC chondrogenesis and cartilage deposition.

*Methodology: A microfluidic system to fabricate microgels of varying diamater was developed using commonly available laboratory consumables, including pipette tips syringe needles, and tubing of various sizes. Cells were dispersed within the precursor solutions for a photocrosslinkable gelatin-norborne and PEG-dithiol hydrogel and encapsulated within microgels prior to crosslinking. Microgel diameter was modulated by varying the nozzle size and flow rate, using image analysis to measure hydrogel diameter. Human bone-marrow MSCs were encapsulated in three microgel diameters (800, 400, and 180 µm) and cultured with chondrogenic supplements to generate engineered cartilage constructs. Cell viability was assessed by Live/Dead staining, cell morphology and distribution by Hoechst and actin staining and chondrogenesis via histology, GAG quantitation and immunohistochemistry. An image analysis program was developed using MatLab to characterize the connectivity of the formed tissues.

*Results: Measurements of microgel diameter showed that it was possible to generate highly monodisperse microgel populations within a range of 100-800 µm. When encapsulated, MSCs retained excellent viability, both after initial encapsulation and for longer term culture. Comparisons were made between assemblies of microgels of 180, 400 and 800 µm diameter. In line with our earlier studies, MSCs migrated to the microgel periphery. For 180 and 400 µm microgels this formed an interconnected tissue structure, but for 800 µm microgels there were still discrete particles. When exposed to chondrogenic supplements, all samples showed increased GAG deposition with the highest levels in 800 µm microgels and decreasing with microgel diameter. Histology showed preferential deposition of this matrix in the voids with substantial differences in tissue structure, where the largest microgels were connected only by thin sections of matrix, whereas the 180 µm microgel samples had a connected matrix, but had collapsed leaving a shrunken construct. Assemblies of 400 µm microgels showed good tissue connectivity without substantial construct shrinkage and the highest levels of type-2-collagen deposition.

*Conclusion/Significance: This study shows clear differences in tissue structure when altering the microgel diameter, where small microgels rapidly degraded resulting in a shrunken tissue whilst the largest microgels did not support tissue connectivity. Overall microgels of 400 µm diameter were optimum for cartilage tissue-engineering. This provides new insights of the interplay between construct architecture, cell migration and matrix deposition in tissue engineering and highlights the importance of considering microgel size and construct porosity in tissue-engineering.

92

93 - Annealed Pvdf-trfe Scaffolds Enhance Piezoelectric Response And Membrane Stiffness For Nerve Repair Applications

M. Krutko¹, H. M. Poling¹, A. E. Bryan², G. M. Harris², L. Esfandiari¹

¹ Biomedical Engineering, University of Cincinnati, Cincinnati, OH,

² Chemical Engineering, University of Cincinnati, Cincinnati, OH

*Purpose/Objectives: Bioelectrical signals regulate broad tissue function and have been shown to aid in regeneration of various tissue such as nerves and skin following injury. Furthermore, an array of electrical stimulation methods has been proven effective in inducing regenerative changes, impacting both excitable cells like neurons and non-excitable cells like Schwann cells (SCs), both pivotal in nerve repair. Next generation scaffolds for tissue repair are engineered with biomimicry in mind, and thus emulate innate tissue properties for enhanced biocompatibility and functional outcomes. Still, many scaffolds have not yet been designed to produce electrical signals directly to neighboring cells and tissue. Piezoelectric scaffolds, such as piezoelectric polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE), possess the ability to produce electrical signals when mechanically stimulated, and offer a solution bridging this limitation. Still, PVDF-TrFE when fabricated as a film produces unideal morphology to guide nerve repair and has a relatively low piezoelectric coefficient. In this investigation, we employ electrospinning to create aligned scaffolds from PVDF-TrFE. Our primary objective is to examine the effects of annealing, a thermal process, on scaffold topographical and mechanical attributes. Furthermore, we investigate its capability to amplify piezoelectric voltage output and facilitate Schwann cell (SC) proliferation, aiming to advance its utility in nerve repair applications

*Methodology: PVDF-TrFE scaffolds were thermally analyzed through differential scanning calorimetry to generate a crystallization curve of the polymer, which signified crstallization occurs at 140°C. PVDF-TrFE were then subsequently annealed at 140°C for 1.5 hours and underwent scanning electron microscopy to depict surface topography changes compared to control. Both scaffold groups were then stimulated through tensile forces to reach maximum deformation, and resultant piezoelectric voltage output was measured through an oscilloscope. Mechanical properties were characterized through atomic force microscopy. Schwann cells were seeded on both scaffold groups and fixed after two days to stain for DAPI, GFAP, and KI67. Confocal microscopy was used to analyze the percent of KI67 positive cells to indicate percent of cells proliferating in annealed and control groups.

*Results: Annealing PVDF-TrFE at 140°C resulted in increased presence of lamellar like structures, which improved overall porosity. The annealed scaffold group produced about double maximum piezoelectric voltage output and resulted with a membrane stiffness approximately triple that of control group. Moreso, it was found that proliferation of Schwann cells on the annealed group was improved by approximately 40% compared to control group.

*Conclusion/Significance: Annealing, as a post-processing technique, has the potential to modify the material properties of polymer scaffolds, expanding their adaptability and utility as biomaterials. Notably, annealing PVDF-TrFE at 140°C has demonstrated the ability to enhance porosity, membrane stiffness, and piezoelectric output. Furthermore, the development of a proliferative phenotype in Schwann cells on annealed substrates highlights the promising applications of annealed PVDF-TrFE in nerve grafting and other broad tissue engineering applications.

94 - Giving It A Twist: Boosting Ultrastructural Biomimicry Of Collagen

V. Priebe¹, Z. Lamberger¹, H. M. Hermanns², J. Groll¹, G. Lang¹, M. Ryma¹

¹ Department for Functional Materials in Medicine and Dentistry, University of Würzburg, Würzburg, GERMANY,

² Medical Clinic II, Hepatology, University Hospital Würzburg, Würzburg, GERMANY

*Purpose/Objectives: For the production of collagen-mimetic polymer fibril bundles, our group recently developed Melt Electrofibrillation (MEF). This technique is based on melt electrowriting of a compatible polymer blend, where an electrical field guides phase separation of the polymers. Subsequent selective dissolution of one polymer leads to the formation of fibrous bundles. In many tissues of the human body fibrillar collagens are not only highly organized in bundles but also twisted. This is especially the case for tissues with high demand for tensile strength like skin or bladder. Therefore, mimicking such twisted collagen structures would be of high interest for engineering these tissues. With techniques existing so far, the production of scaffolds composed of twisted fibers required at least 3 different fabrication steps. Here, we introduce a pioneering one-step fabrication method for the generation of twisted fibril bundles by combining MEF with syringe rotation.

*Methodology: Twisted PCL fibrils were produced via melt electrowriting of polycaprolactone (PCL)/ polyvinyl acetate (PVAc) blends with different ratios under syringe rotation followed by selective dissolution of PVAc. We further varied rotation speed to influence the angle of the twisted fibril bundles. Additionally, mechanical properties of PCL fibrils with varying twisting angle were measured via a tensile testing machine. To examine cell orientation on different twisting angles, C2C12 cells (murine myoblasts) were cultured on PCL fibrils and investigated regarding cell angle and cell aspect ratio. Twisted PCL fibrils were compared to natural collagen fibrils of the murine bladder, uterus, and carotid artery via scanning electron microscopy.

*Results: By changing the syringe rotation speeds during MEF, fibril twist angles of 0° to 30° were achieved. Remarkably, the twisted PCL fibril bundles can be precisely deposited as scaffolds without delamination of the fibril bundles in comparison to non-twisted fibril scaffolds. We further showed that the twisting angle can be changed within one scaffold, leading to even more sophisticated and functional scaffold geometries. Moreover, fibril twisting leads to an increase in yield strength with a mechanically advantageous twist angle of approximately 16°. Furthermore, our twisted fibrils can withstand extreme plastic deformation prior to failure. C2C12 cells exhibited both orientation and elongation along the fibril direction following all different fibril twist angles. This was shown for low and even high cell densities. Additionally, highly twisted fibril bundles induced a significantly stronger elongation of the myoblasts compared to non-twisted fibrils, which can be attributed to the higher fibril density in the twisted fibril bundles. Finally, our twisting angles match those of naturally twisted collagen bundles from stretchable tissues like bladder, uterus, and carotid artery, which have twisting angles ranging mainly from 0° to 40°.

*Conclusion/Significance: We demonstrated that twisting of PCL fibril bundles for scaffold production via MEF leads to improved mechanical robustness while also preventing delamination of the fibril bundles. Additionally, myoblasts aligned with the resulting twisting angles. Remarkably, we were able to produce PCL fibrils mimicking the twisting angles of collagen in the bladder, uterus, and carotid artery, underlining the biological significance of our twisted PCL fibril scaffolds regarding matrix biomimicry.

95 - Developing A 3D Chronic Wound Model Using Animal-free Products

R. Moses, A. Sloan

University of Melbourne, Melbourne, AUSTRALIA

*Purpose/Objectives: Chronic wounds represent scenarios where the acute wound healing response is impaired, burdening both patients and healthcare systems. Animal models are often used to replicate the human wound repair process, providing information on in vivo wounds and assessing the potential of novel wound healing therapeutics. In these studies, rodent models are typically used to demonstrate chronic wound healing scenarios. However, these rodent models have limitations in their translation to human wound healing situations due to vital differences in skin architecture and wound healing cascade. An alternative to animal models involves the use of 3D organotypic in vitro models, to represent the complex human in vivo wound repair process more closely, whilst providing a greater wealth of information than is available through the widely performed 2D in vitro monolayer cultures.

*Methodology: This novel 3D organotypic model comprises human chronic wound derived fibroblasts and epidermal keratinocytes, cultured within a novel synthetic hydrogel (in collaboration with the University of Manchester) over 14 days at the air-liquid interface, resulting in epidermal differentiation and production of the protective outer cornified layer. Use of chronic wound derived cells is key for accurately representing the impaired wound healing environment, due to phenotypic differences in cell function, compared to healthy dermal cells.

*Results: This novel 3D wound model underwent H&E and Masson’s Trichrome staining to demonstrate the histological structure and compared to histological structures observed in skin biopsies. In addition, fluorescent immunostaining was performed to demonstrate the presence of key epidermal and dermal layer components, including keratin 10, keratin 14, involucrin and fibronectin, within the novel 3D wound model.

*Conclusion/Significance: Most cell-based research utilises animal-derived products, whereas this model is cultured using entirely synthetic, animal-free products, resulting in better repeatability in the studies, due to the batch variability associated with animal product use. Many industries and funders are already expressing an interest in replacing animal use in research, but a viable option is required to sufficiently replace currently used models. This novel 3D chronic wound model will more accurately represent the human wound healing scenario, is more cost-effective and without the ethical considerations associated with rodent models and animal-product use.

98 - Hydrogel Microcarriers Reduce Development Of Msc Senescence In Small And Large Scale Culture

Z. Mote, E. Ross, H. Jilani, J. Temenoff

Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA

*Purpose/Objectives: Mesenchymal stromal cells (MSCs) have great potential as a regenerative medicine therapy due to their immunomodulatory capacity. However, in order to reach therapeutic doses, large scale culture in bioreactors is required. One challenge hindering MSC scale-up is development of replicative senescence during expansion, which leads to growth arrest and decreases the quality of the cell product. Our lab has observed that MSC cultured on flat poly(ethylene glycol) diacrylate (PEGDA) hydrogels show reduced senescence-associated beta galactosidase staining over progressive passaging compared to cells on tissue culture plastic. Therefore, the hypothesis of this study is that PEGDA-based microcarriers can serve as substrates to reduce senescence of MSCs cultured in suspension bioreactors.

*Methodology: PEGDA microcarriers (150μm diameter) were fabricated using a microfluidic system and crosslinked using lithium phenyl (2,4,6-trimethylbenzoyl) phosphonate photoinitiator and UV light. The integrin engaging peptide RGD was incorporated at 1mM into the polymer to provide cell adhesion sites. Commercially available tissue culture plastic microcarriers (Corning Synthemax, 120-212μm diameter) were used as a control. MSCs were seeded at 5000 cells/cm² and cultured for 6 days in agitated 6-well plates (10 cm² SA) or in duplicate PBSmini 0.1L bioreactors (450 cm² SA). Media was assayed for lactate (n=2-3), and cells were stained for senescence-associated beta galactosidase (β-gal) and quantified using flow cytometry (n=4-6). Picogreen DNA quantification was used for well plates (n=3), and manual cell counts were performed bioreactors (n=2). Cells from bioreactors were plated into activated macrophage co-culture experiments and TNF-α concentrations were assayed after 24 hours using an ELISA (n=5). Statistical comparisons with multiple comparisons use one-way ANOVA and Tukey’s post-hoc test performed in GraphPad.

*Results: In well plate culture, similar trends in both lactate accumulation (1A) and cell yields (1B) were seen between PEGDA and Synthemax microcarrier formulations. When stained for β-gal, cells cultured on Synthemax showed a significant increase in staining compared to cells on PEGDA (1C). In bioreactors, lactate accumulation was similar between carrier types (1D), but cell yields indicate that PEGDA may support improved growth at larger scale (1E). Significant differences in β-gal staining were seen, similar to the well plate studies (1F). After bioreactor expansion, all cells significantly suppressed secretion of TNF-α from macrophages, but PEGDA-cultured cells suppressed to a degree that was not significantly different than the negative control (1G).

*Conclusion/Significance: From these studies, PEGDA microcarriers were shown to serve as an equivalent or superior surface for the growth of MSCs at both small and larger scale. At bioreactor scale, cells from PEGDA microcarriers also show significant immune suppression in activated macrophage co-cultures, indicating that PEGDA carriers support the growth MSCs that retain a key indicator of immunomodulatory capacity. β-galactosidase results indicate that PEGDA based microcarriers can be used to reduce development of senescence in MSC culture. Excitingly, these material carriers were tested at well plate scale (1000s of cells/well) and validated with a relevant bioreactor platform (millions of cells/bioreactor). Translation of this biomaterial strategy to industrially and clinically relevant scales is essential for the future of cell and tissue engineering therapies.

99 - Biofabrication Of A 3d In Vitro Model Recapitulating The Structure Of An Ovine Intervertebral Disc

E. Carrot

RMeS - UMR 1229, Nantes, France

*Purpose/Objectives: The intervertebral disc (IVD) is a fibrocartilaginous structure composed of an outer fibrous ring of collagen (Annulus Fibrosus, AF) surrounding a soft gelatinous core (Nucleus Pulposus, NP). IVD degeneration is a widespread cause of lower back pain for which there is currently no effective treatment and accurate models for assessing the effectiveness of therapeutic strategies are lacking. Current cultures of IVD cells fail to recapitulate the complex IVD structure. Conversely, in vivo animal models are restricted by ethical regulation, limiting research possibilities. Three-dimensional (3D) models offer greater biological complexity, high-volume production, and high reproducibility. Herein, we sought to develop a complete ovine in vitro IVD model that would reproduce the spatial organization of IVD cells and matrix, including the fibrous and gelatinous structures of AF and NP, respectively.

*Methodology: Alginate methacrylamide (AMA) hydrogel was used to encapsulate AF or NP cells. We first optimized AMA properties by testing various concentration and photocrosslinking conditions. Then, the model was designed based on histological sections from an ovine IVD, reproducing its relative dimensions scaled down at 60%. The AF lamellar structure was reproduced by melt electro-writing (MEW) of 13 concentric circles, each comprising 50 layers of polycaprolactone (PCL) fibers. MEW reproducibility and PCL scaffold stability were assessed by measuring the model size when stored at room temperature. Between each PCL circle, AMA hydrogel containing 4 x 10⁶ ovine AF cells/mL (P3) was deposited by extrusion. To complete the model, AMA hydrogel containing 4 x 10⁶ ovine NP cells/mL (P3) was extruded in the center of the engineered AF part. Once assembled, the model matured over 28 days in culture. Then, model stability, cell viability, and cell organization were assessed at different time points.

*Results: The AMA hydrogel was optimized by adjusting AMA concentration (2%) and photocrosslinker agent concentration (0.29% LAP) to obtain a stiffness matching that of the native NP tissue while ensuring cell viability and limiting UV dose (12 mJ/mm²) (Figure 1A). The AF scaffold was designed using MEW. MEW parameters ensured consistent models with no size variability, demonstrating high reproducibility of the method used (Figure 1B). When stored at room temperature, PCL scaffolds remained stable for at least 21 days (Figure 1B). Despite PCL hydrophobicity, 2% AMA penetrated effectively between PCL circles, forming a cohesive construct (Figure 1C). The cultured model remained stable for 28 days with over 80% cell viability (Figure 1D), and without cell sedimentation. AF cells adhered and proliferated along the PCL fibers, mimicking IVD architecture (Figure 1E). Histological, biochemical, and transcriptomic analyses are underway to assess matrix content, cell organization, and phenotype.

*Conclusion/Significance: Our study presents the development of an in vitro ovine IVD model. Combining melt electro-writing and extrusion methods, we developed a reproducible 3D model that replicated the architecture of IVD. The model demonstrated stability, good cell viability, and effective AF cell adhesion over 28 days in culture. This opens the way for the evaluation of new therapeutic strategies, providing a tool for advancing research on IVD degeneration treatments.

100 - Multiscale Patterning Of 3D Microvascular Scaffolds Via Image Stack-guided Photodegradation

S. Yang¹, L. Orr¹, T. L. Rapp², W. Xie¹, A. K. Glaser³, J. T. Liu¹, C. A. DeForest¹

¹ University of Washington, Seattle, WA,

² University of Oregon, Eugene, OR,

³ Allen Institute, Seattle, WA

*Purpose/Objectives: Vasculature is essential in every organ system, playing critical roles in tissue regeneration and cellular responses. To fully understand physiological cellular functions and engineer tissues for transplantation, recapitulating such vasculature features, especially at the capillary scale, is in great need. While several manufacturing methods have been explored to generate vascular constructs in vitro using different polymeric materials and printing techniques, existing efforts have largely failed to replicate the essential size ranges and complexity of the vascular networks, particularly of smallest capillaries (<10 μm diameter) across large distances (>mm). In this work, we exploit image stack-guided two-photon (2P) lithography in conjunction with photodegradable hydrogel materials to create large-scale, physiologically relevant microvasculature networks in vitro.

*Methodology: A highly photoresponsive crosslinker (Rubpy) was synthesized and used due to its hypersensitivity in both single-photon (1P) and 2P space and has 2P cleavage of 750-850 nm. PEG-based hydrogels crosslinked with Rubpy were formed for 2P patterning. High-resolution vasculature image stack “blueprints” (e.g., fluorescent z-stacks acquired by light-sheet microscopy in cleared tissues, with varying capillary sizes) were computationally processed into binary image stacks for image-guided 2P degradation. Patterned hydrogels were then perfused with fluorescent dextran solutions to verify the channel connectivity.

*Results: Using a 2x2x2 mm³ 3D vasculature image stack acquired from a neonatal mouse brain, microvascular channels with various diameters (3 - 100 µm) were rapidly patterned (∼1 hour) at native complexity within photodegradable hydrogels. Channels were demonstrated to be patent via FITC-dextran perfusion and could be subsequently seeded with living endothelial cells.

*Conclusion/Significance: Following the success in 2P vasculature patterning, our current progress focuses on seeding human endothelial cells into patterned channels and testing the vascularization results by subsequent perfusion and staining experiments. Overall, this “subtractive manufacturing” technique enables natively complex microvoids to be optically sculpted within otherwise solid biomaterials at unprecedented resolutions and at high speed. We expect these hydrogel scaffolds to find great utility for studying vascularized systems in vitro, including 3D disease modeling, therapeutic screening, and many other applications that can accelerate current clinical trials.

102 - Volumetric Bioprinting Of Multi-scale Vasculature Via Photopolymerization-induced Phase Separation For Vascularized Engineered Tissues

O. Dudaryeva¹, M.-B. Buchholz², G. Größbacher¹, P. Català Quilis¹, M. Tibbitt³, R. Levato^1,4

¹ University Medical Center Utrecht, Utrecht, Netherlands,

² Princess Máxima Center for Pediatric Oncology, Utrecht, Netherlands,

³ ETH Zurich, Zurich, Switzerland,

⁴ Utrecht University, Utrecht, Netherlands

*Purpose/Objectives: Volumetric bioprinting (VBP) rapidly constructs centimeter-scale structures from polymeric materials, facilitating the generation of functional in vitro tissue models with encapsulated living cells. While VBP captures many aspects of hierarchical organ architecture producing channels and vessels in the 0.1-1 mm range, achieving microvasculature (∼10 microns) required for efficient nutrient exchange over large volumes remains challenging. Despite continued improvements in light-assisted biofabrication, the field faces a major challenge in the vascularisation of engineered tissues, which is likely to determine the success or failure of centimeter-scale tissue fabrication. Phase separation in polymeric systems is an emerging tool for producing macroporous materials. This study explores light-triggered phase separation in norbornene-functionalized poly(ethylene glycol) (PEG) or gelatin constructs via VBP, aiming to create controllable hierarchical vasculature for nutrient delivery across complex structures.

*Methodology: Macroporous hydrogel constructs were prepared using norbornene-functionalized PEG or gelatin and thiolated linkers with photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP). Using VBP, 3D light fields were used to control the local phase separation kinetics, allowing the formation of 3D geometries with porosity gradients. Human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) were seeded into the porous space at a 5:1 ratio. The vascular structures were characterised using immunochemistry. The constructs were perfused at a rate of 1 μl/ml.

*Results: We have determined different formulations of PEG and gelatin-based materials to form macroporous hydrogels through phase-separation. Using VBP we regulated local rate of photopolymerization using irradiation intensity controlling the pore formation to yield different porosity from single formulation. For example, manipulating light intensity between 6 and 10 mW/cm², generated pores of 145.1 ± 61 and 72.3 ± 24.7 μm respectively (Figure 1A-C). The porous space of casted or printed gels was populated with HUVECs and hMSCs generating vascular structures narrowing down to ∼10 μm. The formed structures stained positively for common vascular markers CD31 and αSMA and remained stable for over 10 days (Figure 1D, E).

*Conclusion/Significance: Applying phase separating systems in VBP facilitated the generation of complex structures with macroporosity, allowing the formation of stable vasculature in the porous space. These structures retained stability for over 10 days and perfusion further enhanced the stability of the formed vasculature.

103 - A 3D Bioprinted Kidney Proximal Tubule Model For The Investigation Of Ammoniagenesis

J. Tashman, B. Coffin, J. Lashway, E. Aranda-Michel, C. Boyd-Shiwarski, D. Shiwarski

University of Pittsburgh, Pittsburgh, PA

*Purpose/Objectives: Ammoniagenesis, the generation of ammonium from glutamine, is a key mechanism in the kidney for excretion of acid and conservation of potassium ions [K+]. While hypokalemia (low [K+]) is associated with upregulated ammoniagenesis, the underlying mechanism is poorly understood and challenging to study in vitro due to the limitations of current kidney proximal tubule (PT) models. Permeable supports (Transwell, Costar) utilized to study kidney physiology lack tubule-like structural constraints, luminal flow, or a 3D microenvironment, that are required to induce and maintain cellular maturity. In this study, we developed an innovative proximal tubule model that combines traditional orbital shakers used in permeable support studies with physiologically relevant circumferential channels 3D bioprinted from collagen-I.

*Methodology: Flat and circumferential channel 3D models were designed using Fusion 360 and the shear stress on the surface was simulated using Ansys Fluent simulations. Freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting was employed to fabricate microporous flat and channeled 3D collagen scaffolds and inspected for dimensional accuracy using optical coherence tomography (OCT, ThorLabs Vega) (Fig. 1A-F). Printed scaffolds were placed into Transwell inserts and seeded with opossum kidney proximal tubule (OK) cells to assess the growth and transport compared to a conventional 2D Transwell model. Seeded scaffolds were cultured for 4 days under static conditions then subjected to rotational shear for an additional 4, 7 and 10 days. At these timepoints the functionality of OK cells were assessed by albumin uptake (Fig. 1I). The viability of OK cells was assessed with calcien-AM and EthD-1 fluorescent staining 4 days after seeding onto 3D bioprinted inserts compared to 2D Transwell controls.

*Results: For flat 3D bioprinted and 2D transwell models the optimal shear stress was observed at 73 rpm at the edge of the transwell (0.18 - 0.41 dyne/cm²). However, at the same rpm, the shear for channeled 3D models was significantly lower (Fig. 1G, 0.005 - 0.018 dyne/cm²). Equivalent shear stress was generated within the channeled model by doubling the orbital rotation. OK cells adhered well to both printed models during static culture. Rotational shear stress further promoted OK cell adhesion and proliferation in the circumferential channel model but caused cell detachment from the flat model (Fig. 1H). Interestingly, the OK cells not only formed a monolayer along the channel surface but also displayed evidence of ECM remodeling and growth into the bulk of the 3D bioprinted collagen. To ensure appropriate morphology and maturation of OK cells within our system we utilized fluorescence and electron microscopy to quantify expression and morphology by probing for SN1, NHE3, NBCe1a, phalloidin and α-tubulin.

*Conclusion/Significance: In conclusion, this 3D bioprinted microfluidic proximal tubule model will facilitate studying kidney cells in a more physiologic environment to identify the key mechanisms controlling ammoniagenesis. Our initial data suggests that collagen-I is sufficient for OK cell adhesion and proliferation; however, by changing the ECM composition and architecture, we can vary our models mechanical properties, adhesion molecules, growth factors, and other elements that are essential to optimize this proximal tubule model.

104 - 3d Bioprinted Liver-on-a-chip For Drug Cytotoxicity Screening

J. Huh, J. R. Parra, C. Bishop, S. Soker, S. Murphy, T. Shupe, J. Yoo, A. Atala, S. Lee

Wake Forest University School of Medicine, Winston-Salem, NC

*Purpose/Objectives: Tissues on a chip are sophisticated 3D in vitro microphysiological systems designed to replicate human tissue conditions within dynamic physicochemical environments. However, the current fabrication methods for tissue spheroids on a chip require multiple parts and manual processing steps, including the deposition of the spheroids onto prefabricated “chips.” These challenges also lead to limitations regarding scalability and reproducibility. To overcome these challenges, this study employed the utilization of 3D bioprinting technology to fabricate a liver tissue spheroids on a chip (LOC) system, leveraging its precise deposition capabilities for polymeric materials and cell-laden hydrogel in a controlled manner with simultaneous automated printing and delivery of liver spheroids onto the chip (Figure 1).

*Methodology: 3D printing techniques were utilized to automate the fabrication process of tissue spheroids on a chip. This allowed for the simultaneous high-throughput printing of human liver spheroids and their surrounding polymeric flow chamber “chips” containing inner channels in a single step. The fabricated LOC were subsequently subjected to dynamic culturing utilizing a peristaltic pump, enabling assessment of cell viability and metabolic activities.

*Results: The 3D printed liver spheroids within the printed chips demonstrated high cell viability (>80%), increased spheroids size, and consistent ATP activity and albumin production for up to 14 days. Furthermore, we conducted a study on the effects of acetaminophen (APAP), a nonsteroidal anti-inflammatory drug, on the LOC. Comparative analysis revealed a substantial decline in cell viability (<40%), diminished ATP activity, and reduced spheroid size after 7 days of culture within the APAP-treated LOC group compared to the non-treated groups.

*Conclusion/Significance: We developed a one-step printing method for the fabrication of human tissue on a chip system, utilizing human liver spheroids embedded within the hydrogel-based bioink. The automated bioprinting approach enhanced chip-to-chip consistency, enabling more precise assessments of liver spheroid viability and metabolic activity in hepatotoxic microenvironments. Ultimately, the one-step 3D printed LOC system has the potential to serve as a high-throughput screening in vitro model, facilitating accurate investigations of in vivo biological processes and monitoring tissue responses to administered drugs.

107 - A Culture Platform With Alveolar Geometry For Induced Pluripotent Stem Cell-derived Distal Lung Organoids

D. Brinson^1,2, C. Lei¹, G. Karoubi^1,2, T. Waddell^1,2

¹ University of Toronto, Toronto, ON, Canada,

² Toronto General Hospital Research Institute, Toronto, ON, Canada

*Purpose/Objectives: Current human induced pluripotent stem cell (hiPSC)-derived distal lung organoids (DLOs) resemble fetal maturity and lack key cell types. They do not adequately resemble the adult lung, which limits their utility. We must improve existing DLOs to better resemble the human lung in terms of their cell composition, functionality, and maturity.

There is a lack of understanding of the signalling processes responsible for cell fate selection in the developing lung. Mechanical cues such as geometry, extracellular matrix (ECM), and stiffness are increasingly being recognized for their importance in directing cell behaviour and fate. Existing DLOs are traditionally cultured as self-organizing spheroids embedded in very soft gel that does not resemble the native microenvironment. We hypothesize that culturing DLOs in a biomimetic microenvironment will influence key signalling pathways throughout differentiation, and thus affect cell fate.

*Methodology: We designed a culture platform with tunable geometry, stiffness, and ECM composition to investigate the roles of these parameters on DLO development. We fabricated a mold using two-photon lithography 3D printing. Then, PDMS or hydrogel was replica molded to create a cell culture substrate with a dimples resembling alveolar shape and size.

We screened 36 ECM compositions to identify those that support hiPSC-derived lung progenitor cell attachment over six hours. Next, the ECM compositions that best enhanced cell attachment will be coated on the dimpled substrate. Lung progenitors will be cultured for 10 days to determine which ECM composition promotes alveolar epithelial cell (AEC) differentiation.

Lastly, lung progenitor differentiation will be compared among three culture platforms: the dimpled hydrogel, flat hydrogel, and traditional 3D spheroids embedded in soft gel. Mechanical signalling pathways have been linked to AEC fate choice, and differential cell fate selection has been shown to depend on the epigenome. Therefore, single cell RNA sequencing and epigenomics will be combined to identify active signalling pathways in each cell type within the heterogeneous DLO cell population. The three culture platforms will be compared to investigate the effect of the mechanical microenvironment on signalling pathway activation.

*Results: We identified several candidate collagen type I-based ECM combinations that improved cell attachment. A parallel plate rheometer was used to assess the stiffness of a collagen type I hydrogel, and collagen concentration was tuned to achieve a stiffness resembling healthy lung tissue (2 kPa).

The dimpled substrate was imaged using scanning electron microscopy to ensure accurate dimensions (200 um diameter, 100 um depth). This culture platform is compatible with downstream analysis techniques such as confocal microscopy, flow cytometry, and transcriptomics.

*Conclusion/Significance: This project will uncover microenvironmental cues that guide distal lung development, ultimately providing insight into which biophysical parameters must be leveraged to improve the fidelity of DLOs as an in vitro model of the adult lung. HiPSCs provide an unlimited source of patient-specific cells for in vitro modelling, cell therapy, and tissue engineering. Through this research we will gain a deeper understanding of which signalling pathways must be activated to better guide cell fate towards desired cell types for future applications.

108 - Biofabrication And Stimulation Of Magnetically Augmented Callus Assembloids Accelerates Bone Formation In Vivo

K. Ioannidis¹, A. Dimopoulos¹, L. Storozhuk², M. Guilliams³, R. Oliveira-Silva¹, S. Basov¹, S. Mourdikoudis⁴, M. J. Van Bael¹, B. Smeets⁵, D. Sakellariou¹, I. Papantoniou¹

¹ KU Leuven, Leuven, Belgium,

² University College London, London, United Kingdom,

³ KU Leuven, Heverlee, United Kingdom,

⁴ University of Chemistry and Technology Prague, Prague, Czech Republic,

⁵ KU Leuven, Heverlee, Belgium

*Purpose/Objectives: The rise of organoid-based tissue engineering presents an efficient strategy for skeletal defect regeneration, utilizing cartilage intermediate templates. Implants composed of callus organoids have demonstrated the ability to form bone ossicles following endochondral ossification and successfully regenerating critical size tibial defects. While conventional biofabrication methods like bioprinting exist, their limitations, such as the use of additional hydrogel-based materials and a lack of post-biofabrication control, hinder their applicability for critical size implants. In response, we aimed to develop a novel, nozzle-free, scaffold-free, and rapid magnetic-driven biofabrication approach for assembling callus organoid-based implants. Although initial studies have shown the capacity of magnetic fields to aid spheroid assembly processes there is currently minimal investigation in the influence of magnetic stimulation in chondrogenic differentiation for prolonged time periods as well lack of in vivo evaluation data. Additionally, we developed, trained, and validated a computational model to predict the outcome of the magnetic biofabrication process, paving the way for more complex applications.

*Methodology: Human periosteum-derived cells (hPDCs) were seeded in AggreWell™400 microwells to form 3D spheroids which differentiated in chondrogenic medium for 7 days. Subsequently callus organoids were incubated with iron oxide nanoparticles (IONPs) before the biofabrication process. A neodymium magnetic plate (24-WELL BIO ASSEMBLER™) provided a strong magnetic field to guide spheroid assembly and stimulate the formed assembloids for 14 and 21 days respectively leading to the formation of callus assembloids. Samples were obtained on Day 1,7,14,21 points during in vitro maturation under magnetic stimulation for RNA seq characterization while Non-magnetically stimulated callus assembloids served as control. Ectopic implantation was performed for Day7 and Day21 assembloids. Explants analysis included histology, confocal microscopy, SEM, and µCT analysis. Liver and spleen biopsies assessed IONPs accumulation or toxicity. The magnetic field of the neodymium magnetic plate was characterized, and IONPs content measured using SQUID magnetometer analysis. Data from these analyses, combined with videos collected during the magnetic biofabrication process, were used to develop the agent based computational model.

*Results: Cell viability and toxicity were unaffected by IONPs throughout biofabrication and maturation. Histological analysis revealed matured hypertrophic chondrocytes and ECM components (SafO+) in samples undergoing chondrogenic differentiation under magnetic stimulation. RNA seq analysis indicated an acceleration of the chondrogenic differentiation process and the maturation trajectory towards hypertrophy for the magnetically stimulated samples. SEM-EDX and SQUID magnetometer analysis localized and quantified IONPs within ECM compartments for the entire period. Ectopic implantation showed early bone formation in magnetic-stimulated assembloids. Both stimulated and non-stimulated groups formed bone compartments in the late time point, with early non-stimulated explants exhibiting poor bone-forming capacity. The computational model efficiently recapitulated the magnetic assembly experiments and quantified the forces exerted on assembloids.

*Conclusion/Significance: By harnessing IONPs' magnetic properties and combining them with organoids and magnetic fields, we created magnetically augmented callus organoids for bottom-up magnetic-driven biofabrication. Post-biofabrication, implants maintained magnet properties, allowing external manipulation and stimulation. Moreover we observed a clear indication of enhanced chondrogenic maturation of stimulated assembloid implants which resulted in significantly accelerated the endochondral ossification process and bone forming capacity.

109 - Knowledge-generation Platform For Unravelling Bone Regeneration Mechanisms Induced By Biophysical Stimuli

S. Gabetti¹, F. Dahou², B. Masante¹, E. Scatena³, E. Zenobi³, A. Sanginario¹, S. Israel¹, A. Canova¹, R. Bardini¹, A. Benso¹, L. Rimondini², C. Bignardi¹, A. Cochis², D. Massai¹

¹ Politecnico di Torino, Turin, Italy,

² University of Piemonte Orientale, Novara, Italy,

³ Hypatia Research Consortium, Rome, Italy

*Purpose/Objectives: Bone fractures represent a growing socio-economic burden worldwide. Although orthopaedic treatments based on biophysical stimuli, such as mechanical loading or externally applied pulsed electromagnetic field (PEMF) stimulation, are known to promote bone healing and regeneration, the triggered biological mechanisms remain partially unknown, leading to empirical therapies. Indeed, there is no agreement on the optimal stimulation parameters (frequency, intensity, and duration) that can promote bone growth and healing and standardized guidelines are missing. In this context, we are developing a pioneering knowledge-generation platform for unravelling the cell- and tissue-scale effects induced in bone tissue by specific biophysical stimuli.

*Methodology: The parallelized platform (Fig.1) is based on three independent perfusion bioreactors combined with a PEMF stimulator (IGEA Clinical Biophysics) and designed for culturing and investigating in vitro three-dimensional (3D) biomimetic bone tissue models under tuneable and combinable biophysical stimuli (e.g., shear stress, intermittent pressure, and PEMF stimulation). Concerning the bone tissue models, we seeded 3D-printed polylactic acid (PLA) scaffolds mimicking the trabecular bone microarchitecture with human mesenchymal stem cells (4 x 10⁶/scaffold). The models were then housed in the bioreactors to be cultured under direct perfusion (flow rate = 0.3 ml/min) for 21 days, with or without PEMF stimulation (intensity = 1.5 mT, frequency = 75 Hz) for 4 h/day (n = 3). In parallel, the control group was statically cultured (n = 3).

*Results: The qPCR analysis showed that direct perfusion combined with PEMF stimulation strongly upregulated the expression of collagen type-I (∼6.5-fold change) and type-II (∼4.3-fold change) with respect to static conditions, revealing a synergic pro-osteogenic effect of flow-induced shear stress and PEMF stimulation. Interestingly, transcriptomic analysis pointed out the activation of immune response pathways and increased expressions of angiogenesis and osteogenesis upstream regulators triggered by the combined biophysical stimuli. Currently, we are developing a tuneable PEMF stimulator and running tests with intermittent pressure mimicking the physiological bone loading and with co-culture of endothelial cells. Lastly, we will setup a Machine Learning pipeline to analyze the collected transcriptomic data to decrypt complex regulatory interactions as well as to extract relationships between gene expression patterns and applied physical stimuli.

*Conclusion/Significance: The proposed knowledge-generation platform represents a powerful tool for investigating in vitro the biological response of 3D bone tissue models to defined biophysical stimuli. Combined with high throughput screening and machine learning tools, it will enable reaching a thorough understanding of the correlations between the applied biophysical stimuli and the bone regeneration mechanisms at the cell- and tissue-scale, with the final aim to define the rationale basis for improved biophysical stimulation treatments, in view of precision orthopaedic medicine. This study was carried out within the project BIGMECH - funded by the Ministero dell’Università e della Ricerca - within the PRIN 2022 program (D.D.104 -02/02/2022). This manuscript reflects only the authors’ views and opinions, and the Ministry cannot be considered responsible for them.

111 - Multiple Stimuli-Responsive Injectable Hydrogel For Functional Tissue Regeneration

S. Panseri¹, A. Rossi¹, G. Bassi¹, A. Lunghi², D. Gardini¹, P. Galizia¹, C. Baldisserri¹, C. Cunha³, F. Teran⁴, M. Bianchi⁵, G. Canu⁶, M. Montorsi⁷, M. Labardi¹, E. Mercadelli¹, A. Piperno⁸, M. Montesi¹

¹ National Research Council of Italy, Faenza, Italy,

² Istituto Italiano di Tecnologia, Ferrara, Italy,

³ Instituto de Investigação e Inovação em Saúde, Porto, Portugal,

⁴ Nanotech Solutions, Villacastin, Spain,

⁵ Università degli Studi di Modena e Reggio Emilia, Modena, Italy,

⁶ National Research Council of Italy, Genova, Italy,

⁷ National Research Council of Italy, Pisa, Italy,

⁸ University of Messina, Messina, Italy

*Purpose/Objectives: Cellular alignment is fundamental in several tissues such as the spinal cord, muscle, tendon, and cartilage, and plays a crucial role in their functionality. Well-oriented scaffolds have been produced by bioprinting, electrospinning, and freeze-drying, but very few have shown translational applications due to the invasive surgery needed. Injectable hydrogels can be transplanted via minimally invasive injection, but usually without displaying any aligned structure. In addition, the use of electrical stimulation can significantly influence cell behavior facilitating tissue regeneration. The ability to control and optimize the bioelectric environment offers a significant opportunity to enhance tissue regeneration modulating cellular signalling, promoting growth factor activation, and regulating cell proliferation and/or differentiation. Attempts have been made regarding the design of electroconductive scaffolds capable to amplify in situ the efficacy of the electrical stimulation therapy. However, literature regarding the in vivo effect of the combination of electroactive materials with external stimulation, is still lacking and no conclusions can be made on the utility of this approach for many tissue treatments. In the present work, we developed an injectable hydrogel that can be transplanted via minimally invasive injection, with a dual feature activated by applying a static or alternate magnetic field (SMF/AMF): anisotropic architecture and electric stimulation to better mimic the characteristics of the native muscle. The system is based on gellan gum (GG), hyaluronic acid (HA) and magnetic piezoelectric collagen bundles (MPCollB) for tissue regeneration.

*Methodology: Magnetite and Barium Titanate nano-, microparticles were coupled through hetero-coagulation to obtain MP particles, which were then blended with collagen type I to obtain MPCollB. MPCollB were incorporated into the 1%GG and 0.3%HA hydrogel before it gelled under physiological conditions. MPs and MPCollB underwent comprehensive characterization through XRD, DLS, AFM and PFM (Piezoresponse FM), SEM and magnetometer analyses. MPCollB alignment was induced by a low-intensity SMF. Hydrogel’s mechanical properties were evaluated by rheology and DMA. 3D in vitro cell culture was performed to study fibroblast and muscle cell viability, proliferation and murine macrophage immunomodulation (qRT-PCR). The hydrogels were then subcutaneously implanted in Wistar-Han rats and the hydrogels and surrounding tissue were processed for histological analyses. Potential systemic toxicity was also evaluated.

*Results: We developed a cutting-edge bioactive injectable hydrogel that can be easily transplanted via minimally invasive injection (30G-needle) and functions as a versatile multi-step therapy. The MPCollB can be initially aligned using a SMF (<60 mT), creating an anisotropic extracellular matrix-like structure. Subsequently, these aligned MPCollB can generate an electrical signal when exposed to mechanical deformation induced by AMF (<300kHz; <2mT), providing a novel tool for a wireless method of electrical stimulation of the endogenous cells. The hydrogels were able to sustain cell proliferation and modulate pro-regenerative macrophage polarization. Furthermore, hydrogels were easily injected in vivo, and no local or systemic immune reactions were detected.

*Conclusion/Significance: We were able to develop a versatile stimuli-responsive injectable hydrogel that can find application in several minimally invasive surgical interventions.

113 - Bioreactor Design To Achieve Immune Cell Perfusion In Liver Models For The Study Of The Extracellular Matrix-immune Cell Interactions In Liver Fibrosis

S. Campinoti¹, M. Accursi¹, D. Gao¹, B. Almeida¹, S. Gallinea¹, L. Caciolli², L. Wei¹, C. McQuitty¹, A. Riva¹, A. Pellegata³, S. Mantero³, S. Chokshi¹, L. Urbani¹

¹ The Roger Williams Institute of Hepatology, London, United Kingdom,

² UCL Great Ormond Street Institute of Child Health, London, United Kingdom,

³ Politecnico di Milano, Milan, Italy

*Purpose/Objectives: Liver fibrosis is caused by progressive dysregulated accumulation of extracellular matrix (ECM) coupled with chronic inflammation. Advanced liver fibrosis correlates with an increased risk of liver cancer and liver failure, resulting in the need for liver transplantation. More studies of the mechanisms that promote fibrosis are necessary to understand this multi-faceted disease and for the development of novel therapeutic targets. Traditional cell culture models often lack both the immune cell compartment and the ECM, failing to recapitulate the complex fibrotic microenvironment, which is highly immune-mediated. Bioengineering allows for the development of advanced disease models to study complex diseases, such as liver fibrosis, where both cellular and extracellular microenvironment components contribute to the pathology.We have successfully developed two bioengineered models which incorporate the dynamic culture of circulating immune cells within decellularised liver ECMs, supported by two custom-made perfusion bioreactors, and we demonstrated how these bioengineered models allow to explore immune responses to fibrotic liver ECM.

*Methodology: We developed two custom-made bioreactors for vascular perfusion of decellularised whole rat livers or confined perfusion of human tissue segments (named WL and HuTS bioreactors respectively). Decellularised normal and fibrotic rat and human liver tissues were obtained and characterised following established detergent and enzymatic treatment protocols. Human peripheral blood mononuclear cells (PBMCs) from healthy donors, were perfused in WL or HuTS bioreactors in the absence (baseline characterization) or presence of decellularised normal or fibrotic liver ECM-scaffolds. Circulating cell viability, phenotype, and cytokine production were assessed in comparison to static culture conditions, using flow cytometry and multiplex cytokine analysis assays (Luminex). The phenotype of PBMCs colonising the ECM scaffolds was examined via immunofluorescence staining.

*Results: The custom-made bioreactors supported the perfusion of PBMCs for up to 7-days without altering cell viability and phenotype in comparison to conventional static culture conditions. Bioreactor culture also improved cell distribution inside the scaffolds compared to static cultures, suggesting that perfusion culture better promotes cell-ECM interactions.FACS analysis of circulating PBMCs showed that co-culture with both healthy or fibrotic liver matrix induced changes in the relative proportion of NKT cells and B cells and that this was fibrosis-dependent. When cultured with fibrotic scaffolds, the number of circulating T cells decreased and interestingly, monocyte sub-populations changed in response to healthy or fibrotic liver matrices. Immunofluorescent staining on sections of perfused ECM matrices revealed that immune cells infiltrated the ECM scaffolds. These were mostly composed of monocytes and macrophages, with a higher relative proportion in fibrotic livers, indicating fibrotic ECM-induced homing of monocyte-derived macrophages, an event that recapitulates the in vivo fibrotic microenvironment.

*Conclusion/Significance: Here we show the validation of two innovative bioreactor-based systems which allow for the perfusion of immune cells through decellularised liver matrices. This perfusion system proved to be suitable for the study of immune cell interaction with normal or fibrotic ECM, and more in general, with bioengineered multi-cellular liver constructs, and showed that mechanisms of chronic inflammation observed in fibrosis can be replicated in vitro.

116 - Tissue Engineered Fibroblastic Reticular Network Regulates Antibody Production By PBMCs

M. ElGindi, S. Karaman, J. Teo

New York University Abu Dhabi, Abu Dhabi, United Arab Emirates

*Purpose/Objectives: Three-dimensional (3D) human lymph node models serve as crucial tools in advancing immunological and therapeutic research. Fibroblastic reticular cells (FRCs) play an essential role in orchestrating immune responses within a lymph node, yet most lymph node models, as of date, overlook their significant role. In this study, we aim to engineer a 3D lymph node model that incorporates FRCs as well as peripheral blood mononuclear cells (PBMCs) in order to achieve a more physiologically relevant and functional representation of a human lymph node.

*Methodology: To construct this model, FRCs are derived from adipocyte-derived stem cells (ADSCs) and cultured in a collagen-based 3D gel. These FRCs are subsequently co-cultured with PBMCs and mature dendritic cells (DCs) primed with specific antigen. Subsequent analyses included confocal microscopy for high-resolution visualization of the cells and flow cytometry for quantitative assessments to determine changes in cell populations and activation, chemokine and cytokine secretion profiles, and production of IgM and IgG antibody.

*Results: The ADSC-derived FRCs exhibit an upregulation of known markers such as ICAM-1 and podoplanin. In addition, there is an upregulation in the secretion of pro-inflammatory cytokines such as TNFα and IFNγ form the co-culture of FRCs and PBMCs. The resultant model exhibits a dynamic interplay between FRCs, PBMCs, and antigen-primed DCs that leads to changes in cell activation, release of cytokines and chemokines consistent with those in in vivo human lymph nodes, and antibody production.

*Conclusion/Significance: This study not only enhances the fidelity of current lymph node models by incorporating FRCs, but provides a platform to investigate more cellular interactions, vaccine efficiency assessment, and antibody production dynamics. The ADSC-derived FRCs introduces a unique aspect to this model, allowing for its customization for patient-specific therapeutic applications and a step forward in immunotherapy precision medicine.

117 - Magnesium Ions-releasing Bioactive Hydrogels For In Situ Tissue Regeneration Via Macrophage Polarization

J. Kim, K. Park

Incheon National University, Incheon, Korea, Republic of

*Purpose/Objectives: Bioactive hydrogels are attractive biomaterials for tissue regeneration due to their ability to promote biological processes in surrounding tissues through various biophysical and biochemical stimuli in vivo. Various strategies have been used for fabricating bioactive hydrogels, including modifications to their physicochemical properties and encapsulation of biological molecules within hydrogels. Among these factors, magnesium ions (Mg²⁺) are crucial for tissue regeneration by M2 macrophage polarization and neovascularization. Herein, we present bioactive Mg²⁺-releasing hydrogels (Mg-Gels) for enhanced in situ tissue regeneration.

*Methodology: Thiolated gelatin (GtnSH) and maleimide-conjugated gelatin (GtnMI) were synthesized using EDC/NHS chemistry to introduce thiol and maleimide groups into the gelatin backbone, respectively. We fabricated Mg-Gels by mixing GtnSH, GntMI, and magnesium peroxide (MgO₂) solutions. The phase transition time was measured by the vial-tilting method with varying MgO₂ concentrations. Next, we measured elastic modulus (G') using a rheometric fluid spectrometer (HR-1, TA instruments). We measured Mg²⁺ and H₂O₂ released from Mg-Gels using a Mg²⁺ detection kit and Cu(II)-neocuproine assay. The inner structure and atomic % were analyzed using a scanning electron microscope-energy dispersive spectrometer (JSM 7800F, JEOL) on day 0 and day 28. For the in vitro test, human dermal fibroblasts (HDFs), human umbilical vein endothelial cells (HUVECs), and pre-osteoblasts (MC3T3-E1s) were cultured with Mg-Gels and the viability was evaluated by live/dead assay and WST-1 assay. To assess tissue compatibility, Mg-Gels were implanted under the mouse subcutaneous tissue. After 4 weeks, major organs (heart, liver, spleen, lung, and kidney) were harvested for histological analysis. To investigate the in vivo biological response of Mg-Gels, we performed mouse subcutaneous implantation, wound healing test, and gene expression analysis in a mouse critical skin wound model.

*Results: Our Mg-Gels were fabricated by thiol-ene reaction and MgO₂-meditated disulfide bond formation. With increasing MgO₂ concentration, the phase transition time was shortened (76-10 sec) and elastic modulus (G') was increased (910-1580 Pa), showing controllable phase transition time and mechanical strength. Our Mg-Gels released Mg²⁺ for 24 hours (30-977 μM) and residual Mg²⁺ remained in the matrices for up to 28 days. In addition, Mg-Gels showed excellent cytocompatibility (> 75% compared to the non-treated group) and no pathological changes in major organs. In histological analysis, Mg-Gels increased the number of M2 macrophages and CD31⁺ cells in vivo. Furthermore, our bioactive hydrogels facilitated wound healing at day 7 (DPBS, 72.0%; Mg0, 73.8%; Mg0.25, 79.6%) and enhanced type I collagen deposition at day 14 (DPBS, 16.6%; Mg0, 16.2%; Mg0.25, 26.3%). Interestingly, the results of gene expression of Mg-Gels treated wound showed enhanced M2 macrophage polarization on day 3 (IL-10: 1.6-folds; VEGF: 2.7-folds) and upregulated expression of M2 macrophage and collagen maturation related genes (TGF-β1: 2.1-folds; IL-10: 2.0-folds; VEGF: 2.3-folds) on day 7 compared to Mg²⁺-free group (Mg0).

*Conclusion/Significance: We developed bioactive Mg²⁺-releasing hydrogels (Mg-Gels) via thiol-ene reaction and MgO₂-mediated crosslinking reaction. Our Mg-Gels facilitated wound healing by promoting Mg²⁺-induced M2 macrophage polarization and neovascularization in vivo. Therefore, we expect that our Mg-Gels can serve as bioactive matrices for tissue regeneration through easy fabrication, long-term Mg²⁺ encapsulation, and bioactivities.

119 - In Vitro Modulation Of Macrophage Phenotype By 3D Custom-made Micro Scaffolds

C. Martinelli¹, F. Bonetto¹, T. Baldissera¹, C. Conci¹, G. Chirico², G. Cerullo¹, R. Osellame¹, E. Jacchetti¹, M. T. Raimondi¹

¹ Politecnico di Milano, Milan, Italy,

² University of Milano Bicocca, Milan, Italy

*Purpose/Objectives: Upon in vivo implantation of a micro-structured device, a complex physiological process called foreign body reaction (FBR) takes place, characterized by a cascade of events involving many key players. An essential role is carried out by immune cells, specifically macrophages, that can polarize towards the M1 (pro-inflammatory) or M2 (pro-healing) phenotype. Optimizing 3D micro scaffolds design for prompting an anti-inflammatory response of macrophages is a fundamental strategy for achieving successful implant integration and tissue regeneration in vivo.

*Methodology: 3D micro scaffolds were fabricated by 2-photon polymerization of the SZ2080 biocompatible photosensitive resin. Different pore sizes were tested for their ability to polarize mouse macrophages, ranging from 10x10x15 μm³ to 50x50x20 μm³. M0 macrophages were stimulated with 1µg/mL Escherichia coli lipopolysaccharide (M1, pro-inflammatory) or 40 ng/mL interleukin-4 (M2, anti-inflammatory) for 24 hours. Their polarization was evaluated after 48 h, in the presence of the micro scaffolds and on a flat glass surface, by confocal laser scanning microscopy (CLSM) and Western blot, quantifying the expression of specific biomarkers (inducible nitric oxide synthase, iNOS, and Arginase 1, Arg1). These analyses enabled us to determine which micro scaffold and to what extent contributed to pushing macrophages toward an M2 pro-healing phenotype.

*Results: The in vitro experiments allowed us to evaluate the contribution of the 3D micro scaffolds’ and their different pore sizes in directing the polarization of macrophages. Quantification of protein expression levels by immunofluorescence and Western blot showed that the presence of micro scaffolds does not promote M1 polarization (iNOS expression), while to favor an M2 pro-healing phenotype, it is necessary to choose larger pore scaffolds, such as 50x50x20 μm³. Moreover, the presence of the micro scaffolds showed a significant increase in Arginase 1 expression, maximizing macrophage polarization toward M2.

*Conclusion/Significance: The experiments showed that different 3D micro scaffold geometries can influence macrophage behavior, favoring their polarization to a pro-inflammatory or pro-healing phenotype and confirming their role in determining macrophage response. This finding suggests that the design of microstructures for decorating devices used for in vivo implantation needs to consider specific tridimensional features for preventing fibrotic encapsulation and favoring a successful integration. An in-depth analysis of the role of 3D micro scaffolds in influencing rearrangements in the macrophage cytoskeleton with activation of mechanotransduction pathways will be performed, together with an evaluation of specific bioactive molecules released into cell culture supernatants.

Acknowledgements: European Commission (EU, FET-OPEN project IN2SIGHT, G.A. 964481); European Research Council (ERC, project BEACONSANDEGG, G.A. 101053122). Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them.

120 - Liver-targeting Microparticles For Hepatocyte Transplantation

I.-N. Lee¹, V. Gadd², C. Ashmore-Harris², S. Walker², S. Forbes², S. J. Forbes², L. J. White¹

¹ University of Nottingham, Nottingham, United Kingdom,

² University of Edinburgh, Edinburgh, United Kingdom

*Purpose/Objectives: Hepatocyte engraftment is a critical challenge for liver cell therapies. The majority of transplanted cells are cleared from the liver by proinflammatory cytokines, chemokines or receptors following instant blood-mediated inflammatory reaction or after the activation of resident macrophages. The addition of immunomodulatory cytokines, such as IL-10, or etanercept, a TNF blocker which specifically binds to TNF-α, has potential to reduce inflammation and improve cell transplantation. Encapsulation of these molecules within microparticles (MPs) for co-transplantation with hepatocytes provides controlled release strategies to increase cell engraftment. However, MPs typically fabricated with commercially available polymers are non-specific to the liver, resulting in inefficient delivery of encapsulated molecules. The asialoglycoprotein receptor (ASGPR) exhibits high affinity as a galactose receptor and is the only liver-specific receptor identified on hepatocytes. We demonstrate targeted drug delivery to the liver via microparticles fabricated with galactose ligands to achieve targeted release profiles. Using suitable mice model, co-transplantation of MPs and hepatocyte can promote hepatocyte survival.

*Methodology: Galactosylation of poly(lactic-co-glycolic acid) (Gal-PLGA) was synthesised in-house. In vitro functional release was measured using ELISAs. Immunomodulatory response of MPs in vitro was performed using THP-1 differentiated macrophages. In vitro MP-cell attachment was performed using HepG2. In vivo biodistribution was performed using wild type mice through hepatic portal vein and intrasplenic injections. Evaluation of in vivo response to IL-10 and etanercept loaded microparticles was performed using CCl4 and Ah^CreMdm2^fl/fl mice models that induce liver injury and injury + senescence respectively.

*Results: MPs fabricated from Gal-PLGA demonstrated an enhanced MP retention (>85%, compared to 40% with conventional PLGA) in vitro. In vivo biodistribution studies showed superior MP retention in the liver (>70%) compared to commercial PLGA MPs (<30%), when MPs were delivered via hepatic portal vein or intrasplenic injections. Gal-PLGA MPs exhibited a mean controlled daily release for up to 7 days, providing suitable formulations for etanercept and IL-10 release. In vitro functional etanercept release showed binding with TNF-α and reduced inflammation observed by down-regulated pro-inflammatory genes (CXCL2, IL-1β) and reduced release of soluble inflammatory cytokines (TNF-α, IL-6). In vivo dose response performed in CCl4 mice showed no apparent safety concerns with 0.1 mg MP dose. CCl4 mice showed no apparent difference in regulation of inflammatory cytokines. Preliminary dose response performed in Ah^CreMdm2^fl/fl mice showed detectable down regulation of inflammatory cytokines (IL-1β and IL-6) (Figure 1).

*Conclusion/Significance: We have developed novel synthesis routes for galactosylation of polymers and subsequently fabrication of Gal-PLGA microparticles. MPs were preferentially retained in the liver in vivo via both hepatic portal vein and intrasplenic injection methods; this suggests that the inclusion of galactose moieties in MPs sustained robust liver retention. MPs had controlled functional release kinetics of etanercept and IL-10 and in vitro reduced inflammation. There were no adverse safety impacts when etanercept and IL-10 MP formulations were delivered to CCl4 and Ah^CreMdm2^fl/fl mice. Downregulation of inflammatory cytokines was achieved in Ah^CreMdm2^fl/fl mice. Ah^CreMdm2^fl/fl mice are a suitable model for testing immunomodulatory cytokine loaded microparticles co-transplanted with hepatocytes.

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128 - Physical Stimulation Of Stem Cells Using Surface Acoustic Waves

K. Witte¹, C. Witte², J. Reboud², M. Salmeron-Sanchez², J. M. Cooper²

¹ University of Strathclyde, Glasgow, United Kingdom,

² University of Glasgow, Glasgow, United Kingdom

*Purpose/Objectives: In vivo cells are subjected to myriad of physical stimulations which are crucial for cell signalling and cell development. While the individual influence of these on cellular behaviour, particularly stem cell differentiation, is not well understood, in vitro and in vivo trials investigate a range of technologies to impose stimuli and regulate cell fate. We propose the use of surface acoustic wave (SAW) transducer as a versatile tool to deliver physical cues for stem cell stimulation. We introduce a platform that generates mechanical stimuli in form of travelling wave induced nanometer surface deformations, radiation pressure induced compression and rarefaction, acoustic streaming induced shear stress as well as electrical stimuli through piezoelectrically induced surface potentials and associated electric fields (Figure 1a).

*Methodology: Single phase unidirectional transducer (SPUDT) fabricated onto a 128° Y-cut lithium niobate wafer was used to generate pulsed surface acoustic waves at 7 MHz, 14 MHz, 21 MHz and 34 MHz (Figure 1b). Culture wells were seeded with human MSCs from bone marrow and stimulated 30 mins a day for 14 days. Positive control cells (no SAW) were supplemented with growth factor (BMP2). Negative controls (no SAW), positive control and stimulated samples were cultured in DMEM with 1 % FBS. Cells were stained for osteopontin (OPN) and qPCR for gene expression levels of various osteogenic markers was carried out.

*Results: Stem cells were seeded in commercially available culture wells. Surface acoustic waves were coupled via mode conversion into the substrate of the culture well, subjecting cells to power and frequency dependent surface deformations from 100 pm to 5 nm (Figure 1c), with corresponding pressure amplitudes of 15 kPa to 850 kPa. Viability (>95 %) of cells was maintained using a short pulsed (250 ms, 250 mHz repetition rate) SAW actuation cycle of 30 mins a day (Figure 1d). Stimulation over a course of 14 days at 7 MHz, 14 MHz and 21 MHz showed significant up regulation of the osteogenic marker OPN compared to untreated samples (Figure 1e and f). Comparison of the frequency dependent surface deformation amplitudes with the OPN content in the cells suggested a correlation, where higher amplitudes resulted in higher protein expression. Moreover, relative gene expression analysis showed an increase in OPN (Figure 1g) and osteocalcin (OCN) RNA for positive control and all frequencies except 7 MHz, which showed a decrease compared to untreated controls. This can be understood as an earlier onset of RNA expression resulting in a higher concentration of OPN as seen in samples treated with 7 MHz as compared to other frequencies. Therefore, considering results from OPN staining and time dependent expression of the corresponding gene, 7 MHZ stimulation could accelerate the commitment towards osteogenic differentiation.

*Conclusion/Significance: Surface acoustic waves can deliver a range of physical stimulation phenomena to cells cultured on surfaces without compromising viability. Our first results suggested an early osteogenic commitment when MSCs were stimulated with high frequency SAW.

129 - Multi-material 3D Bioprinting Of Human Stem Cell Based Corneal Structures With Stroma And Epithelium

S. Huhtanen¹, P. Puistola¹, M. Kauppila¹, K. Hopia¹, S. Miettinen^1,2, H. Skottman¹, A. Mörö¹

¹ Tampere University, Tampere, Finland,

² Tampere University Hospital, Tampere, Finland

*Purpose/Objectives: There is an immense need for corneal transplants to restore vision after injury or disease. Due to the shortage of donor corneas, three dimensional (3D) bioprinting has emerged as a solution with tremendous potential to manufacture artificial corneas. The cornea consists of three cellular layers, with the stroma and epithelium forming the two outermost layers. Therefore, multi-material 3D bioprinting of different bioinks and appropriate cell types is needed. Harnessing the potential of human pluripotent stem cell-derived corneal limbal epithelial stem cells (hPSC-LSCs) holds promise for the epithelial layer. Meanwhile, corneal stromal keratocytes derived from human adipose stem cells (hASC-CSKs) exhibit high clinical relevance and the potential to form the stromal layer. To achieve fully matured corneal structures comprising both stroma and epithelium, a vital step is to establish successful co-culture of the bioprinted structures. However, the complex composition of co-culture mediums affecting the stability of bioprinted structures poses a challenge for long-term co-culture of these structures, crucial for tissue formation and maturation. Previously, hASC-CSKs have been successfully bioprinted in HA-based bioink. In this study, the HA-based bioink was optimized for hPSC-LSCs. Furthermore, an additional crosslinking method was explored for the HA-bioink used for the stromal layer to enhance the stiffness and stability in the complex co-culture condition.

*Methodology: The same HA-based bioink backbone was used for bioprinting both the epithelial and stromal layers. A methacrylated hyaluronic acid (HAMA) component was incorporated into the stromal bioink for additional crosslinking. The HA-based bioinks with and without HAMA were compared by analyzing the printability, shape fidelity, viscosity, swelling behavior, handling, mechanical properties, and transparency of the bioinks in hPSC-LSC medium. Thereafter, the cytocompatibility of the bioinks with and without HAMA was assessed with hASC-CSKs. Finally, multi-material bioprinting of hASC-CSKs and hPSC-LSCs was carried out to manufacture human corneal structures with stroma and epithelium.

*Results: The HA-bioink with HAMA had shear-thinning properties, and demonstrated good printability, shape fidelity and transparency. Importantly, the addition of HAMA did not hamper these properties when compared to HA-bioink without HAMA. The HA-bioink with HAMA showed good stability in hPSC-LSC medium and had improved characteristics regarding handling, swelling, and shape fidelity. Interestingly however, the HA-bioink with HAMA lacked the necessary cytocompatibility required for bioinks and demonstrated inferior cytocompatibility with hASC-CSKs compared to the HA-bioink without HAMA. Thus, the final multi-material 3D bioprinting was carried out without HAMA. These structures with stroma and epithelium were successfully bioprinted and co-cultured for seven days.

*Conclusion/Significance: This study underlined the importance of conducting cytocompatibility tests as part of bioink characterization and the challenge of long-term co-culture of different cell types needed for tissue maturation after bioprinting. To the best of our knowledge, corneal structures with stroma and epithelium have not been previously 3D bioprinted using extrusion-based bioprinting technology. Therefore, this study advances the development of bioprinted corneal structures combining multiple corneal layers and provides valuable information in the field of bioprinting multiple cell types into one structure.

131 - Development Of A Multi-layered Dynamic In Vitro Trabecular Meshwork Model For Comprehensive Glaucoma Drug Screening

M. Z. Gladysz

University of Groningen, Groningen, Netherlands

*Purpose/Objectives: Glaucoma, a leading global cause of irreversible blindness, is caused by impaired aqueous humor drainage through the trabecular meshwork (TM) - a complex tissue comprising three layers within the eye's anterior chamber. Existing TM models often fall short, emphasizing only the juxtacanalicular tissue (JCT) while neglecting the nuanced architecture of the complete TM. This study aims to pioneer an in vitro TM model, faithfully replicating its hierarchical structure, encompassing all TM layers (JCT, corneoscleral meshwork, and uveal meshwork) for comprehensive glaucoma drug screening studies.

*Methodology: Utilizing the melt electrowriting (MEW) technique, artificial TM scaffolds were fabricated from polycaprolactone, featuring three distinct designs that mimic the layered architecture and sizes of the TM (Figure 1A). Mechanical properties were assessed through tensile testing. Employing a dynamic Fluigent perfusion system, coupled with custom-made inserts, we conducted studies under flow conditions with real-time control. Primary human TM cells were cultured on the printed scaffolds in both static and dynamic conditions. The results validation included outflow facility measurements, cell viability and metabolic activity assays, immunofluorescence staining for specific markers such as myocilin and αB-crystallin, scanning electron microscopy for cell morphology, and Latrunculin B exposure to study cellular response to drug administration (Figure 1 B, C, and D).

*Results: MEW-printed scaffolds successfully replicated the intricate TM layers, incorporating varying pore sizes, geometries, fiber, and layer thickness, closely resembling native tissue architecture. Real-time outflow facility measurements in the dynamic system faithfully mirrored native conditions. Primary human TM cells demonstrated high viability (>90% over 15 days) and increased metabolic activity over 13 days of culture. Cells subjected to dynamic conditions exhibited distinct behaviour, evidenced by altered myocilin and αB-crystallin expression, pivotal proteins associated with TM and glaucoma. Given that αB-crystallin expression is linked to mechanical stresses, its expression was notably lower than that of myocilin. Furthermore, myocilin expression varied between static and dynamic conditions, hinting at further investigation, particularly due to its known mutations associated with primary open-angle glaucoma.

*Conclusion/Significance: Outflow facility measurements for JCT-only scaffolds closely mirrored in vivo values, while the three-layered scaffold exhibited different trends attributed to low initial seeding density, currently being corrected in ongoing studies. Initial Latrunculin B tests displayed desirable actin skeleton disruption, suggesting its potential for increasing aqueous humor outflow. Ongoing drug exposure studies in dynamic conditions aim to further investigate changes in the outflow facility, as the early results suggest promising therapeutic outcomes.

132 - Tissue-engineered Human Corneal Endothelium

K. D. Brown¹, J. Palmer¹, J. R. Finnegan², J. Montenegro¹, G. G. Dusting¹, P. A. Gurr³, G. Qiao³, M. Daniell¹

¹ Surgical Research Unit, Centre for Eye Research Australia, East Melbourne, AUSTRALIA,

² Department of Chemical Engineering, University of Melbourne, Parkville, AUSTRALIA,

³ Department of Chemical and Biomolecular Engineering, University of Melbourne, Parkville, AUSTRALIA

*Purpose/Objectives: There is a global shortage of donor corneas. 12.7 million people are waiting for a corneal transplant. Endothelial keratoplasty (EK), replaces the most posterior tissue layer and leave remainder of patient cornea intact. EK accounts for >50% of keratoplasties in Australia. Expanding donor corneal endothelial cells (CEC) as a monolayer on a thin transparent strong permeable biocompatible carrier film would create a tissue engineered endothelial keratoplasty (TEEK) compatible with existing EK techniques thereby expanding the tissue supply. Previously, we demonstrated in vivo safety and efficacy of TEEK incorporating a sheep CEC expanded on a novel patented poly(ethylene glycol) based hydrogel film (PHF). In this work human cells were used to compare various functionalised PHFs.

*Methodology: PHFs were functionalised with carboxylic acid (PHF-COOH) or arginine-, guanine-, aspartate- peptide (two formulations PHF-RGD1 and PHF-RGD2). All surfaces used were 13 mm diameter. The B4G12 corneal endothelial cell line, used to screen the formulations before proceeding to donor cells, was seeded at 200 cell/mm² was cultured on PHF (non-functionalised control), PHF-RGD1, PHF-RGD2, PHF-COOH, and tissue culture plastic (positive control) for 14 days, then fixed in 4% PFA and immunofluorescence for zonula occuludens-1 (ZO-1), a marker for CEC, was performed. Morphometrics including confluence, cell area, and cell circularity was performed on immunofluorescent images using ImageJ. Cell/mm² was calculated from cell area and confluence. A lead candidate formulation was selected for validation with human donor CEC seeded at 200 cell/mm². Tissue culture plastic acted as a positive control surface. After 14 days of culture cultures cells were fixed in 4% PFA and immunofluorescence for zonula occuludens-1 (ZO-1), was performed.

*Results: On all surfaces B4G12 cells by immunofluorescence demonstrated that cells expressed ZO-1 and had polygonal morphology indicating the cells maintained corneal endothelial phenotype. As hypothesised, functionalised hydrogels produced superior cultures with higher confluence, smaller cells, and therefore higher cells/mm² than non-functionalised hydrogels. Mean results (confluence, area, calculated cell/mm², circularity): non-functionalised hydrogel (50 %, 317 µm², 1578 cell/mm², 0.74), PHF-RGD1 (93%, 305 µm², 3065 cell/mm², 0.71) PHF-RGD2 (93%, 241 µm², 3877 cell/mm², 0.71), PHF-COOH (87 %, 368 µm², 2357 cell/mm², 0.72), tissue culture plastic (100 %, 263 µm², 3805 cell/mm², 0.71). A donor corneal endothelium had a circularity of 0.76. Eyebanks typically require donor tissue to have greater than 2500 cell/mm². PHF-RGD1 was selected as the lead candidate formulation for validation. Donor cells (age 4 years, female, cause of death hypoxic brain injury) in culture. Cultures reached confluence <9 days. After culture on PHF-RGD1 immunofluorescence demonstrated cells expressed ZO-1 and showed polygonal morphology, indicating that the cells maintained corneal endothelial phenotype.

*Conclusion/Significance: Our results demonstrate functionalisation of the PHF improves cell confluence while retaining CEC phenotype. Human donor cells seeded at low density were able to expand to confluence on PHF-RGD1 of a diameter suitable for keratoplasty. These results indicate that a PHF-based TEEK could alleviate donor cornea shortages.

133 - Transplantation Of CGMP Compliant IPCS-derived Retinal Progenitor Cells

L. R. Bohrer, L. A. Wiley, A. T. Wright, L. Affatigato, R. F. Mullins, E. M. Stone, I. C. Han, B. A. Tucker

University of Iowa, Institute for Vision Research, Iowa City, IA

*Purpose/Objectives: Induced pluripotent stem cell (iPSC)-derived retinal progenitor cells are promising for restoring vision in patients with retinal degeneration. The purpose of this study was to evaluate local tolerability and cellular integration of human retinal cells generated under cGMP in a rat model of retinal degeneration following subretinal transplantation.

*Methodology: Patient-derived iPSCs were generated via Sendai viral (Cytotune 2) reprogramming of dermal fibroblasts. Retinal organoids were derived using a stepwise 3D differentiation protocol testing different cGMP compliant reagents and oxygen tension (5-20%) in a cGMP compliant Biospherix cell culture isolator. Organoids were characterized via light and confocal microscopy. At day 160, organoids were dissociated with papain and retinal progenitor cells were subretinally transplanted into immune suppressed Pde6b-/- rats with advanced disease (i.e., no photoreceptor cells remaining at the time of transplantation) (N=22). Immune response in rats and human donor cell survival, cellular identity, and synaptic integration were assessed at 3- and 30-days post-injection.

*Results: IPSC reprogramming efficiency is highest under 5% oxygen tension, however, retinal organoid production was inefficient at this oxygen level. Increasing oxygen tension to 20% resulted in decreased pluripotency and increased ectoderm gene expression by day 7 of differentiation. By day 30, laminated retinal organoids were abundant in cultures differentiated under 20% oxygen tension, while they were smaller and less consistent at 5% oxygen tension. By 160 days of differentiation, retinal organoids generated at 20% oxygen tension under cGMP conditions contain abundant rod and cone photoreceptor precursor cells expressing markers such as NRL and cone arrestin, respectively. Human donor cells were identified in 100% of immunosuppressed rats. At both 3- and 30-days post-injection, cells expressing donor cell antigen (HNA) and photoreceptor cell markers (e.g., recoverin) were abundant at the injection site (Figure 1). Widespread neurite extension and expression of synaptic markers in conjunction with host bipolar cells was observed (Figure 1).

*Conclusion/Significance: We have generated a cGMP compliant iPSC-derived retinal organoid differentiation protocol suitable for derivation of transplantable retinal progenitor cells.

134 - A Pre-Vascularized Structured Hydrogel Membrane To Rescue The Outer Blood-Retina Barrier In Retinal Dystrophies

C. Dujardin¹, W. Habeler², O. Goureau³, C. Monville², D. Letourneur¹, T. Simon-Yarza¹

¹ Université Paris Cité, Université Sorbonne Paris Nord, Laboratory for Vascular Translational Science - LVTS - INSERM U1148, Paris, France,

² INSERM U861, Institute for Stem Cell Therapy and Exploration of Monogenic Diseases – I-Stem, AFM, Corbeil-Essonnes, France,

³ Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France

*Purpose/Objectives: The outer blood-retina barrier (oBRB), composed of the retinal pigment epithelium (RPE), on the Bruch’s membrane (BM) and overlying the vascularized choroid, is disrupted in several retinal dystrophies, leading to barrier leakage and vision loss. Tissue engineering techniques have been explored for a few decades to cope with these diseases (Dujardin et al. 2023). One therapeutic strategy, currently under clinical trial, relies on the implantation of a RPE cultivated on a biomaterial mimicking the BM (Ben M’Barek et al. 2017). However, this approach does not include the choroid or any pre-vascularization, which can alter the graft integration and survival upon implantation. Here, we aim to develop a 3D structured membrane, for cellular therapy, mimicking the entire oBRB, with both the RPE on top of the graft and a pre-vascularization within the membrane to optimize its integration.

*Methodology: A polysaccharide 200 µm thick hydrogel was synthesized from a solution of pullulan and dextran, and freeze-dried (FD) to tailor its porosity to mimic the oBRB structure. The membrane was then coated with collagen to enhance cellular adhesion and further freeze-dried. The membrane was characterized in terms of porosity, permeability, along with mechanical and degradation properties. After an additional laminin coating of the membrane, the RPE cells derived from human induced pluripotent stem cells were seeded on top of the hydrogel for up to a month. For co-culture experiments, human retinal microvascular endothelial cells (HRMVEC) were seeded on the opposite side, 10 days after the RPE cells, to form the pre-vascularization over 3 weeks (Fig. A). Pericytes were also incorporated as support cells. Immunostainings and protein quantification were conducted to characterize the oBRB model.

*Results: After FD optimization, we obtained membranes with, on one side, a smooth non-porous surface intended for the RPE monolayer and, on the other side, a porous surface connected to the inner porosity for the pre-vascularization (Fig. B). Permeability studies showed that molecules under 130 kDa are able to diffuse across the membrane, enabling exchanges between the different cell types. Thanks to the coatings, the RPE cells adhered and proliferated on the smooth side, forming a tight monolayer (Fig. D) within a week, without entering the porous network. After 10 days of RPE culture, the HRMVEC were seeded on the porous side and were able to enter the inner structure, where they adhered, proliferated and formed tight junctions (Fig E). Each cell type remained located on its dedicated side (Fig. C). Cellular survival and expression of typical markers increased in co-culture showing beneficial interactions between the two cell types. Addition of pericytes along with the HRMVEC allowed for a more complex pre-vascularization.

*Conclusion/Significance: In conclusion, we designed a membrane mimicking the oBRB structure and suitable for co-culture with precise localization of each cell type. Co-culture experiments showed positive interactions between RPE and ECs, thus demonstrating the importance of including the choroid in an oBRB model. In vivo experiments are on-going to study the graft immunogenicity and the benefits of the ECs and pericytes presence on the graft vascularization.

135

137 - Expansion Of Hematopoietic Stem Cells On Engineered Human Liver Cultures For Regenerative Medicine

A. Michell, G. Brown, K. Pajcini, S. Khetani

University of Illinois Chicago, Chicago, IL

*Purpose/Objectives: The need for bone marrow (BM) transplants and blood transfusions in the clinic remains unmet with 17K+ Americans requiring BM transplants yearly. Hematopoietic stem cells (HSCs) are the stem cell population critical for blood reconstitution. The main limitation to improving HSC transplants is the inability to expand functional HSCs in vitro without differentiation. The fetal liver is the site of hematopoiesis where HSCs undergo massive expansion and differentiation before homing to their quiescent BM niche. In cases of pathological BM damage, HSCs return to the liver for hematopoiesis, suggesting that the adult liver retains its hematopoietic capability. Therefore, we sought to test the novel hypothesis that adult HSCs can be expanded on functionally stable micropatterned co-cultures (MPCCs) of primary adult hepatocytes and fibroblasts and can repopulate damaged BM in mice. We also sought to develop a fetal liver model to expand HSCs, hypothesizing that the liver’s age is a critical variable in the expansion fold potential and functionality of HSCs.

*Methodology: Multi-well plates were subjected to soft lithography to pattern collagen into circular domains. Primary adult hepatocytes were seeded onto the micropatterned collagen and supportive 3T3-J2 murine embryonic fibroblasts were subsequently seeded around the hepatocyte islands to form MPCCs. MPCCs were then allowed to stabilize for 7 days before switching to a serum-free media. Next, the BM from 6-8-week-old mTmG transgenic mice, in which the hematopoietic compartment is labeled by dTomato, was isolated. Long-term HSCs were purified using flow cytometry for LSK markers (Lin^-Sca1⁺cKit⁺) and SLAM markers CD150⁺CD48^- and 50 HSCs/well were seeded onto MPCCs (Fig. 1A) for 2 weeks. Additionally, hepatoblasts from fetal mouse livers at E14.5 were isolated and either used to form MPCCs or seeded into microwells to form spheroids (Fig. 1B). HSCs were seeded onto fetal cultures as described above. Flow cytometry was used on cultured HSCs to determine the retention of LSK and SLAM markers. Cultured HSCs were also transplanted into lethally irradiated mice, and engraftment and differentiation were tracked for 3 months.

*Results: Cultured HSCs maintained their small morphology (Fig. 1C). Long-term HSCs expanded 10-fold, while HSCs cultured on fibroblast-only and hepatocyte-only wells had little to no expansion (Fig. 1D). In vitro expanded HSCs on MPCCs could rescue lethally irradiated mice following transplantation (Fig. 1E). Fetal hepatoblasts retained high albumin production in HSC expansion media only in spheroid cultures, while albumin declined in MPCCs (Fig. 1F); high expression of hepatic genes for the hepatoblasts was retained across all conditions (Fig. 1G). Cells expanded significantly over time on spheroid cultures (Fig. 1H, I); however, long-term HSCs showed declining frequency of the live population and fluctuating counts (Fig. 1I).

*Conclusion/Significance: We demonstrate for the first time that HSCs can be expanded on liver cultures of either fetal or adult cells, and the expanded HSCs are functional during transplantation assays. Our next steps include increasing HSC stability in fetal liver cultures by splitting the cultured HSCs after Week 1 of expansion, incorporating fetal liver sinusoidal endothelial cells, and comparing fetal and adult liver cells’ hematopoietic potential.

138 - Crispr Activation Of Il-10 In Asc By Hybrid Baculovirus For Neuropathic Pain Attenuation

S.-W. Yang

National Tsing Hua University, Hsinchu, Taiwan

*Purpose/Objectives: Neuropathic pain is one of the major neuronal diseases worldwide and treatment has remained a challenge in clinical settings due to its pathological complexity, one of which is attributed to hyperinflammatory homeostasis. Anti-inflammatory cytokines are, therefore, potential targets for inflammation alleviation and pain control. Adipose-derived mesenchymal stem cell (ASC) has proven effective for various cell therapy applications thanks to its immunomodulatorary, multipotency, abundance, and accessibility; but its potential for neuropathic pain attenuation has not been demonstrated. In this study, we aimed to engineer ASC with enhanced secretion of anti-inflammatory by CRISPR activation (CRISPRa) platform and employ the engineered ASC to reduce inflammation and alleviate neuropathic pain.

*Methodology: We constructed the CRISPRa system consisting of (1) the catalytically inactive Cas9 from S. aureus fused with the tripartite transcriptional activator domain VP64-p65-Rta (dSaCas9-VPR) and (2) a 4 single guide RNA (sgRNA) array targeting the anti-inflammatory cytokine IL-10. The two components were subsequently flanked with 2 loxP sequences, facilitating episomal DNA minicircle formation of the transgenes and enhanced expression in the presence of Cre recombinase. dSaCas9-VPR, 4-sgRNA array and Cre expression cassettes were packaged into baculoviral vectors (Bac-VPR, Bac-IL-10 and Bac-Cre) for transduction. ASC was isolated from SD rat subcutaneous fat pad and cultured in 15-cm dish with αMEM supplemented with penicillin-streptomycin, serum and bFGF. Transduced ASC was collected for RNA extraction and qRT-PCR for differential gene expression analysis while culture medium was harvested for ELISA IL10 quantitation. Alternatively, the transduced ASC was subsequently co-cultured with LPS and IFNγ-stimulated RAW 264.7. The co-cultured RAW 264.7 was daily imaged for morphology monitoring or subjected qRT-PCR analysis.

*Results: IL-10 transcript level was robustly activated (∼ 615-fold), leading to an increase in secreted protein level (∼403 pg/ml) in transduced ASC up to 13 days post-transduction. Co-culturing with IL-10-secreting ASC led to the phenotype change in stimulated RAW 264.7. More importantly, transduced ASC induced can upregulation in Cd206, Il4ra, and Arg1 and a downregulation in Cd80, Tnfa, and Il6 expression in RAW 264.7.

*Conclusion/Significance: Altogether, these data strongly suggested the application of CRISPRa-engineered ASC facilitated in inflammation mitigation and neuropathic pain treatment.

139 - 3D PRINTED PULMONARY HEART VALVES FOR PEDIATRIC PATIENTS

A. Jafari, H. Savoji

University of Montreal, Montreal, QC, Canada

*Purpose/Objectives: Congenital and acquired valvular heart diseases are significant causes of mortality worldwide. With valve replacement being the primary solution for valvular heart diseases, current options display shortcomings, including calcification, thrombogenicity, and hemodynamic alteration, leading to repetitive surgeries. Tissue engineering, however, has shown great potential for fabricating heart valves with fewer complications. The goal of this study was to formulate novel inks for the 3D printing of tissue-engineered heart valves. Special considerations have been made so that the engineered inks, with suitable physicochemical, mechanical, and biological characteristics, can be printed in air to fabricate complex tissue-sized structures with tissue-specific features (i.e., tissue-engineered heart valves).

*Methodology: Composite inks were formulated from synthetic and natural polymers, namely poly(vinyl alcohol) (PVA), gelatin (Gel), and κ-carrageenan (CG). The inks/hydrogels were investigated to characterize their physico-chemical, morphological, mechanical, and rheological characteristics. In vitro and in vivo biocompatibility, immune response, hemolysis, and thrombogenicity of the inks/hydrogels were also evaluated. Moreover, in vitro hydrodynamics of the tissue-engineered heart valves under physiological conditions were reported.

*Results: The aim of this study was to develop a new ink capable of high-precision 3D printing of complex structures such as heart valves. The selected materials provided the mechanical, rheological, and biological characteristics essential for this purpose. Collectively, this research introduced new tailored inks to 3D print large, sophisticated constructs without the need for a supporting bath and strands. Rheological characteristics, including shear-thinning behavior, high-yield stress, and fast recovery, were the main aspects contributing to this feature. Large human-sized heart valves were successfully 3D printed and shown to have high fidelity compared to the initial digital file (i.e., CAD design) used for 3D printing. Moreover, the mechanical properties were shown to be comparable to the native valve leaflets, and the hydrogels are capable of withstanding cyclic compressions with similar frequencies to a native heart (i.e., 1 Hz). From the biological point of view, composite scaffolds revealed high biocompatibility assessed by in vitro cell culture using inhabitant valvular cells (VICs). From the structural point of view, VICs showed expected fibroblastic phenotype with elongated and spindle-like morphology that expressed Vimentin and α-SMA. In vitro hemocompatibility assessments have proved minimal hemolysis of the proposed hydrogels with low thrombogenicity. Furthermore, subcutaneous implantation showed that fabricated hydrogels induced a non-chronic inflammation response and underwent in vivo resorption and remodeling, reconfirming the biocompatibility of the hydrogels in physiological conditions. Finally, in vitro hydrodynamic assessment of 3D-printed heart valves confirmed that they are capable of withstanding aortic conditions. Leaflets' opening and closing during cyclic pulsatile flows were observed, and printed valves could function properly with no regurgitation and stenosis for the bioprosthetic-like valve and no regurgitation and mild stenosis for the tri-leaflet valve.

*Conclusion/Significance: The aim of this study was to develop a new ink capable of high-precision 3D printing of complex structures such as heart valves. Altogether, the 3D-printed tissue-engineered heart valves can be a promising alternative for valve replacement to solve the problems associated with the current options.

141 - Human Lung Bioinks Embedded With Induced Basal Stem Cells Can Be Used To 3d Bioprint Pediatric-sized Airways

H. Wanczyk¹, L. Asarian², D. Weiss³, C. Finck⁴

¹ Uconn Health, Farmington, CT,

² Helmholtz Munich, Munich, Germany,

³ University of Vermont, Burlington, VT,

⁴ Connecticut Childrens, Hartford, CT

*Purpose/Objectives: Ex vivo bioengineering of functional respiratory tissues remains a challenge despite numerous efforts over the past few decades. However, new and emerging technologies such as 3D bioprinting, are increasingly being applied to the printing of trachea and large airways for treatment of pediatric respiratory conditions. For this purpose, biological matrices derived from native tissue can be used as bioinks to aid in the development of functionally mature 3D printed lung tissues. We demonstrate the ability of decellularized human lung tissue (dECM) to support the differentiation, maturation and function of embedded induced basal stem cell (iBCs) organoids and show its utility in bioprinting pediatric sized airways.

*Methodology: Human induced pluripotent stem cells (hiPSCs) differentiated to iBCs and expressing a dual fluorescence reporter system (NKX2-1-GFP, TP63-tdTomato) were embedded in 8 mg/mL Matrigel, 30 mg/mL bulk lung, or 15 mg/mL and 30 mg/mL airway and cultured in Pneumacult Ex medium over 23 days. Organoid morphology was evaluated via microscopy as well as airway marker expression via IF and qPCR analyses. For 3D bioprinting feasibility, lung CT scans were converted into 3D files using 3D Slicr and bioprinted with 4% alginate + airway bioinks.

*Results: iBC organoids embedded in bulk lung and airway matrices exhibited excellent proliferation over 23 days of culture and expressed markers associated with ciliated (FOXJ1), club (SCGB3A2, SCGB1A1), and goblet (MUC5AC, MUC5B) expression, while also maintaining TP63+ and NKX2.1+ protein expression in a subset of cells, indicated by IF. By Day 9, iBCs in bulk and airway matrices exhibited elevated MUC5AC and MUC5B expression compared to a human lung control and were absent in Matrigel alone. Cells cultured in 30 mg/mL airway presented with larger, self-organizing structures compared to those in 15 mg/mL airway. Additionally, iBCs cultured in 2D over 1 month in submersed culture demonstrated functionally beating cilia capable of mucus transport. Finally, 30 mg/mL airway + 4% alginate bioinks exhibited excellent shear thinning behavior as evidenced by successful 3D bioprinting of pediatric sized airways.

*Conclusion/Significance: This study demonstrates the utility of decellularized lung matrices to support the differentiation, proliferation and maturation of embedded iBCs. Additionally, when combined with alginate, bioinks exhibit excellent shear thinning behavior, which allowed for 3D bioprinting of pediatric-sized airways. This work paves the way for improved treatment options for individuals suffering from chronic respiratory diseases and congenital defects of the lung.

143 - Rat Liver Extracellular Matrix And Perfusion Bioreactor 4d Culture Promote Human Amnion Epithelial Cell Differentiation Towards Hepatocyte-like Cells

S. Campinoti¹, B. Almeida¹, N. Goudarzi¹, S. Bencina², F. Grundland Freile¹, C. McQuitty¹, D. Natarajan¹, J. Cox¹, A. Le Guennec³, V. Khati², G. Gaudenzi², R. Gramignoli², L. Urbani¹

¹ The Roger Williams Institute of Hepatology, London, United Kingdom,

² Karolinska Institutet, Stockholm, Sweden,

³ NMR Facility King's College London, London, United Kingdom

*Purpose/Objectives: Mortality caused by liver disease is on the rise, representing a significant global health issue. Transplantation is the only efficient treatment for end-stage liver disease but is limited by the shortage of organ donors. Human amnion epithelial cells (AEC) are a widely available source of cells with the plasticity of multipotent stem cells and immunomodulatory properties of perinatal cells. AEC can differentiate towards hepatocyte-like cells and rescue animals with metabolic disorders; however, they showed limited metabolic activities in vitro. Decellularised extracellular matrix (ECM) scaffolds have gained recognition as adjunct biological support. Here, we combined ECM-scaffolds and primary human AEC in a custom-made bioreactor culture and evaluated AEC differentiation into HCL.

*Methodology: Human AEC were seeded into decellularised rat liver scaffolds obtained via established protocols. AE cell-seeded scaffolds were maintained either in static condition (used as control), or in a custom bioreactor, in which the medium was perfused through the vasculature of the scaffold. For the first 10 days of culture, cells were exposed to expansion medium, and switched into differentiation medium for additional 20-30 days. Metabolite production and hepatic activity were monitored at different time points via NMR, ELISA and EROD assays. Transcriptome and proteomic analysis at intermediate and end-time points were used to characterise AEC differentiation into functional hepatocyte-like cells.

*Results: We confirmed appropriate decellularisation and production of whole rat liver ECM scaffolds assessing cell removal and preservation of ECM components. AEC viability post-cryogenic storage was >80% and 99% of cells were E-Cadherin⁺CD49f⁺EpCAM⁺. Cell retention after seeding was 91%, and sterility was preserved throughout the culture with the bioreactor.AEC-seeded scaffolds cultured for up to 40 days in both static and bioreactor conditions showed comparable cell number and homogeneous distribution. Immunofluorescence analysis showed AEC expressed higher levels of CK18 and CK19 and hepatocyte functional markers (albumin, CYP3A4 and HNF4α) in bioreactor compared to static condition.Expression for early-stage CYPs and other hepatic genes (A1AT, UGT1A1 and HNF4α) was higher in bioreactor compared to static conditions, confirming that the perfusion aids differentiation to hepatocyte-like cells . Hepatic function was assessed longitudinally in both culture conditions: albumin secretion and urea concentration in the culture media were significantly higher in the bioreactor in comparison to static conditions. CYP1A activity measured by EROD assay increased during the culture, with higher conversion detected in bioreactor compared to static cultures.

*Conclusion/Significance: Rat liver scaffolds were successfully generated and used to promote the ability of AEC to differentiate into HLC. Our approach included using a custom 4D-bioreactor to provide constant oxygenation and media perfusion to cells in 3D-cultures over time. We successfully generated HLC positive for hepatic markers such as ALB, CYP3A4 and CK18. AEC-derived HLC displayed early signs of hepatocyte phenotype, secreted albumin and urea, and CYP activities. The combination of liver-specific ECM and bioreactor provides a system able to aid differentiation into HLC, indicating that the perfusion ECM-scaffold technology may support the functional improvement of tissue-engineered liver constructs for regenerative medicine.

144 - The Anti-inflammatory Effect Of Decellularized Extracellular Matrix Hydrogel Microparticles On Lps-induced Acute Lung Injury In Rats

C.-Y. Kao, H.-Y. Chin

National Taiwan University of Science and Technology, Taipei, Taiwan

*Purpose/Objectives: There is no standard treatment to mitigate the acute respiratory disease syndrome caused by the COVID-19 virus. One emerging supplementary therapy is mesenchymal stem cells (MSCs). However, whether the MSCs circulate throughout the body or migrate directly to damaged lung tissue still needs to be addressed. The inability of stem cells to stay in the lungs long enough may affect the therapeutic effects of stem cells. The extracellular matrix (ECM) hydrogels have been shown to support many stem cells’ growth and differentiation ability by providing a unique dynamic microenvironment. Therefore, it can be hypothesized that decellularized ECM hydrogel from porcine organs can serve as a promising candidate to create a scaffold for MSCs to stay long enough to exert their immunomodulatory effects on immune cells, which could be exploited to be an adjuvant therapy for COVID-19 patients. The aim of this study is to develop a novel pulmonary delivery system for the delivery of dECM hydrogel microparticles and study its anti-inflammatory effect on LPS-induced rats.

*Methodology: In this study, dECM hydrogels were prepared from porcine lungs as previously described. The decellularized efficiency of tissue was confirmed by using dsDNA quantification, DAPI staining, and DNA electrophoresis. The amount of collagen and sGAG retention was studied using ELISA kits. The dECM hydrogel particles were prepared by sonication from dECM hydrogel and were characterized by using SEM and DLS. The in vivo evaluations of the anti-inflammatory effects of the dECM hydrogels were evaluated by using an LPS-induced acute lung injury model of rats. TNF-alpha and IL-10 levels in the bronchoalveolar lavage fluid(BLAF) were analyzed for anti-inflammatory effects.

*Results: After treatment with 1% SDS and 10% FCS, the porcine lungs were decellularized effectively, with no nuclei being detected in DAPI staining and less than 50 ng dsDNA per 1 mg dried tissues. DNA fragments were also less than 100 base pairs in the electrophoresis test. The decellularized lung tissue retained 97% of collagen and 94.5% GAGs from native lungs. The resulting dECM hydrogel microparticles range from 0.4 to 2 μm. Histological analysis of lung inflammation was used to study the protective effect of dECM hydrogel microparticles in LPS-induced acute lung injury rats. The LPS-challenged group showed severe lung injury characterized by alveolar wall thickening, lung edema, and infiltration of neutrophils in the lungs. In contrast, the dECM hydrogel-treated group showed no significant inflammation or alveolar wall thickening. The dECM microparticles have significantly reduced protein level and TNF-alpha concentration in the BALF in rats.

*Conclusion/Significance: In summary, we generated a novel pulmonary delivery system for the delivery of dECM hydrogel microparticles. The dECM hydrogel microparticles were successfully delivery to the rat lungs and the dECM microparticle group exhibited significant anti-inflmammatory effects both in vitro and in vivo. We anticipate dECM hydrogel microparticles could serve as a new adjuvant therapy for COVID-19 patients.

145 - Development Of 3d Printed Human Amnion Based Bioactive Wound Dressings For Full Thickness Chronic Ulcers Management

S. Sarkar, A. Choudhari, A. Poundarik

Indian Institute of Technology Ropar, Ropar, India

*Purpose/Objectives: Chronic ulcers represent a substantial global health challenge, impacting approximately 40 million individuals globally and often leading to amputations, especially among the comorbid population. In developed countries, 1 to 2% of the entire population is susceptible to chronic wounds. Within India, the prevalence of chronic wounds stands at around 4.5 per 1000 population, a figure that is escalating rapidly with the rising diabetic population. In response to this challenge, we have innovated a 3D printed bioactive dressing utilizing human amniotic membrane. Leveraging the excellent regenerative potential of amnion and integrating 3D printing technology, the dressing aims to expedite healing and provide a customized fit for wound-specific geometries.

*Methodology: Fresh amnion was procured from Tata Memorial Hospital tissue bank, Mumbai. We postulated a methodology comprising novel decellularization combinations, trehalose lyopreservation followed by lyophilisation for developing graft with improved translational potential. The bioactivity of the processed grafts was assessed using chronic diabetic rat wound model (wound size 10 mm diameter, blood glucose level >300mg/dL) against standard of care dressing as comparator. Wound healing efficacy were assessed through longitudinal monitoring of healing indicators and ex vivo analysis of wound biopsies. For bioink preparation, processed graft was cryomilled, mixed with other suitable excipients and crosslinker. Rheological parameters were evaluated to optimize the printability and shape fidelity. Extrusion based printer (Alfatek systems, India) was used for printing the prototype dressing with nozzle diameter 0.25 mm, printing speed 10 mm/s.

*Results: à Physicochemical characteristics of processed graft: • Quantitatively 90% reduction in DNA content in processed graft compared to fresh membrane. Histological assessments unveil the absence of cellular populations and retain the compactness of the lyopreserved membrane substantially. • ECM components like total acid-soluble proteins, collagen and sulphated glycosaminoglycans are well-preserved. • Lyopreserved graft contained growth factors PDGF-BB (2.021±0.11 pg/mg) and VEGF-A (39.03± 1.27 pg/mg), essential for chronic wound healing. • Thermogram recorded for the processed graft showed thermal behaviour similar to collagen. à Animal trial data of processed graft: • Complete healing was achieved in the amnion-treated group after 9 days of treatment, whereas the SOC treated group required more than 21 days for complete healing. • Ex vivo evaluation of the wound biopsy specimen through histopathology affirmed improved healing attributes in amnion treated groups in terms of epithelialization, restoration of rete pegs, collagen fibre arrangements and neovascularization. à Printable bioink characteristics: • The storage modulus (G′) of the amnion bioink was higher than the loss modulus (G′′), indicative of a dominant elastic behaviour. • Amnion ink was subjected to alternating cycles of high (100%) and mild strain (0.1%) at a constant frequency. Shape recovery was observed after the high strain was removed. It regained its storage modulus each time after the withdrawal of maximum strain, for up to nine cycles.

*Conclusion/Significance: à These findings affirmed that the processing method adopted did not impede the bioactivity of amnion; rather, they contributed to enhanced physical properties. à 3D printing technique broadens the applicability of amnion to both partial and full-thickness wounds, overcoming its minimal thickness, limited mechanical resilience and inability to manage wound exudate.

147 - Evaluating Effects Of The Protein Components Of An Infusible Decellularized Extracellular Matrix Material For Targeting And Treating Leaky Vasculature Following Myocardial Infarction

M. Nguyen, A. Chen, C. Luo, K. Christman

University of California, San Diego, La Jolla, CA

*Purpose/Objectives: Current treatments for myocardial infarction (MI) and ischemia reperfusion (I/R) injury fail to address the resulting overreactive inflammatory response, resulting in negative left ventricular (LV) remodeling and heart failure. Decellularized extracellular matrix materials have demonstrated pro-regenerative effects, but require direct administration for delivery. Our lab has previously developed an infusible ECM material (iECM) derived from porcine LV myocardium, which can be delivered through intracoronary infusion following percutaneous coronary intervention, targets the leaky vasculature of the infarct, and exerts immunomodulatory and pro-regenerative effects. To better understand the mechanism of action of iECM, components were characterized and their biochemical properties, in vitro biological effects, and in vivo targeting ability were assessed.

*Methodology: iECM was manufactured by digesting decellularized porcine LV myocardium, separating large particulates through centrifugation, then filtering the supernatant through a 0.2 µm filter. Size exclusion chromatography (SEC) was used to quantify the molecular weight of iECM components. Based on SEC results, a molecular weight cut off (MWCO) of 10kDa was used to separate iECM components into two fractions, sub-10kDa and super-10kDa. Protein and peptide composition of iECM fractions were analyzed through SDS-PAGE and morphology was characterized by transmission electron microscopy (TEM). Fractions were analyzed for their biochemical properties, including free amines and thiols, which can mediate reactive oxygen species scavenging, and evaluated for their ability to promote survival of cardiomyocytes undergoing oxidative stress in vitro. For in vivo biodistribution, iECM and iECM fractions (10 mg/mL) were delivered through simulated intracoronary infusion following reperfusion in a rat I/R model of MI. 1 and 3 days post infusion, rats were euthanized and the heart and satellite organs were collected for analysis through near infrared imaging and immunohistochemical staining.

*Results: SEC analysis of iECM revealed a bimodal distribution with either high molecular weight components (>100 kDa) or peptide sized components (<5 kDa). Following fractionation against a 10 kDa MWCO, biochemical analysis demonstrated differences in free amines and free thiols between sub-10kDa and super-kDa fractions, with the sub-10kDa components of iECM containing more free amines and thiols. Protein composition analysis of iECM fractions also showed differences, with super-10kDa iECM components being predominantly fibrillar collagens based on findings from SDS-PAGE and TEM. Despite differences in structure and biochemical properties, the iECM fractions’ ability to promote cell survival was the same when tested in vitro. However, significant differences in the iECM fractions’ ability to localize to the site of infarct were observed, with super-10kDa components localizing primarily to the infarct, similar to complete iECM, while sub-10kDa components were spread throughout the heart, with no preference to the infarcted LV.

*Conclusion/Significance: Based on studying the components of iECM, a possible mechanism of targeting to the LV following MI has been elucidated. High molecular weight proteins with characteristics akin to fibrillar collagen appear to more readily target the infarct compared to the peptide sized components of iECM, suggesting their importance in the reduction in vascular permeability, and immunomodulatory and pro-regenerative effects previously observed. Ongoing studies will evaluate the in vivo biological effects of iECM fractions following administration.

149 - The Effect Of Platelet Activating Factor Acetyl-hydrolase On Matrix Bound Nanovesicle Immunomodulatory Activity

S. O. El-Mossier^1,2, S. F. Badylak^1,2, G. S. Hussey^1,2

¹ McGowan Institute For Regenerative Medicine, Pittsburgh, PA,

² University of Pittsburgh, Pittsburgh, PA

*Purpose/Objectives: Despite their widespread clinical use, the biologic mechanisms by which ECM bioscaffolds mediate functional tissue restoration are largely unknown. Mounting evidence shows that enzymatic degradation of the ECM scaffold material and subsequent release of Matrix Bound Nanovesicles (MBV) are critical for activation of the reparative and anti-inflammatory immune cell phenotype. MBV are nanometer-sized (∼50-200nm diameter), lipid membrane vesicles that are bound within the collagen network of the ECM within soft tissues and organs of all mammals. However, given their release into inflammatory environments, the effect inflammatory hydrolases on MBV lipids and downstream bioactivity remain unexplored. One such inflammatory hydrolase is Platelet Activating Factor Acetyl-Hydrolase (PAF-AH), a potent inflammatory mediator.The objective of this study was to investigate the effect of PAF-AH on the bioactivity of the MBV. Herein, we hypothesize that PAF-AH can directly hydrolyze MBV lipids, thereby altering their biologic activity. Previous studies showed that PAF-AH can bind directly to phospholipid vesicles. Our lab has previously shown that MBV are a rich reservoir of polyunsaturated fatty acids (PUFA) phospholipids. PUFA phospholipids can be hydrolyzed by PAF-AH, resulting in the release of free PUFA and lysophospholipids (LPLs) that can be further used to produce pro or anti-inflammatory lipid mediators.

*Methodology: MBV were isolated from porcine Small Intestine Submucosa (SIS) and pretreated with 1ug/1E^11 particles PAF-AH for 1 hour at 37C. After removal of PAF-AH via buffer exchange, the MBV were used (1E^11 P/mL) to-treat bone marrow-derived macrophages and RAW-264.7 cells to compare the effect of PAF-AH-treated MBV on the expression of pro and anti-inflammatory cytokines. After treatment for 24 hours, cells were washed with PBS and total protein was extracted.

*Results: The data showed that pre-conditioning the MBV with PAF-AH increased the expression CD206 in both primary macrophages and Raw-264.7 cells compared to control groups. These results were confirmed by pre-challenging macrophages with pro-inflammatory cytokines before treatment. Results showed that pre-conditioned MBV with PAF-AH significantly increased their capability to regulate/decrease the inflammatory markers compared to naïve MBV. These results suggest that the environment that the MBV is exposed to is important for their bioactivity, and that the initial inflammatory stage during injury is crucial in providing the necessary conditions for MBV bioactivity in immunomodulation and tissue remodeling.

*Conclusion/Significance: MBV are a rich reservoir of polyunsaturated fatty acids (PUFA) phospholipids. PUFA phospholipids can be hydrolyzed by PAF-AH, resulting in the release of free PUFA and lysophospholipids (LPLs) that can be further used to produce pro or anti-inflammatory lipid mediators. Recognition of the presence of MBV within ECM bioscaffolds and an understanding of their biologic activity provides opportunities for new therapeutic approaches that are otherwise untenable. In order to maximize the clinical potential of MBV, and EV in general, a firm understanding of the complex biology is warranted, and will inform on the rationale design and use of EV-based therapeutics.

150 - Microparticles Based On Solidified Stem Cell Secretome (MIPSOS) For The Treatment Of Diabetic Chronic Wounds

A. Blocki ^1,2

¹ The Chinese University of Hong Kong, Hong Kong, Hong Kong,

² Center for Neuromusculoskeletal Restorative Medicine, Hong Kong, Hong Kong

*Purpose/Objectives: Non-healing wounds are characterized by dysregulated microenvironment, resulting in delayed healing, thereby severely limiting patients’ quality of life. In an attempt to address limitations of current established and experimental treatment approaches, we engineered a functional biomaterial capable of capturing and concentrating the pro-reparative activities of mesenchymal stem cells (MSCs) in a rapid and scalable manner. MSCs were chosen due to their pro-angiogenic and anti-inflammatory properties and cultured in the presence of ascorbic acid and dextran sulfate (DxS). We have previously shown that DxS aggregates and co-precipitates with major extracellular matrix (ECM) components, thereby amplifying ECM deposition. Furthermore, the heparan sulfate mimetic character of DxS facilitated binding and thus accumulation of pro-angiogenic factors during ECM deposition. Upon decellularization, this dextran sulfate-ECM hybrid material, enriched in pro-angiogenic factors, was processed into MIcroparticles of SOlidified Secretome (MIPSOS), exhibiting enhanced pro-angiogenic bioactivity when compared to naïve MSC-derived ECM (cECM). Consequently, skin wounds treated with MIPSOS demonstrated accelerated revascularization and healing superior to treatment with cECM. In order to pave the way for clinical translation of MIPSOS we set out to further improve the manufacturing process of MIPSOS and identify functional components in MIPSOS responsible for the enhanced therapeutic effect.

*Methodology: Various xeno- and serum-free media were screened for their suitability to produce MIPSOS with enhanced pro-angiogenic properties. The manufacturing process was further improved by introduction of hypoxia during ECM synthesis. The therapeutic potential of improved MIPSOS was then evaluated in a full thickness skin wound model in diabetic mice. Comparative proteomic analysis identified IGFBP7 as one of the mostly enriched components in MIPSOS over cECM. We thus conducted knock-down experiments to produce MIPSOS without IGFBP7 to investigate the role of IGFBP7 in MIPSOS’ pro-angiogenic potential.

*Results: MIPSOS could be successfully manufactured under hypoxic conditions in chemically defined medium. Although overall amounts of deposited ECM remained the same, MIPSOS synthesized under hypoxic conditions in chemically defined medium exceeded the pro-angiogenic of standard MIPSOS, as evident by its ability to promote endothelial proliferation and sprouting. Consequently, improved MIPSOS formulation accelerated skin wound closure in diabetic mice and exceeded the therapeutic potential of standard MIPSOS and even the clinical gold standard GraftJacket^TM. To investigate the role of IGFBP7 in MIPSOS’ pro-angiogenic bioactivity, IGFBP7 was successfully knocked down for the whole duration of ECM synthesis. Removal of IGFBP7 had no effect on MIPSOS ability to promote endothelial proliferation, but reduced its ability to promote endothelial sprouting.

*Conclusion/Significance: MIPSOS is a suitable off-the-shelf material that could become available to a large population of patients. Since MIPSOS contains an enriched, complex portfolio of pro-reparative factors, while being of human origin and cell-free, its utilization can address major limitations of soluble factor-based, tissue-derived ECM-based, as well as cell-based therapies. IGFBP7 is partially responsible for the pro-angiogenic potential of MIPSOS and can serve as a functional quality control biomarker during future clinical translation.

151 - Fundamental Material Differences Between Hydrogel And Infusible Versions Of Decellularized Ecm Biomaterials

A. Chen¹, M. Nguyen², K. L. Christman²

¹ Materials Science and Engineering, University of California San Diego, La Jolla, CA,

² Bioengineering, University of California San Diego, La Jolla, CA

*Purpose/Objectives: Naturally derived, decellularized extracellular matrix-based biomaterials have been increasingly investigated for tissue engineering due to their ability to assemble into a hydrogel in situ, and their intrinsic regenerative properties. Previously, we developed a decellularized, porcine myocardial extracellular matrix hydrogel (ECM hydrogel) and an intravascularly infusible extracellular matrix-based formulation (iECM). While they differ in processing and delivery modality, both materials are derived from the same tissue source and have shown efficacy in small and large animal models of acute myocardial infarction; however, there has been limited characterization from a fundamental materials perspective. The goal of this study was to evaluate and compare the fundamental and mechanistic materials properties of ECM hydrogels versus infusible ECM.

*Methodology: The ECM hydrogel and iECM were manufactured as follows. Porcine left ventricle myocardium was chopped, decellularized, milled into a powder, and digested with pepsin for 48 hours to make the liquid form of the ECM hydrogel. For the iECM, this liquid was subjected to high-speed centrifugation and filtration to remove the submicron particulate components. . Fourier transform infrared spectroscopy (FTIR) was used to analyze the overall organic content and thermal gravimetric analysis (TGA) was used to evaluate combustion temperature. Transmission electron microscopy (TEM) and second harmonics generation (SHG) were used to evaluate the fibrillar content and mass spectrometry to identify protein content.

*Results: The largest FTIR peaks for both materials were Amide A, B, I, II and III, and the TGA revealed predominant combustion around 300°C, all pointing to a majority protein content. Via TEM, we saw several fibers within the ECM hydrogel with an average of 43.49±6.58 nm diameter (Fig. 1a) and sparse nanofibrillar structure within the iECM (Fig. 1b). This showed that a dominant mass of the iECM consists of low molecular peptides not resolved at lower magnifications. After incubation, via TEM, we found a significant increase in type I collagen fiber diameters for the ECM hydrogel to over 140 nm and the appearance of type I collagen fibers within the iECM similar to pre-gel ECM hydrogel fibers. Via SHG, we investigated type I collagen spatial abundance post-incubation and revealed a dense network of type I collagen in the ECM hydrogel (Fig. 1c.) while the iECM reveals sparse, thinner fibers (Fig. 1d). These results were reflected in the global mass spectrometry where the iECM had an average Col1α1 and Col1α2 total intensity 2 orders of magnitude lower than the ECM hydrogel. Moreover, the global proteomics for the iECM also identified a variety of other components including, but not limited to, 6 laminin subunits, nidogens, and other collagens (type II, III, IV, V, VI, XXII).

*Conclusion/Significance: These data revealed that type I collagen lateral self-assembly drove hydrogel formation and a major fraction of the iECM was low molecular weight peptides with compositional differences compared to the ECM hydrogel. The methods employed provide the groundwork for evaluating these materials from a fundamental perspective and the results highlight fundamental differences between decellularized materials produced from the same source, showing their flexibility in tissue engineering applications.

152 - Cornea-specific Human Adipose Stem Cell -derived Extracellular Matrix For Corneal Stroma Tissue Engineering

P. Puistola¹, A. Kethiri¹, A. Nurminen¹, J. Turkki¹, K. Hopia¹, S. Miettinen^1,2, A. Mörö¹, H. Skottman¹

¹ Tampere University, Tampere, Finland,

² Tampere University Hospital, Tampere, Finland

*Purpose/Objectives: Biologically relevant biomaterials are needed for replicating complex compositions of native tissues, and tissue-specific extracellular matrices (ECMs) have high potential in developing these materials. However, most tissue-specific biomaterials are based on human or animal tissue derived ECMs. These sources have several limitations, including availability, risk of disease transmission, potentially high immunological reaction, batch-to-batch variation, and low customizability. Cell-derived ECMs hold tremendous potential as an alternative for engineering tissue-specific biomaterials, such as bioinks for bioprinting. 3D bioprinting technology addresses the vast global need for donor tissues and organs, including human donor corneas. Donor corneas are needed for treating corneal blindness which is often a result from damage in corneal stromal microstructure. Human adipose tissue is abundant and easy to access, and adipose-derived stem cells (hASCs) have several advantages for tissue engineering due to their accessibility, easy expansion in vitro and immunomodulatory properties. Furthermore, hASCs are studied in clinical trials for treating corneal stromal pathologies, and they have been shown to differentiate towards corneal stromal keratocytes (hASC-CSKs).

*Methodology: Here, we developed a novel approach of engineering corneal stroma -specific ECM without the need for donor corneas and applied it to bioprinting of corneal stroma structures. ECM was derived from hASC-CSKs during their differentiation and decellularized. Thereafter, ECM was utilized as a component for corneal stroma -specific hyaluronic acid -based bioink. The decellularization efficacy was studied by DNA concentration measurements. The stroma-specificity of ECM was evaluated with Western Blot and immunofluorescence staining. The ECM bioink was characterized with viscosity measurements, printability and shape fidelity evaluation, and studying its mechanical properties with a rheometer. Immunogenicity was assessed based on the proliferation of human peripheral blood mononuclear cells on the bioink with and without decellularized ECM. Finally, the cytocompatibility of the ECM bioink was evaluated by bioprinting hASC-CSKs to produce corneal stroma structures.

*Results: Efficient decellularization was seen with significant decrease in DNA concentration after the decellularization process. The presence of three proteins expressed in native human corneal stroma, collagen type I, lumican and keratocan, were detected in Western Blot. The immunofluorescence staining of decellularized ECM showed high amount of lumican and fiber-like appearance of collagen types I and V. Keratocan was present in lower amounts along collagen I fibers. The ECM bioink demonstrated shear-thinning properties, great printability and shape fidelity, and its immunogenicity was at similar level as clinically used fibrin sealant. The hASC-CSK viability in the ECM bioink was high, and the cell morphology after 10 days post-printing was stellate, resembling native hCSKs. Furthermore, bioprinted hASC-CSKs expressed connexin 43, indicating cellular interactions.

*Conclusion/Significance: The novel approach developed in this study eliminates the need for donor corneas, making treatments for corneal blindness more accessible and efficient in the future. To the best of our knowledge, hASC-CSK derived decellularized ECM containing corneal stroma -specific proteins has not been previously applied in 3D bioprinting corneal stroma structures. This research contributes to addressing the current bottleneck of developing cost-effective methods for cell-based therapies and elevates the state of bioprinting cornea-specific bioinks to a higher level.

153 - Bioactive Matrix Bound Nanovesicles From Decellularized Bovine Pericardium For Regenerative Medicine Applications

D. Di Francesco¹, C. Di Varsavia², S. Casarella², D. Mantovani¹, F. Boccafoschi²

¹ Universitè Laval, Québec, QC, Canada,

² Università del Piemonte Orientale, Novara, Italy

*Purpose/Objectives: Matrix bound nanovesicles (MBVs) are a recently discovered subtype of extracellular vesicles (EVs). They are distinguished from other EVs because they are found entrapped in the collagenous extracellular matrix (ECM), are 20 to 400 nm in size, contained in a lipidic membrane and have tissue specific surface markers and content. They have been identified in a number of decellularized ECM (dECM) biomaterials and have proven to confer dECM its signature biological and immunomodulatory potential. This is due to the highly bioactive content made up from peptides, nucleic acids and lipids. These characteristics allowed to better understand the mechanisms underlying the biological potential of dECM materials, as MBVs are retained in dECM matrix, and to explore a new type of biomaterial which could represent a minimally invasive injectable regenerative therapy. To date, MBVs extracted from bovine pericardium dECM (dBP) have not yet been reported. Therefore, the aim of this research was to characterize the physico-chemical properties, and the biological role of MBVs isolated from bovine pericardium dECM.

*Methodology: The isolation protocol of MBV from dBP was optimized. To characterize concentration, size and morphology of MBVs, nanoparticle tracking analysis and transmission electron microscopy (TEM) were performed. The composition was assessed with silver staining through SDS-PAGE gel, cytokine array assay and mass spectroscopy. The biological potential was evaluated by viability assay and immunofluorescence staining with different cell lines, while the immunomodulatory potential was characterized by evaluating macrophage polarization with human macrophages. Finally, the angiogenic potential was assessed using an in vivo hind limb ischemia model.

*Results: MBVs were successfully isolated from dBP at a mean concentration of 2*10⁸ ± 0,22*10⁸ MBVs/mL and a mode size of 142 ± 1,45 nm. TEM imaging confirmed the presence and size of MBVs, as irregular shaped MBVs were spotted, characterized by an external lipidic membrane. Protein content characterization also shows a rich bioactive content of the MBVs, consistent throughout MBVs isolated from different dBP batches. Cytokine array evidenced the presence of a variety of immunomodulatory cytokines contained in MBVs. Cell viability evaluation showed that MBVs at different concentrations are not cytotoxic and significantly enhance cell viability. MBVs showed to induce a regenerative phenotype in human macrophages and to recapitulate the angiogenic and regenerative potential of dBP.

*Conclusion/Significance: Herein, the presence of bioactive MBVs retained in dBP was demonstrated. MBVs showed a tissue-specific, rich protein content, including a set of immunomodulatory cytokines, which confers biological potential to dBP. This translates in the ability of MBVs from dBP to enhance cell viability, induce an anti-inflammatory and regenerative phenotype in immune cells and induce angiogenesis. Therefore, MBVs from dBP show high potential for regenerative medicine applications.

154 - Convergence Of Extracellular Matrix (ECM)-based Biomaterials And Biomimetic Culture Methods To Bioengineer Humanized In Vivo Bone Models

A. Bessot^1,2,3, J. Gunter^1,4, D. Waugh^1,5, J. McGovern^1,2,3, D. W. Hutmacher^2,3,6, N. Bock^1,2,3

¹ School of Biomedical Sciences, Faculty of Health, Queensland University of Technology / Translational Research Institute, Woolloongabba, AUSTRALIA,

² Max Planck Queensland Centre, Brisbane, AUSTRALIA,

³ Centre for Biomedical Technologies, Brisbane, AUSTRALIA,

⁴ Australian Prostate Cancer Research Centre - Queensland (APCRC-Q), Brisbane, AUSTRALIA,

⁵ Centre for Cancer Biology, University of South Australia, Adelaide, AUSTRALIA,

⁶ School of Mechanical Medical and Process Engineering, Engineering Faculty, Queensland University of Technology / Translational Research Institute, Brisbane, AUSTRALIA

*Purpose/Objectives: Modelling the human bone microenvironment is facilitated by a variety of biomaterials, with advantages and limitations depending on applications. For the past decades, polycaprolactone (PCL)-based melt electrowritten (MEW) scaffolds, coated with calcium phosphate (CaP) were used to engineer human osteoblast-derived mineralized tissues for the study of bone tumors, both in vitro and in vivo. In vivo, the shortcomings of traditional rodent models lacking a human bone microenvironment was addressed by ectopic humanized bone niches formed using the MEW-PCL-derived mineralized microtissues, which served to study bone tumor mechanisms and treatments. Although successful, limitations included the long-term in vitro culture prior to implantation (6-12 weeks in osteogenic medium (OM)) and a limited humanization of the bone graft. In this study, we hypothesized that semi-synthetic gelatin methacrylyol (GelMA)-based hydrogels combined with a CaP-enriched in vitro culture would address these issues due to closer ECM recapitulation than CaP-coated PCL scaffolds.

*Methodology: CaP- PCL scaffolds and GelMA hydrogels were loaded with primary human osteoprogenitors and cultured in vitro for up to four weeks using different biomimetic cultures; 1) osteogenic medium (OM) control; 2) OM after three initial days using a mineralization medium, previously validated by our group, (OM+) and 3) growth medium supplemented with the initial 3-day mineralization medium treatment (named GM+). After one or four weeks in vitro, microtissues cultured in OM and OM+ were implanted subcutaneously into immunodeficient mice and kept for 11 weeks to form humanized bone organs.

*Results: The in vitro results showed higher osteogenic differentiation and calcium uptake in microtissues cultured in OM+. Mineralization was enhanced by the mineralized medium, with higher mineralization in GelMA hydrogels compared to CaP-PCL scaffolds after four weeks of culture. In vivo, microcomputed tomography (µCT) demonstrated earlier mineralization and higher trabecular network in OM+ implants. Higher vascularization and hematopoietic bone marrow infiltration was observed in GelMA constructs cultured after OM+ vs OM. These data indicate that mineralizing the constructs before implantation could enhance mature bone formation in vivo. Compared to CaP-PCL-derived explants, GelMA constructs showed slower bone formation but higher bone volume fraction, superior cortical shell and the presence of cartilaginous-like tissue, correlating with endochondral ossification, which is more physiologically relevant to bone formation with definitive hematopoiesis. Human cells were maintained in vivo and contributed to bone formation, especially in GelMA, while in CaP-PCL scaffolds, they were concentrated in fibroblastic areas, which could indicate that embedding human cells in a soft matrix could lead to a slower but more effective ossification compared to seeding them on fibers. Further analysis via histology, SEM, EDS, qBEI and nanoindentation informed on human osteocyte network, collagen fibers and mineral density and quality.

*Conclusion/Significance: Our current findings demonstrated that faster and superior humanized bone tissue formation could be achieved in rodents using GelMA hydrogels combined with an initial mineralization medium treatment, leading the path towards advanced preclinical humanized models.

155 - Comparative Analysis Of Extracellular Matrix Architecture And Matrisome In Decellularized Brain Scaffolds And Matrigel®

U. Rende^1,2, A. Igrunkova^1,2,3, M. Vaezzadeh¹, B. Heng¹, A. Guller^1,2

¹ Macquarie University, Sydney, Australia,

² University of New South Wales, Sydney, Australia,

³ Sechenov University, Moscow, Russian Federation

*Purpose/Objectives: Matrigel® is a popular gel-like scaffold obtained via proprietary processing of a highly vascularised malignant mouse tumour (angiosarcoma). It is commonly used in the 3D cell culture, including in vitro models that employ various types of brain cells. However, it is not clear to what extent the extracellular matrix (ECM) of Matrigel® is representative of and relevant to the ECM of the brain. Decellularized brain scaffolds (DBS) represent an alternative platform for 3D in vitro culture of normal and tumour cerebral cells. A better understanding of the scaffolds’ properties is essential for deciphering the variability of biological responses observed in 3D cell culture models.This study elucidates the architecture and protein composition of brain extracellular matrix (ECM) using decellularized lamb brain scaffolds (DBS), comparing it with Matrigel®, to enhance understanding of 3D cell culture environments for brain tissue and tumour models, brain tissue engineering and regenerative medicine.

*Methodology: Lambs’ brains were sourced from a local butchery and decellularized using an adapted protocol (Guller, SPIE, 2015). The brain hemispheres were dissected, split into smaller fragments, decellularized and then analysed using histology and scanning electron microscopy (SEM). Image analysis was performed using ImageJ (Fiji), and statistical analysis was carried out using GraphPad Prism software. For the comparative study, the 50 µL plugs of high protein concentration Matrigel® were prepared and analysed similarly. Proteomic characterization of the matrisome of DBS was conducted via LC-MS/MS and analysed using UniProt and Matrisome databases and MaxQuant and Matrisome Annotator software. The Matrigel® proteins of high confidence identified in the published study (Wang et al., 2022) were searched in the same databases to identify ECM proteins. Then, the matritecture and matrisome properties of DBS and Matrigel® were compared quantitatively.

*Results: Matrigel® had a homogenous organisation with thin and short randomly oriented fibres bordering the roundish pores of regular size. The DBS ECM revealed two compartments with distinct patterns with predominantly membrane-like and fibrous structures, respectively. DBS displayed hierarchically arranged filaments of various thicknesses, contrasting the homogenous organisation of Matrigel®. In particular, thick collagen bundles intersected at the angles of 90° and 45° were found only in the DBS. While both materials showed similar overall porosity, their pore sizes differed significantly. Proteomic analysis uncovered a significantly richer matrisome in DBS (107 ECM proteins) compared to Matrigel® (24 ECM proteins), with only 8 shared components. DBS contained 33 collagens, 26 ECM glycoproteins, 19 secreted factors, 15 ECM regulators, 11 ECM-affiliated proteins, and 3 proteoglycans.

*Conclusion/Significance: This study underscores the complexity and region-specific architecture and richness of the brain ECM, achievable through decellularization. The stark contrast in structural and matrisome complexity between natural brain scaffolds and Matrigel® suggests for potential significant implications for their use in 3D cell cultures and tissue engineering. These findings pave the way for future research into the functional roles of these structures and the biological significance of the observed differences, potentially influencing the design of more effective biomaterials for brain tissue engineering, tumour modelling and regenerative medicine.

156 - Urinary Bladder Matrix Hydrogel Improves Anastomotic Healing In A Standardized Rat Model Of Rectal Resection

V. P. Anto, C. Patterson, S. Johnson, D. Medich, H. George, S. Badylak

University of Pittsburgh, Pittsburgh, PA

*Purpose/Objectives: Anastomotic leaks are a dreaded source of morbidity following surgical resections for rectal cancer. Despite advancements in surgical techniques, the rates of rectal anastomotic leaks remain elevated. Considering the prevalence of anastomotic leaks, temporary diverting ostomies are created to reduce leak morbidity. Unfortunately, diverting ostomies have their own complications. A substantial reduction in anastomotic leaks could negate the need for routine prophylactic fecal diversion. Surgical devices derived from extracellular matrix (ECM) have shown promise in enhancing healing in gastrointestinal applications. Urinary bladder matrix (UBM) represents a versatile form of acellular ECM that promotes constructive remodeling. However, its potential efficacy in colorectal anastomoses has not been evaluated. The absence of reliable small animal models for colorectal anastomotic leaks has been a hindrance to product development. The primary objective of this study was to establish a reproducible rat model of distal colorectal resection to assess the effectiveness of UBM ECM adjuncts in reducing anastomotic leaks.

*Methodology: Female Sprague-Dawley rats underwent standardized distal colonic resections and sutured anastomoses below the peritoneal reflection. Rats were randomly assigned to control, UBM extraluminal wraps, or UBM hydrogel application after anastomotic creation. In the initial experiments, necropsy was performed on post-operative day seven with a multi-modal assessment of anastomotic leak. The perianastomotic tissue underwent a comprehensive histologic assessment to evaluate the quality of healing. Additional experiments compared intraluminal UBM hydrogel to inert synthetic copolymer gel. Further experiments compared control animals to intraluminal UBM hydrogel treated animals over a series of timepoints (days 1, 3, 5, 14, 28, 56).

*Results: Anastomotic leaks reliably occurred in 46% of control animals with a significantly lower leak rate of 8% in UBM hydrogel treated rats on post-operative day 7 (Table 1). UBM extraluminal wraps did not result in a reduction of anastomotic leaks. Fewer perianastomotic adhesions were seen in animals receiving hydrogel treatment. Histologic evaluation demonstrated significant improvements in composite healing scores for anastomoses treated with the hydrogel. Improved anastomotic healing was demonstrated throughout the series of timepoints in anastomoses that received intraluminal hydrogel applications.

*Conclusion/Significance: This small animal model of a low rectal anastomosis presents a dependable platform for surgical device testing. UBM hydrogel exhibits promising potential as an adjunct to decrease rectal anastomotic leak rates through both mechanical and biochemical effects. Multi-modal evaluation of anastomotic healing over time provides insight into mechanisms involved in anastomotic leak and pathways to reduce leak rates.

157 - Towards An Inhalable Tissue-specific Extracellular Matrix-based Substrate For Targeted Laryngeal Tracheal Regeneration

R. Colmon¹, S. White¹, G. Dion², C. Mora-Navarro³, K. Huang¹, D. Freytes¹

¹ North Carolina State University, Raleigh, NC,

² University of Cincinnati, Cincinnati, OH,

³ University of Puerto Rico, Mayaguez, Puerto Rico

*Purpose/Objectives: Annually, an estimated 28 million Americans suffer from dysphonia-related pathologies, including vocal fold (VF) cysts, paralysis, and scarring or fibrosis. These VF injuries arise from various causes, such as VF scarring and laryngeal tracheal stenosis, often triggered by procedures like endotracheal intubation. Laryngeal surgery, which is performed based on lesion severity, frequently leads to additional VF scarring, exacerbating the patient's condition. Current treatments for VF damage are minimally effective, with limited regenerative options available. To achieve glottic closure, therapists often augment the VF using various biomaterials, but these methods yield limited success in the long term. Our laboratory has produced a porcine-derived vocal fold lamina propria extracellular matrix hydrogel (VFLP-ECMh) that retains native proteins and peptides that show anti-fibrotic properties in vitro. Decellularized Injectable ECM (dECM) based hydrogels are advantageous over other natural and synthetic hydrogels for the contribution of their tissue-specific ECM signaling in regenerative efficacy, anti-fibrotic properties, and low immunogenicity via in vivo settings. Although this is encouraging, implantation of these injectable materials involves recurring invasive procedures (endoscopic surgery, tracking of the implant, and even multiple doses). This study aimed to develop a minimally invasive alternate VFLP-ECM substrate that can be nebulized at relative therapeutic doses while retaining its desirable bioactive properties in vivo.

*Methodology: Nebulizable ECM (NebECM) was developed by first manufacturing and subsequent characterization of a solubilized form of the VFLP-ECM, which could be delivered directly to the upper respiratory tract (URT) via a commercially available ultrasonic mesh nebulizer. UV Spectroscopy was used to determine the concentration of solubilized VFLP-ECM formulation before and after nebulization. Dynamic Light Scattering techniques were used to determine ECM particle size. To assess the cytocompatibility of the NebECM, we cultured Human Vocal Fibroblasts (HVOX) with Neb-ECM. We evaluated changes in cell metabolic activity and viability using MTT, Pico Green, and LIVE/DEAD assays. Additionally, we investigated the potential activation of HVOX into myofibroblasts via gene expression in the presence of TGF-β1 for 24 hours. In vivo safety studies are being performed to monitor progressive lung compliance and volume changes following Neb-ECM inhalation, employing the FlexiVent system.

*Results: The final NebECM substrate before nebulization was shown to contain particle sizes around 1 micrometer in diameter. Cytotoxicity studies showed that the NebECM does not affect cell metabolism or contribute to apoptosis. Lung function measurements are still underway, and pressure-volume and flow-volume loops will be used to assess fluctuations in lung compliance and overall pulmonary function in rats treated with NebECM vs a control group of rats with induced pulmonary fibrosis.

*Conclusion/Significance: In vitro assays highlight desirable biological attributes of NebECM, namely, modulation of fibrosis-associated pathways in VF fibroblasts. Changes in rat pulmonary function will serve as determinants of the safety of NebECM as a biomaterial.

158 - Microfluidic Incubation Of Patient Tumor, Lymph Node, And Peripheral Blood Cells Produces Tumor Reactive Cytotoxic Lymphocytes

D. C. Hutchins, C. Schaaf, T. Liu, N. Edenhoffer, M. Kooshki, A. R. Hall, L. D. Miller, P. Triozzi, S. Soker, K. Votanopoulos

Wake Forest School of Medicine, Winston-Salem, NC

*Purpose/Objectives: Isolation, expansion, and re-administration of autologous tumor-infiltrating lymphocytes (TILs) is an increasingly promising therapy for patients with solid tumors. However, despite their potential FDA approval, not all patients can produce sufficient, active TILs for therapeutic use. Oftentimes, TILs are difficult to proliferate and are already exhausted upon collection, leading to failure in the reduction of tumor burden. To improve the therapeutic use of TILS, patients need a source of abundant and reactive T cells as well as an easy method to determine if TIL therapy will reduce tumor burden. In this study, we endeavored to fulfill that need by training peripheral blood cells to respond to tumor in an ex vivo microfluidic device.

*Methodology: Our device houses central chambers containing organoids of human patient tumor (metastatic appendiceal and peritoneal mesothelioma) and antigen-presenting cells (APCs) grown in a biomimetic hydrogel mix of photo crosslinked thiol-modified hyaluronic acid, gelatin, and polyethylene glycol. Autologous peripheral blood mononuclear cells were circulated through the chambers for 7 days. These blood cells were termed Organoid Infiltrating lymphocytes (OILs). We devised this manner of design and duration of incubation to emulate in vivo lymphocyte training. Multiple controls were utilized to determine how each step of the process affected the cells. In addition to OILs, there were tumor-only trained blood cells, blood cells incubated in the device with only the hydrogel mixture, and finally blood cells expanded in a standard cell culture flask. We have conducted flow cytometry analysis as well proteomics analysis to identify phenotype and polyfunctionality of the OILs compared to controls, in addition to cytotoxic function upon reintroduction to tumor cells.

*Results: Overall, the reintroduction of OILs to tumor cells induced significantly higher interferon gamma and granzyme A expression. Furthermore, cytotoxicity analysis of patient-specific tumor cells in the presence of microfluidics-trained peripheral cells showed that for three patients, OILs induced significantly higher tumor cell death compared to controls.

*Conclusion/Significance: These data suggest that this method can produce highly effective and personalized immunotherapy for patients with solid tumors. Further validation with in vivo animal models are needed to confirm therapeutic use.

159 - Using A Blood Brain Barrier (BBB)-on-a Chip To Study How Different Subpopulations Of Glioblastoma Tumor Cells Influence BBB Permeability And Drug Delivery

T. DePalma, E. Stagner, H. Sivakumar, K. Hughes, K. Jones, A. Skardal

The Ohio State University, Columbus, OH

*Purpose/Objectives: Glioblastoma (GBM) is the most common malignant primary brain tumor, and it is considered incurable. This is likely due to the extreme heterogeneity of GBM tumors and a unique tumor microenvironment (TME) that drives invasion, adaptive drug resistance, and recurrence after treatment. Additionally, the blood-brain barrier (BBB) prevents most therapeutics from reaching the tumor. The failure of many promising drug candidates may indicate that current preclinical models do not replicate tumor biology and human BBB function. In these studies, we utilize a novel human BBB-on-a-chip model and heterogeneous patient-derived GBM tumor cells to study how BBB permeability and drug delivery vary across patients.

*Methodology: The BBB-on-a-chip model was produced using a custom 3D-printed mold. Polydimethylsiloxane casts are bonded to glass coverslips. A hydrogel containing primary human astrocytes was injected around a needle to pattern a cylindrical lumen (diameter=300μm). The needle was removed and primary human brain pericytes were seeded into the lumen, followed by human iPSC brain microvascular endothelial-like cells (iBMEC) (Figure A). The hydrogel used was composed of thiolated hyaluronic acid (HA) and gelatin methacrylate (GelMA). Our lab has previously validated that HA-GelMA hydrogels support quiescent astrocyte phenotype and morphology (Figure D). Throughout these studies, confocal microscopy was used to assess the presence of tight junctions, astrocytic endfeet surrounding the vessel, and iBMEC-pericyte and astrocyte-pericyte interactions. BBB permeability was assessed by measuring the diffusion of small molecules across the barrier. Patient GBM tumor biospecimens were obtained from the clinic in accordance with approved protocols.

*Results: We first validated that our BBB-on-a-chip mimics in vivo function. iBMECs formed robust tight junctions (Figure B) and showed low permeability (<2x10^-8 cm/s) to a variety of small molecules including fluorescein (Figure C). After validating the BBB model, RNAseq was conducted on human GBM tumors, and the samples were classified based on the expression of genes related to stemness, invasion, and ECM degradation, all characteristics of more aggressive and drug-resistant tumors. We used these results to select 4 patient cell populations that represent distinct subpopulations of GBM. To study how the BBB changes in the presence of tumor cells, GBM cells were injected into the system behind the BBB. The invasion distance of each population was measured, and their interactions with astrocytes and the vessel structure were visualized using confocal microscopy (Figure E). Permeability and tight junction integrity were measured at 1, 3, and 7 days after injection. The bioavailability of 2 drugs, temozolomide (TMZ) and paclitaxel (PTX), was measured for all samples. Our results indicate that more invasive GBM populations induce greater changes in BBB permeability, translating to increased permeability to various candidate drugs.

*Conclusion/Significance: The BBB-on-a-chip model presented utilizes a defined brain mimetic hydrogel and accurately replicates the function of the human BBB. We have shown that different populations of GBM cause distinct changes in BBB function. Using human in vitro models to study GBM could inform the design of new treatment and drug delivery vehicles helping bridge the gap between current preclinical studies and human trials.

160 - 3d Bioprinting Ultrasound-responsive Tissue Constructs For Remote-controlled Oncogene Patterning In 3d Tumor Models

M. K. Lowrey, H. Day, A. Vasquez, A. Tahayeri, K. T. Huynh, L. Bertassoni, C. E. Schutt

Oregon Health and Science University, Portland, OR

*Purpose/Objectives: 3D bioprinting is a valuable tool for in-vitro cancer modeling, as it can recapitulate tissue architectures and biophysical parameters of the tumor microenvironment. Genetic manipulation of cells within bioprints is a key technique to model how changes in oncogene expression affect cancer progression in the 3D microenvironment. However, thick 3D scaffolds hinder diffusion of traditional transfection vectors, limiting spatial and temporal control of genetic manipulation. Focused ultrasound combined with ultrasound-responsive gene delivery microparticles is a promising trigger for remote-controlled cellular manipulation in engineered tissue constructs, due to its multi-centimeter penetration depth and ability to be tightly focused to small volumes within tissues. To address the challenge of spatiotemporally-controllable genetic manipulation in bioprinted tumor models, we developed a 3D bioprinting platform that enables remote-controlled gene delivery by printing hydrogel bioinks containing cells and ultrasound-responsive microparticles and subsequently activating selected microparticles using focused ultrasound.

*Methodology: An extrusion 3D bioprinter (CellInk BioX6) was loaded with an alginate bioink precursor pre-mixed with either HEK293T cells or MCF10A breast epithelial spheroids and lipid-based ultrasound-responsive microparticles. The microparticles were coupled with either green fluorescent protein (GFP) plasmid or a plasmid coding for HER2 oncogenic protein fused to a fluorescent reporter. Constructs were printed into a support bath containing calcium chloride to promote alginate crosslinking. Post-printing, constructs were exposed to focused ultrasound, then imaged via brightfield and fluorescence microscopy to evaluate particle integrity and cell transfection 48hr post-ultrasound exposure. Calcein-AM and ethidium homodimer staining was used to assess cell viability.

*Results: Successful bioprinting of alginate hydrogel filaments containing cells and lipid-based ultrasound-responsive microparticles was confirmed by microscopy. Following focused ultrasound exposure, bioprinted microparticles ruptured only within the ultrasound focal zone (Fig. 1A-B, 1D-E), leaving remaining particles intact. At 48hr post-ultrasound, GFP-expressing HEK293T cells were observed only in the ultrasound focal zone (Fig. 1C). In bioprints containing breast epithelial spheroids, cells and cell clusters within spheroids in the ultrasound focal zone were seen to overexpress GFP (Fig. 1F). Constructs containing breast epithelial spheroids and HER2-coupled ultrasound-responsive microparticles (Fig. 1G-H) resulted in ultrasound-activated HER2 overexpression in spheroid cells within the ultrasound focal zone (Fig. 1I). Over 80% viability was observed in both HEK293T cell and breast epithelial spheroid bioprints at 48hr. By varying extrusion needle size, microparticle-containing filaments were reproducibly printed with diameters of 200 to 850 µm with microparticles remaining intact and well-dispersed post-printing. These results show the responsive nature of the particles is maintained in multiple printed feature sizes and demonstrates the ability to control localized gene delivery using focused ultrasound.

*Conclusion/Significance: We developed a 3D bioprinting method that allows for noninvasive focused ultrasound-controlled microparticle activation and localized gene delivery within bioprinted constructs. This provides the first demonstration of cell-seeded 3D-bioprinted structures designed for ultrasound-activatable gene delivery, and enables site-specific oncogene expression in bioprinted tumor constructs. Future work will utilize this platform to study cell behavior and signaling upon induction of site-specific oncogene overexpression, and to model how complex tissue microenvironments affect the progression from a single transformed cell to aggressive cancer in the 3D context.

161 - Microfluidic-Driven Engineering Of Complex 3D Cancer Architectures For Quantitative Modeling

C. F. Guimarães¹, L. Gasperini¹, R. R. Rilva¹, U. Demirci², R. L. Reis¹

¹ 3B’s Research Group, University of Minho, Guimarães, Portugal,

² Canary Center for Cancer Early Detection, Stanford University, Palo Alto, CA

*Purpose/Objectives: The functionality of biological tissues, healthy or diseased, is intricately linked to their complex 3D shapes and mechanical properties. Achieving the recapitulation of such characteristics by finely tuning cell/material interactions is crucial for engineering in vitro tissues tailored for biomedical applications. High-throughput and reproducible data generation are additional requisites for enhancing the translational potential of these engineered tissues for in vitro testing. In this study, we investigate the utilization of microfluidic flow to regulate hydrogel precursors and fabricate diverse biological architectures within multi-material, multi-cellular hydrogel microfibers that emulate functional 3D cancer environments. Computational modeling is employed to predict cell/material organization, while optical-driven tools facilitate the efficient digitization of ongoing biological events.

*Methodology: A natural biomaterial toolbox comprising Gellan Gum, Alginate, GelMA, GelTrex, and Collagen was employed to fabricate hydrogel microarchitectures with varying shapes and compositions. Precursor viscosity and flow characteristics were modeled and optimized to modify the 3D arrangement of compartments within hydrogel fibers of multiple configurations (Fig. 1a, b). Wet-spinning with Calcium Chloride was utilized to crosslink the hydrogel precursors into distinct micro-fiber architectures which can approach the intricate 3D architectures of all main cancer types (Fig. 1c-f).

*Results: Diverse hydrogel fiber architectures were successfully produced by manipulating hydrogel precursor viscosity and channel organization within one similar wet-spinning approach. Ribbon-like shapes were utilized to integrate two cellular compartments (cancer and stroma), separated by a thin basement membrane, thus recapitulating the early stages of pre-metastatic layered-like cancer cell invasion, in vitro (Fig. 1c). Core-shell shapes were further fine-tuned to cultivate living cancer fiberoids (fiber-shaped organoids) in continuous fibers approaching solid cancer structures (Fig. 1d). Alternatively, a similar architecture was employed in reverse organization to approach the cellular layer surrounding an acellular lumen present in glandular tumors such as those of breast and prostate (Fig. 1e). Finally, the multi-layered hydrogel coaxial structures were adapted and interfaced with light guidance for quantification of cancer tissue progression and drug susceptibility (Fig. 1f). This leveraged light-cell interactions occurring throughout living optical fibers, enabling unprecedentedly fast, real-time quantification of ongoing events such as cancer 3D growth and drug susceptibility.

*Conclusion/Significance: The flow-driven assembly of hydrogel/cell fiber constructs proves to be an extremely versatile technology, highly adaptable for the high-throughput fabrication of living 3D cancer architectures in varied configurations. Moreover, when combined with optical tools, this approach allows for the direct quantification of biological events with exceptional efficiency and throughput.

162 - Development Of A Novel Goat Model For Osteochondral Defects In The TMJ Condyle

M. Socorro, X. Dong, S. Trbojevic, J. Stover, J. Taboas, A. Almarza

University of Pittsburgh, Pittsburgh, PA

*Purpose/Objectives: Focal osteochondral defects (OCD) are areas of articular cartilage damage and injury of the underlying subchondral bone. Particularly, the OCD of the temporomandibular joint (TMJ) osteoarthritis (OA) constitute a challenging problem to oral surgeons due to a poor ability to self-heal, requiring full joint replacement since there are no other available therapeutics. No suitable large animal model exists for development and preclinical testing of regenerative therapies. Our team has published utility of the goat as a model for treatment of TMJ derangements because the anatomy and biomechanics better models those of humans when compared to dogs and pigs. Another advantage of the goat is that the joint space is easily accessible during surgery. Therefore, the aim of this study was to develop a new animal model of OCD in the TMJ condyle of goats.

*Methodology: We created two types of OCD in the TMJ condyle of 3-year-old female goats, a tunnel defect and a superior defect (Figure 1). In the first group, a hole was drilled using a 3.2 mm drill bit, from the inferior-posterior-lateral aspect of the condyle towards the superior-anterior-medial aspect, until coming out through the articular surface (tunnel defect) (n=6). Because the temporomandibular joint disc articulates with the condylar surface, a metal plate was inserted between the two to avoid any damage to the disc. In the second group, a 5 mm deep and 2.4 mm wide hole was drilled from the superior-posterior-lateral aspect to the inferior -anterior-lateral aspect (superior defect) (n=4). The superior defect decreased the time required to analyze the functional regeneration of the goat condyle. In both models, the TMJ disc was liberated in the lateral aspect by cutting lateral attachments in the anterior and posterior of the disc. This allowed for half the disc to be folded into itself to access the condylar surface. Both defect types were left empty. Blood in the surrounding areas was allowed to clot before the underlying muscular tissue was sutured. The condyles were analyzed by live computed tomography (CT) at day 0, 30 and 60 (post-surgery). After 60 days of healing, the condyles were processed for paraffin embedding. Serial 8 μm sagittal sections were collected and deparaffinized. Histomorphometry of the tissues (condyle cartilage, and bone) was performed using H&E, Toluidine blue, Safranin O and Masson-Goldner’s trichome staining.

*Results: Our analyses showed that both the tunnel and superior defects did not completely heal in 2-months, simulating the clinical presentation of OCD in the TMJ of human patients. All defects were infiltrated with fibrous tissue and fat cells, while woven bone originated from the borders of the defect. Furthermore, the articular fibrocartilage surrounding both defect types showed evidence of degeneration, namely loss of the zonal structure (fibrous, transition, and cartilaginous layers).

*Conclusion/Significance: This study introduces an innovative goat model to study the regeneration of cartilage/OCD of the TMJ, which will allow to develop future therapies on the treatment of patients that suffer from significant pathologies of the mandibular condyle articular cartilage which have no regenerative treatment options.

163 - Application Of Magnesium In Bone Regeneration: From Embryonic Development To Energy Metabolism

S. Lin, X. Jiang

Shanghai Ninth People's Hospital, Shanghai, China

*Purpose/Objectives: Functional repair of craniofacial bone defects via regenerative medicine relies on the osteoinductivity of bone substitutes. Several methods, such as surface modification, application of bone morphogenetic proterin-2 (BMP-2), has been proved to effective for improving inductivity of biomaterials. However, the vascularization of bone substitutes and the side effects of BMP-2 still hamper the repair of large bone defects. To address these problems, a magnesium (Mg) -enriched environment is taken to mimic the bone developmental microenvironment for vascularized bone regeneration, and a Mg-based bioenergetic-driven strategy is put forward to promote the efficacy of low-dose BMP-2. We hope that our biomaterial engineering strategy could provide new ideas for craniofacial bone defects treatment.

*Methodology: 1. The correlation between Mg and bone development is evaluated. Then, Western Blot, CRISPR/Cas9, and other methods are taken to investigate the mechanism underlying the inductivity of Mg²⁺ on stem cells and endothelial cells. Moreover, a magnesium-enriched stem cell 3D culture system is fabricated using microfluidic methods. Finally, the rat skull defect model is used to evaluate the effect of such stem cell deliveries on vascularized bone regeneration. 2. The relation among energy metabolism, BMP-2 and Mg²⁺ is evaluated by techniques, such as Seahorse assay, JC-1 staining, flow cytometry. Moreover, a microgel composite hydrogel as the BMP-2/Mg²⁺ codelivery platform is fabricated via microfluidic devices and 3D printing. The regenerative efficiency of the Mg²⁺-based bioenergetic-driven low-dose BMP-2 delivery platform is detected on the rat cranial bone defects model.

*Results: 1. The upregulation of MagT1 is observed during embryonic endochondral ossification and osteogenic differentiation of stem cells, which implies Mg²⁺ is strongly related to bone development. Mechanistically, Mg²⁺ selectively activates the MAPK/ERK pathway to stimulate osteogenic differentiation and neovascularization through MagT1. A magnesium-enriched stem cell culture system is successfully fabricated. The magnesium-enriched microenvironment not only promotes the osteogenic differentiation of stem cells, but also exerts obvious angiogenic effects. After implantation of such cell delivery vehicles to the rat skull defects, obvious vascularized bone regeneration is achieved. 2. We demonstrate that inadequate metabolic stimulation is shown to be responsible for the ineffectiveness of low-dose BMP-2. Mg²⁺, as an “energy propellant”, substantially increases cellular bioenergetic levels to support the osteogenesis via the Akt-glycolysis-Mrs2-mitochondrial axis, and consequently enhanced the osteoinductivity of BMP-2. Then, microgel composite hydrogels are fabricated as low-dose BMP-2/Mg²⁺ codelivery system, which induce rapid and robust bone regeneration in vivo.

*Conclusion/Significance: Collectively, the results of our studies prove that the novel magnesium-enriched strategy significantly stimulate the bone regeneration, providing a promising alternative for the clinical treatment of craniofacial bone defects.

166 - Enhancing Craniofacial Bone Regeneration: Immunomodulation With Sting Inhibition

S. Jalali, Y. Ouhaddi, E. Harvey, N. Makhoul, J. Barralet

McGill University, Montreal, QC, Canada

*Purpose/Objectives: The management of craniofacial bone defects poses a significant clinical challenge, impacting individuals with conditions such as cleft lip and palate, trauma victims, and those dealing with malignancies. Autogenous grafts are the current gold standard for bone reconstruction. Though effective, autogenous grafts have inherent limitations including donor site morbidity, limited availability, infection risk, and resorption issues. These limitations create a need for synthetic bone graft substitutes, like calcium phosphates, which offer a therapeutic alternative but have not consistently shown superior outcomes. Innate immune cells play a crucial role in bone repair, with inflammation serving as the foundation for subsequent repair and healing. The stimulator of interferon genes (STING) is a critical factor in innate immunity and has been linked to angiogenesis. However, STING stimulation can lead to prolonged inflammatory responses, potentially delaying revascularization and the bone healing process. This study aimed to assess whether STING inhibition could enhance bone formation induced by monetite in critical-sized craniofacial defects.

*Methodology: The surgical protocol for animal research was approved by McGill University Ethics Committee (AUP 7660). Eight adult male Wistar rats were randomly assigned to two groups (N=4); Control and STING inhibitor (SI). An 8 mm diameter defect was created in the superior portion of the skull and filled with prepared monetite granules (dicalcium phosphate). Rats in the SI group received injections of H-151 (a STING inhibitor) every 48 hours for a total of 6 doses, directly to the graft site, starting on postoperative day 4. Control rats received the same volume of normal saline. Following a 5-week post-surgery observation period, the rats were euthanized, and their skulls were removed and fixed for micro-CT analysis. After quantification using micro-CT software, samples were decalcified and prepared for histological staining and immunohistochemistry analysis.

*Results: Micro-CT quantification revealed significantly higher amounts of bone formation within the defect (BV/TV) in the group that was injected with H-151 (P<0.05). Notably, vertical bone formation was more noticeable in the H-151 group. Additionally, there was a notable distinction in the volume of the remaining granules between groups (P<0.05), suggesting higher rates of granule absorption in the H-151 group. Hematoxylin and eosin (H&E) staining confirmed the results by indicating more bone formation in the STING group. Tartrate-resistant acid phosphatase (TRAP staining) and immunohistochemistry against a panel of immune cells confirmed an immunomodulatory effect of STING inhibition.

*Conclusion/Significance: This study provides evidence supporting the potential of local delivery of novel drugs combined with bioresorbable monetite granules as a new therapy to augment and regenerate bone defects in patients undergoing oral and maxillofacial surgeries, including those undergoing orthognathic surgeries and management of periodontal bone defects, as well as bone defects caused by malignancies and trauma. These findings offer a proof-of-concept for further exploration of STING inhibition as a strategy for enhancing craniofacial bone regeneration.

167 - Co-based Artificial Enzyme For ROS Elimination And Periodontitis Treatment

Y. Sun

Sichuan University, Chengdu, China

*Purpose/Objectives: In periodontitis, the accumulation of oxidative damage, especially DNA damage, often leads to stem cell senescence and impedes the repair of tissue defects. Current artificial enzymes have high metal content, low atomic utilization, and poor catalytic performance, making it difficult to achieve good therapeutic effects. To improve the ROS scavenging efficiency and biocompatibility. we designed a cobalt based artificial enzyme.

*Methodology: In this work, we simulate the structure of natural catalase, and constructs ruthenium coordination artificial enzymes (Ru-NC) with three atomic scale catalytic centers (single atom, cluster, and nanoparticles), and studies the relationship between material structure and therapeutic effects. Commercial human bone marrow derived mesenchymal stem cells (hMSCs) are employed in this study. The protective effects of cell survival were studied by reactive oxygen species (ROS) staining, cell viability staining, and immunofluorescence staining. Rat model of periodontitis was established. The in vivo antioxidant effects were assessed by the in vivo imaging system, MicroCT, and blood cell analyzer. The mechanism of material cell interaction is explorered through transcriptome sequencing and functional enrichment of Reactome.

*Results: Artificial enzymes with ruthenium single atom (Ru@SA-NC), ruthenium cluster (Ru@NC-NC), and ruthenium nanoparticles (Ru@NP-NC) have been successfully synthesized and characterized. Among them. Ru@NC-NC possesses the highest reaction efficiency of a single catalytic center because of the three-dimensional coordination structure. Ru@NC-NC show high efficiency in reducing the level of ROS in cells, increasing the survival rate of cells, increasing the spreading area and adhesion focal area. After removing ROS stimulation, the cells in Ru@NC-NC group perform well in osteogenic indicators, such as alkaline phosphatase, alizarin red, IBSP, and adipogenic indicators, such as lipid droplet formation, FABP4, which is able to undergo normal osteogenesis and adipogenic differentiation. In rat periodontitis moedel, Ru@NC-NC can reduce the level of reactive oxygen species in periodontal tissue and reduce the absorption of alveolar bone. Furthermore, Ru@NC-NC can inhibit the expression of senescence-related-secretory-phenotypes, P21 protein, senescence related-β-galactosidase, and the phosphorylation of H2Ax, which prevents cells from senescence under oxdative damage. Ru@NC-NC may protect hMSCs by upregulating the expression of SETDB2, thus maintaining normal cell cycle and inhibiting the expression of senescence related markers.

*Conclusion/Significance: In this study, it has been found that the relationship between the catalytic performance and the atomic scales of single atom, cluster, and nanoparticle is “first rising and then equilibrium”. Among them, cluster (Ru@NC-NC) has a three-dimensional coordination structure, with richer electron clouds, lower energy barriers, better catalytic activity, and higher utilization of metal atoms in catalytic reactions. Ru@SA-NC, Ru@NC-NC, and Ru@NP-NC possess good biocompatibility. Ru@NC-NC reduces intracellular ROS levels, protects the survival, adhesion, and osteogenic and adipogenic differentiation abilities of hMSCs, and reduces tissue ROS levels in the rat periodontitis model. Further mechanism research suggests that, Ru@NC-NC can prevent hMSCs from cell cycle arrest and inhibiting the expression of cellular senescence related markers, thus exert the protective effect.

168 - Transcriptomics Approach: Investigating The Development And Pathogenesis Of Tendinopathy In A Rat Patellar Model

Y. RAO^1,2, R. S. Tuan^1,2,3, D. M. Wang^1,2,3

¹ The Chinese University of Hong Kong, Shatin, N.T. Hong Kong SAR., China,

² Institute for Tissue Engineering and Regenerative Medicine, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong SAR., China,

³ Center for Neuromusculoskeletal Restorative Medicine, Hong Kong Science Park,, Shatin, N.T. Hong Kong SAR., China

*Purpose/Objectives: Calcific tendinopathy (CT) is a debilitating condition characterized by apatite deposits within the tendon, resulting in chronic pain, functional decline, and reduced exercise tolerance among patients. Despite various clinical treatments, outcomes have been inconsistent, highlighting the need for a better understanding of the underlying pathogenesis. The pathogenesis of CT is multifactorial and complex, with one potential mechanism being the erroneous differentiation of tendon stem/progenitor cells (TSPCs) into chondrocytes or osteoblasts. Additionally, immune-stem cell crosstalk may also play a role in this process, necessitating further investigation. Hence, the objective of this study is to establish a dynamic landscape of tendinopathy development and explore the mechanisms underlying TSPC differentiation and immune-cell crosstalk in CT.

*Methodology: To establish a rat model of patellar tendinopathy, 10-12-week-old, Sprague Dawley (SD) rats were used in this study. These rats were randomly divided into three groups: (1) phosphate buffer solution (PBS) injection group; (2) collagenase (250 U)-treated group (COL); and (3) the contralateral tendon served as an intact group. The tendinopathy model was characterized via gross morphology, histopathology, and gene expression analysis. Additionally, transcriptomics investigations were performed on 2-, 4- and 8-week post-surgery to study the mechanisms involved in cell lineage switch (Figure 1A).

*Results: Our findings show that (1) The COL groups exhibited swelling, yellowish tendons, as well as an abundant secretion of mucus compared to the PBS groups (Figure 1B); (2) Histologically, the COL groups showed a complete loss of architecture, irregular fibers, and abnormal tenocytes at week 2. Chondrocyte-like cells, indicated by round cells with lacunar space, were first observed at week 4. At week 8, a large area of calcification and more chondrocyte-like cells were observed (Figure 1B); (3) The rat patellar tendinopathy group displayed distinct gene expression patterns compared to the control groups. Specifically, significant enrichment in gene ontology (GO) patterns and pathways associated with chondrogenesis, osteogenesis, and inflammation within the tendinopathy group were observed. Moreover, an increase in the proportion of certain immune cells was noted, indicating the presence of a cartilaginous transcriptional program and chronic inflammation in rat patellar tendinopathy (Figure 1C).

*Conclusion/Significance: The rat tendinopathy model we established demonstrated a phenotype characterized by tendon matrix degradation and heterotopic ossification, which aligns with existing literature. Preliminary transcriptomic data further validated the presence of a cartilaginous transcriptional program and noticeable changes in immune cell populations in the rat patellar tendinopathy. For future investigations, we intend to employ multiparametric flow cytometry analysis to explore the underlying mechanisms associated with immune-stem cell crosstalk as well as cell lineage switch.

169

171 - Biofabrication Of Region-Specific Meniscus Microtissues As Biological Building Blocks To Engineer Anisotropic Meniscal Grafts

K. Chattahy, G. Soares Kronemberger, A. S. Karam, D. J. Kelly

Trinity Centre for Biomedical Engineering, Trinity College Dublin, Dublin, Dublin, Ireland

*Purpose/Objectives: Meniscus injuries are a prevalent clinical problem, affecting 66/100,000 individuals annually worldwide. Meniscus regeneration remains a great challenge due to their poor intrinsic healing potential, particularly in avascular lesions, resulting in tear propagation and subsequent tissue deterioration. As the meniscus is critical for joint function, ineffective repair of meniscal injuries typically leads to the development of osteoarthritis. Common meniscal injury repair interventions, such as meniscectomy and meniscal repair, do alleviate pain and restore some level of function, but ultimately do not prevent the development of osteoarthritis. This drives the need for effective meniscal tissue engineering solutions to reconstruct the structural inhomogeneity and anisotropy of meniscus tissue, including its zonal composition, structure and biomechanics, particularly the circumferentially aligned collagen fibers that provide tensile strength and stiffness. One promising emerging approach in the field of tissue engineering is the use of cellular spheroids or microtissues as “biological building-blocks” for the biofabrication of complex tissue and organs. Furthermore, such microtissues can be integrated into 3D bioprinting workflows, enabling the engineering of meniscal grafts with user defined geometries and complex biomimetic architectures. The aim of this study is to develop a new “scaffold-free” biofabrication platform that leverages the capacity of meniscal progenitor cell-derived microtissues to self-organize into complex anisotropic meniscal fibrocartilage tissue grafts.

*Methodology: Meniscal progenitor cells (MPCs), known for their strong regenerative potential, were isolated from both inner (iMPCs) and outer (iMPCs) regions of the meniscus based on preferential adhesion to fibronectin. The clonogenicity and multi-lineage differentiation of these cells were characterised. The MPCs were then employed to generate uniform cellular microtissues. We then assessed the effect of different physical boundaries (specifically hydrogel boundaries with different channel widths; see Fig 1.iii) on microtissue bioink fusion and subsequent tissue deposition and organization.

*Results: MPCs isolated from the inner and outer regions of the meniscus demonstrated clonogenicity and sustained multi-potentiality across passages (Fig 1. i), demonstrating their potential as a progenitor cell source for meniscus tissue engineering applications. Microtissues generated from iMPCs and oMPCs were rich in both collagen and sGAGs. Increasing the cell density in MPC derived microtissues was associated with increased production of sGAG and distinct regional differences in collagen accumulation (Fig 1.ii), suggesting a complex relationship between microtissue cell density and resultant matrix composition. We then cast inner and outer MPC derived microtissues into spatially confining channels of differing widths to assess how such boundary conditions would impact tissue development. A fibrocartilage-like tissue was generated in all channels irrespective of their width, as evident by sGAG and collagen deposition (Fig. 1.iii). Notably, channel width was found to influence the extent of collagen alignment, with more alignment observed in the smaller diameter channels. The findings demonstrate how physical boundary conditions can be used to direct the organization of tissues generated via the fusion of multiple microtissues.

*Conclusion/Significance: Overall, these findings represent a significant advancement in the development of innovative meniscal regeneration solutions by combining biofabrication techniques with the regenerative capacity of MPCs, paving the way for enhanced patient outcomes and joint health.

172 - A High-throughput In Vitro Platform For Electrical Characterisation Of Biohybrid Electrodes

M. Boulingre, R. Green, R. Portillo Lara

Imperial College London, London, United Kingdom

*Purpose/Objectives: Implantable neural interfaces (NIs) have emerged as promising tools to treat several conditions, but their clinical translation is hindered by poor long-term stability. This is mainly due to the biomechanical mismatch at the device-tissue interface, which leads to a foreign body response (FBR) and fibrotic encapsulation of the device. Biohybrid NIs aim to improve biological integration by adding a tissue-engineered layer between the electronics and the host tissue. It is hypothesized that the embedded cells may establish synaptic connectivity with the endogenous neural circuitry, resulting in increased selectivity and biointegration. Electrical stimulation (ES) has been shown to influence neural cell fate and could therefore be leveraged to modulate the maturation and synaptic integration of biohybrid NIs. However, standardised, and reliable platforms to study the influence of ES on biohybrid devices in vitro are still lacking.

*Methodology: This study focused on the development of an in vitro platform for the electrical characterisation of biohybrid interfaces. Monolithic biohybrid electrodes were fabricated from platinum (Pt) disks insulated with PDMS. The exposed active site was then coated with a conductive hydrogel (CH) made of poly(3,4-ethylenedioxythiophene)/poly(vinyl alcohol) (PEDOT/PVA)-taurine, and subsequently a PVA-gelatin hydrogel cell carrier was photopolymerized on top. Impedance spectroscopy and cyclic voltammetry were used to characterize the electrochemical properties of the electrode. Cytocompatibility was evaluated in vitro using a SCL4.1/F7 Schwann cell line and a commercial LIVE/DEAD cell viability assay. Following this, an electrode system for in vitro culture with a 24-well plate format was designed using SolidWorks and 3D-printed using poly(lactic acid) (PLA) for rapid prototyping. The device was then assembled, and the impedance of the electrodes was measured with a multimeter. The stability of the circuit and the performance of the electrode system after multiple autoclave cycles was also assessed.

*Results: Results showed that the addition of the PVA-gelatin carrier did not alter the electrochemical characteristics of the CH-coated electrodes and that no cytotoxic responses were elicited by the electrode assemblies. The impedance of the electrodes on the multi-well device and the ability to deliver specific ES paradigms remained stable after multiple autoclave cycles.

*Conclusion/Significance: This platform will enable the study of the influence of ES on neural cell fate towards the in vitro biofabrication of tissue-engineered NIs. Moreover, this will also increase our understanding on the mechanisms that underlie neural development and function in health and disease.

173 - Electroconductive Collagen-pedot:pss Hydrogel For Attenuation Of Post-infarct Cardiac Arrhythmias And Enhancement Of Hipsc-cardiomyocyte Functionality

K. Roshanbinfar¹, M. Schiffer², E. Carls², M. Angeloni¹, M. Koleśnik - Gray¹, S. Schruefer¹, D. W. Schubert¹, F. Ferrazzi¹, V. Krstić¹, B. K. Fleischmann³, W. Roell², F. B. Engel¹

¹ Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany,

² University of Bonn, Bonn, Germany,

³ Univeristy of Bonn, Bonn, Germany

*Purpose/Objectives: Myocardial infarction (MI) causes cell death, disrupts electrical activity of the heart, triggers arrhythmia, and results in heart failure. Importantly, 50-60% of MI patients die from sudden cardiac death (SCD). Due to a limited renewal potential of the adult cardiomyocytes, the cardiomyocyte loss caused by MI leads to the development of fibrotic tissue and subsequent ventricular wall dilation. The fibrotic tissue has a low conductivity and thus is electrically insulating. This, in conjunction with hetero-cellular interactions in the scar border zone, contributes to electrical decoupling of the remaining viable cardiomyocytes in the infarcted region; thereby causing arrhythmia. The most effective therapy for SCD prevention is implantable cardioverter defibrillators (ICDs). However, ICDs do not prevent arrhythmia and contribute to adverse remodeling and disease progression. Notably, there is a gap in designing an electrically conductive hydrogel that prevents post-MI arrhythmia and can be combined with cardiomyocyte delivery. Here, we developed an injectable collagen-PEDOT:PSS hydrogel that protects infarcted hearts against ventricular tachycardia (VT) and can be combined with hiPSC-cardiomyocytes to promote partial cardiac remuscularization (Figure 1).

*Methodology: We have generated an injectable collagen-PEDOT:PSS hydrogel and studied gelation kinetics, electrical conductivity, as well as the hydrogel micromorphology by scanning electron microscopy. Moreover, we generated engineered cardiac tissues based on hiPSC-derived cardiomyocytes and studied cell survival, subcellular organization of the contractile machinery, and calcium handling of these cells for up to 40 days. We performed bulk RNA-sequencing analyses to elucidate changes in hiPSC-cardiomyocytes at gene level and identify dysregulated pathways. Eventually, we transplanted the hydrogels in a cryoinfarction model in mice analyzing five treatment groups: sham, MI, MI + collagen, MI + collagen-PEDOT:PSS, and MI + collagen-PEDOT:PSS containing hiPSC-derived cardiomyocytes.

*Results: PEDOT:PSS improves collagen gel formation and results in faster gelation. It transforms single-fibrillar collagen morphology into entangled thick helical microfibrous morphology in collagen-PEDOT:PSS and increases electrical conductivity from ∼41 mS/cm for collagen to ∼65 mS/cm for collagen-PEDOT:PSS. hiPSC-cardiomyocytes exhibit near-adult sarcomeric length (∼2 µm) in collagen-PEDOT:PSS hydrogels and improved compared to cells in collagen hydrogels contractility by ∼500% and conduction velocity by ∼700% accompanied by enhanced calcium handling. RNA-sequencing data revealed an upregulation of genes associated with conduction, structure, energetics, oxidative phosphorylation and fatty acid oxidation, calcium handling, and cell-ECM interactions, indicating enhanced maturation and improved cell-matrix interactions in hiPSC-cardiomyocytes within collagen-PEDOT:PSS hydrogels. Injecting collagen-PEDOT:PSS hydrogels in infarcted mouse hearts reduced the occurrence of ventricular tachycardia upon burst stimulation from∼80% to ∼20%, which corresponds to the occurrence rate in healthy hearts. Furthermore, hiPSC-cardiomyocyte implementation results in improved heart function (fraction shortening: 25% vs. ∼18% in MI).

*Conclusion/Significance: Collectively, the here-developed hydrogel showed great potential to promote stem cell-derived cardiomyocyte maturation and to protect the infarcted heart against ventricular tachycardia. Thus, collagen-PEDOT:PSS hydrogels offer a versatile platform for treating injuries in hearts and possibly other electrically sensitive tissues.

174 - Overcoming The Blood-brain Barrier: A Novel Approach Using A Conductive Bioadhesive Coating For Voltage-driven Drug Delivery In Glioblastoma Multiforme

G. Miceli¹, A. Lee¹, E. Cuttaz¹, J. Killilea¹, C. Chapman², R. Green¹

¹ Imperial College London, London, United Kingdom,

² Queen Mary University of London, London, United Kingdom

*Purpose/Objectives: Glioblastoma multiforme stands out as the most prevalent form of malignant brain tumour among adults, marked by a disheartening 15-month median survival rate, and less than 5% of patients managing to survive beyond 5 years. A pivotal obstacle is the blood-brain barrier (BBB), which regulates the entry of chemicals into the brain. To address this challenge, this study aims to develop a conductive bioadhesive coating, designed to be strategically inserted into the intricate microenvironment of the tumour, to control the drug concentration by leveraging the application of voltage. Moreover, this strategy aims to overcome chemoresistance and unnecessary exposure to healthy tissues by circumventing the BBB. Furthermore, our investigation seeks to delve into the intricate relationship between the applied voltage and the efficacy of the delivered drug, unravelling crucial insights to optimize tumour treatment strategies.

*Methodology: Poly(3,4-ethylenedioxythiophene) (PEDOT) doped with polystyrene sulfonate was incorporated inside a bioadhesive polyurethane urea matrix to develop a conductive bioadhesive coating. The final polymer was characterised by electrochemical impedance spectroscopy, in both wet and dry conditions, and cyclic voltammetry. Then, paclitaxel (PTX) was incorporated into the polymer matrix and porous titanium rods (Ti-rod)were dip-coated into the loaded polymeric solution. These devices were designed around the neurosurgical toolkit to perform a biopsy and will be supported by a cartridge during the insertion. The evaluation of the release was conducted at 37°C inside Franz’s cells, under stirring, in a 1:1 mixture of FBS and PBS buffer and the in vitro effects were evaluated using a neuroblastoma cells line (SH-SY5Y).

*Results: The release study of PTX from the devices was performed in enhanced conditions applying a constant voltage equal to -1V and in passive condition at 0V (Figure 1-a).

Figure 1 a) Schematic representation of the release conditions, b) Bode plots of the impedance magnitude and phase angle of the conductive polymer matrix (CB) compared with platinum, c) Cumulative comparison of the % of PTX released between enhanced and passive conditions, d) Live/dead staining of SH-SY5Y 48h after the conditioning (scale bar 300μm). The comparison of drug release demonstrates a substantial increase when voltage is applied (Figure 1-c). This dynamic interplay between the enhanced and passive release profiles highlights the effectiveness of applying voltage in promoting controlled and increased drug release, presenting valuable insights for optimizing drug delivery systems. Notably, when voltage is applied in conjunction with PTX, it becomes evident that the cytotoxic effect is more pronounced when the drug is administered before the application of voltage (Figure 1-d). This finding supports our hypothesis that applied voltage alters the permeability of the cell membrane, facilitating enhanced drug uptake. Only a few cell clusters survived in the case of 1h passive release from the devices, underscoring a notable cytotoxic effect. Contrastingly, 1h enhanced release induced a complete cytotoxic effect.

*Conclusion/Significance: These results highlight the effectiveness of the devices and the potential of combining voltage application with drug delivery systems to augment therapeutic efficacy, demonstrating the synergistic effects of drug administration and voltage application for optimized cytotoxic outcomes.

175 - Mxene Functionalized Collagen Biomaterials For Cardiac Tissue Engineering Driving Ipsc-derived Cardiomyocyte Maturation

M. G. Monaghan

Biomedical Engineering, Trinity College Dublin, Dublin, IRELAND

*Purpose/Objectives: Electroconductive biomaterials are gaining significant consideration for regeneration in tissues where electrical functionality is of crucial importance, such as myocardium, neural, musculoskeletal, and bone tissue. Traditionally one considers metals as imbibing this property however electroconductive polymers and/or functionalising bulk materials with nanomaterials are emerging as an exciting option in tailoring electroconductive biomaterials. In this work, conductive biohybrid platforms were engineered by blending collagen type I and 2D MXene (Ti3C2Tx) and afterwards covalently crosslinking; to harness the biofunctionality of the protein component and the increased stiffness and enhanced electrical conductivity (matching and even surpassing native tissues) that two-dimensional titanium carbide provides.

*Methodology: Etched Ti3C2Tx MXene was suspended in a 4 mg/ml gelatin solution and combined with porcine type I collagen to form hybrid platforms. These were also crosslinked using EDAC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-Hydroxysuccinimide) to covalently crosslink the collagen structure. Extensive physiochemical testing (nanoindenter, FTIR, degradation, swelling, conductivity) was performed to assess the impact of MXene % on the platform's properties. Additionally, the impact of % was assessed with fibroblasts, neonatal rat cardiomyocytes, induced pluripotent stem cell derived cardiomyocytes and Staphylococcus aureus.

*Results: These MXene platforms were highly biocompatible and resulted in increased proliferation and cell spreading when seeded with fibroblasts. Conversely, they limited bacterial attachment (Staphylococcus aureus) and proliferation. When neonatal rat cardiomyocytes (nrCMs) were cultured on the substrates increased spreading and viability up to day 7 were studied when compared to control collagen substrates. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were seeded and stimulated using electric-field generation in a custom-made bioreactor. The combination of an electroconductive substrate with an external electrical field enhanced cell growth, and significantly increased cx43 expression.

*Conclusion/Significance: These findings are promising in the development of highly biocompatible and conductive biohybrid platforms that may be applied to a variety of biological applications requiring electrical conductivity. Furthermore, this work has demonstrated the excellent features of MXene and its prospective usage in the future as a component with natural polymers. Indeed it could be implanted into electrically conductive tissues to serve as an electrically conductive bridge or scaffold, as well as an in vitro platform to grow electrically sensitive cells outside the body.

177 - Synthetic Matrix Fibers Promote Vascular Assembly In Dense Fibrin Hydrogels

F. S. Midekssa, C. D. Davidson, M. E. Wieger, J. L. Kamen, B. M. Baker

University of Michigan, Ann Arbor, MI

*Purpose/Objectives: Vasculogenesis is the de novo formation of vascular networks during embryonic development, organogenesis, and adult neovascularization. This process can be harnessed to vascularize engineered tissues. Most successful in vitro approaches to guide and stabilize vessel-like networks in 3D hydrogels require the admixing of stromal cells such as fibroblasts. However, the inclusion of these support cells often leads to undesirable outcomes such as tissue contraction and stiffening. In our prior work (Davidson 2023) using synthetic fibrous matrices, we found that EC self-assembly is facilitated by cell force-mediated matrix reorganization and mechanical intercellular communication via force transmission along fibers. Here, we hypothesized that the inclusion of synthetic matrix fibers in a fiber-reinforced hydrogel composite (FRHC) can provide mechanical and topographical cues that enhance vasculogenic assembly.

*Methodology: We used a previously developed method to embed dextran vinyl sulfone (DexVS) electrospun fibers in a fibrin hydrogel. DexVS fibers were functionalized with cell adhesive and non-adhesive peptides to control integrin-ligand interactions (Fig. 1A). HUVECs were encapsulated (6 million ml^-1) in fibrin (5 mg ml^-1 fibrinogen, 1U ml^-1 thrombin) containing 2.5 vol/vol % fibers (Fig. 1B). Samples were gelled at 37^oC for 20 min before hydration with EGM2 supplemented with fetal bovine serum (FBS; 5% v/v), VEGF; 50 ng mL-1, and phorbol 12-myristate 13-acetate (PMA; 25 ng mL-1), with media replaced every 24 h for up to 3-5 days. To assess transcriptional activity during the early phase of vascular assembly, bulk RNA-seq was run on tissue lysates and Gene Ontology terms were analyzed. Additionally, to investigate if FRHCs support vascular integration in vivo, pre-cultured FRHCs supporting endothelial vascular assembly were implanted in the fat pad of NSG mice for 7 days. Vascular integration of implants with the host was assessed by histology and immunostaining.

*Results: FRHCs led to enhanced vascular network assembly compared to pure fibrin hydrogels lacking DexVS fibers (Fig. 1C). Intriguingly, the inclusion of non-adhesive fibers led to the greatest network assembly, as determined by total network length (Fig. 1C, D). Additionally, non-adhesive fibers supported lumen formation after 3 days in culture. Examining the transcriptional response of ECs to cell-adhesive or non-adhesive fibers, we noted numerous differentially regulated genes and GO terms associated with cell-ECM adhesion, actin cytoskeleton organization, integrin-mediated signaling and focal adhesion assembly (Fig. 1E-F). Vinculin immunostaining and matrix bead displacement studies demonstrated that ECs in FRHCs enhanced their adhesion to the ECM and generated greater matrix deformations. After 7 days of implantation, FRHCs (whether with cell-adhesive or non-adhesive fibers) enhanced host cell infiltration as indicated by H&E staining (Fig. 1G). Furthermore, mouse erythroid marker Ter-119 immunostaining showed that only FRHCs with non-adhesive fibers supported vascular integration and blood perfusion from the host (Fig. 1H).

*Conclusion/Significance: Altogether, we integrated electrospun DexVS fibers into 3D fibrin hydrogel to investigate the role of microscale fibers on the vascular assembly of ECs. We found that the addition of non-cell-adhesive DexVS fibers enhanced EC spreading and vascular assembly, expression of genes involved in cell-cell and cell-ECM interaction, and enabled vascular integration in vivo.

178 - Sustaining Ischemic Tissue Throughout The Healing Process

A. Watt, B. Dallison, P. Azizi-Mehr, H. Shash, M. Gilardino, J. Barralet

McGill University, Montreal, QC, Canada

*Purpose/Objectives: Tissue can become transiently fully or partially ischaemic following surgical repair and replantation and while the vasculature remodels this tissue is at risk of necrosis and infection. Necrosis can spread beyond the region of tissue repair and results in permanent tissue loss. Effective healing requires a sufficient oxygen supply to prevent cell death, maintain activity of oxygen dependent enzymes, allow oxidative phosphorylation and normal development of the inflammatory process. There is some evidence for the use of hyperbaric treatments to raise tissue oxygen concentrations with specific indications, but this process is expensive, cumbersome, and oxygen levels drop to atmospheric concentrations rapidly after the patient leaves the hyperbaric chamber. In this study, a degradable biomaterial-based oxygen releasing dressing was evaluated for efficacy in improving tissue regeneration, mitigating necrosis, and enhancing wound healing in a preclinical ischemic wound model.

*Methodology: The biomaterial composite was prepared from a suspension of CaO₂ and Fe₃O₄ in 10% (w/v) PCL solution in chloroform which was subsequently cast, dried, and embedded in sterile 3% (w/v) sodium alginate and crosslinked with sterilized CaCl₂ (1M). Oxygen and hydrogen peroxide release was measured over 3 days in vitro. In vivo studies were performed using an ischemic rabbit ear full thickness wound mode. Ethical approval for this study was obtained from the Research Institute of the McGill University Health Center (MUHC-8075). Wound vascular supply was observed via fluorescein injection and tissue samples were histologically evaluated. Wound size, healing rate, tissue regeneration quality (epithelialization, granulation), and necrosis incidence for the peroxide treated group were compared to an alginate-based non-oxygen eluting control and conventional wound dressings.

*Results: In vitro findings showed that the peroxide-polymer composite sustained oxygen release for up to 72 hours. In vivo experiments showed that wound closure at day 17 was 54±29% for the group treated with the peroxide-polymer composite versus 31±23% N=15, P<0.05 for the group receiving alginate only. Fluorescein injections for wound vascularization showed no fluorescence beyond the edges of the wound at any time point in the control group, while the experimental group expressed significantly higher levels of fluorescence (**P<0.01) as early as day 7 inside the wound perimeter. Notably, there was decrease in full thickness necrosis from 87% in the control group to 40% in the experimental group. Treated wounds exhibited enhanced cell proliferation, and higher levels of re-epithelialization (0.85±0.4mm² vs 0.47±0.22mm² for control) and granulation tissue (43.7±25.5% vs 27.3±13.9% for control). These results demonstrate that the oxygen-eluting biomaterial dressing supported the healing process and improved outcomes across a variety of parameters after 17 days compared to control.

*Conclusion/Significance: Oxygen releasing calcium peroxide composite successfully maintained tissue viability and reduced necrosis without disrupting the healing process in an ischemic full thickness wound model. Its ability to deliver sustained oxygen directly to the wound site has shown to significantly improve the healing process and enhance tissue regeneration, without the drawbacks inherent in solutions like hyperbaric oxygen therapy. This technology has promising applications in improving diabetic wound care, where peripheral vasculopathy often leads to chronic, ischemic ulcers.

179 - Functionalized Silk Matrices For Single- Or Bi-layered Models Of Barrier Tissue

S. Gkouma, M. Widhe, M. Hedhammar

KTH Royal Institute of Technology, Stockholm, Sweden

*Purpose/Objectives: The need to disengage from animal models has generated increased interest in engineering defined, physiologically relevant tissue models for applications in drug testing, infection studies, or clinical settings (e.g., skin grafts). Recombinant spider silk can self-assemble into nanofibrillar structures (e.g., three-dimensional (3D) network and nanomembrane) when exposed to interfaces. Except for their favorable morphology and mechanical properties, these silk matrices favor cell adhesion due to their functionalization with a fibronectin-derived RGD motif, called FN-silk. In this study, we evaluated FN-silk’s ability to support macrosized models of tissues with an epithelial-mesenchymal architecture (e.g., skin, lung, intestine). The 3D network is proposed as a fibrillar, ECM-like matrix, and the nanomembrane mimics the basement membrane that supports the epithelial layer. The two matrices are standalone but can also be combined into bilayered epithelial-mesenchymal models.

*Methodology: Different FN-silk matrices were constructed by exploiting silk’s self-assembly property at the interface of different media (e.g., air-water). By allowing the silk solution to stand still, a fibrillar nanomembrane is formed at the interface, onto which epithelial cells (e.g., primary human keratinocytes or human alveolar epithelial cells) were seeded to form a confluent layer. For the 3D networks, a silk/media solution containing primary human cells (e.g., fibroblasts, endothelial cells) was briefly mixed with air using a system of two interconnected syringes, to yield a cell-integrated 3D network. The two layers were combined by lowering the nanomembrane onto the 3D network and were cultured as a bilayered construct. The engineered models were evaluated with immunofluorescence or spatial transcriptomics analysis.

*Results: The 3D network is an elastic and fibrillar scaffold suitable for tissue-like cell culture. Within it, cells assume functions similar to those of their in vivo environment. Specifically, fibroblasts evenly populate the scaffold and deposit ECM components (i.e., collagen I and III, and elastin) within 14 days. During the same timeframe, the endothelial cells assemble to form microvascular structures in linear or lumen-like conformations. On the nanomembrane, cells adhere to form a tight layer, as indicated by the formation of a tight junction network. Additionally, epithelial cells can differentiate and form multilayered stratified tissue (e.g., epidermis), mimicking the architecture of the native one. Lastly, the formation of a bilayered construct facilitates the modelling of more complex systems (e.g., dermal-epidermal combination). Gene expression of the cells cultured in these models verifies the presence of subpopulations of skin cells appearing in a depth-dependent manner within the skin model, much like the in vivo tissue.

*Conclusion/Significance: FN-silk matrices allow for 3D cell culture in conditions similar to the cells’ in vivo environment. They can be used to mimic the tissues’ basement membrane or provide a fibrillar scaffold for the cells to deposit ECM. Lastly, they support the formation of microvascular structures under static in vitro conditions. They can be used as standalone single-layered models or combined into one bilayered one.

180 - Reinforced biomaterials for tissue regeneration, stem cell fate commitment, and wound healing

S. Hassan

Khalifa University, Abu Dhabi, United Arab Emirates

*Purpose/Objectives: The effects of self‐oxygenation biomaterials on different aspects of regenerative medicine such as healing of the myocardium after MI or stem cell fate in cellularized hydrogels have not yet been studied. Additionally, although decellularized hydrogels‐based research is gaining attention, however, very little is known about their burn wound healing properties.

*Methodology: Biomaterials were reinforced to give them oxygenating, antioxidative, and tissue‐adhesive properties for three different applications: 1.) Better tissue regeneration in MI, 2.) Stem cell fate commitment for regenerative medicine such as bone regeneration, and 3) Complete healing of burn wounds (Figure 1). Calcium peroxide as the source of oxygen was encapsulated in polycaprolactone to produce oxygenating microparticles with prolonged oxygen release profile. These oxygen‐generating microparticles were incorporated into bioadhesive silk‐alginate or gelatin methacryloyl (GelMA) based hydrogels with or without mesenchymal stem cells (MSCs), for MI and osteochondral differentiation, respectively. For MI, these hydrogels were conjugated with stromal cell derived factor (SDF) to orchestrate chemotaxis and angiogenesis and generate oxygen via oxygenating microparticles (OMPs) to alleviate the cytotoxic anoxic environment. Silk fibroin (SF) was explored to endow elasticity and resilience to the injectable hydrogel. Additionally, tyramine (TA) was conjugated to alginate (TA‐Alg) and SF, producing a mechanically robust and tissue adhesive hybrid hydrogel (TSF) that encourages tissue adhesion and enhances the injectability of the hydrogels. For stem cell fate commitment for bone tissue engineering applications, oxygen‐enerating microparticles wereincorporated into GelMA hydrogels in the presence/absence of osteoinductive silicate nanoparticles (SNPs). A comparative study revealed the osteogenic fate of hMSCs in the designed hydrogels under normoxic (the gold standard for in vitro cultures) and anoxic (common in large bone defects; <0.1% oxygen) conditions. Additionally, hydrogels from decellularized caprine small intestine submucosa (DG‐SIS) were reinforced with vitamin C (Vt‐C) and ZnO NPs for complete burn wound healing in rabbits. This study demonstrated asynergistic effect of ZnO/Vt‐C in the bioactive gel as an effective therapeutic approach for full‐thickness burn wound treatment.

*Results: The hydrogel was shown to continuously release SDF and oxygen for MI applications. The subsequent combinational and synergistic effect results in a significant improvement in vascularization, increasing the cardiomyocyte survival by 30% cardiomyocyte survival and reducing the fibrotic scar formation in an MI animal rodent model. To study the role of oxygenating biomaterials in driving fate commitment in MSCs, in line with the literature, under standard laboratory conditions (normoxia), stem cells exposed to osteoinductive SNPs most effectively were differentiated into bone‐forming osteoblasts. Bulk RNA‐sequencing confirmed that OMPs possessed a stronger expression of osteogenic gene transcript fingerprint than SNPs or SNPs in combination with OMPs. The addition of OMP also increased the number of vessels and potentially increased vessel diameter and thus vessel maturation. Decellularized caprine scaffolds encapsulating vitamin C and ZnO nanoparticles showed complete healing of a burn wound in a rabbit.

*Conclusion/Significance: Taken together, we present innovations in biomaterials in their oxygenating capacity, bioadhesion, and antioxidating and wound healing properties to treat a wide variety of medical conditions in small and bigger animal models.

182 - Mechanotransduction In Magnetic Hydrocups: A Hollow Magnetic Electrospun Scaffold For Hydrogel Stiffening

W. Chen¹, M. Kalogeropoulou¹, P. H. Kouwer², L. Moroni¹

¹ MERLN – Institute for Technology-Inspired Regenerative Medicine, Maastricht, Netherlands,

² IMM – Institute for Molecules and Materials, Nijmegen, Netherlands

*Purpose/Objectives: Cells mechanically respond to their local microenvironment, and actively remodel the mechanical properties of the extracellular matrix. Mechanotransduction is often used to direct cell fate, regulate tissue regeneration, and plays a key role in pathologic changes.¹ However, these dynamic behaviors are extremely challenging to replicate in matrices for in vitro study. We develop a new approach by electrospinning, where hydrogels are injected into a hollow electrospun scaffold with magnetic responsiveness to enable dynamic, spatiotemporal and remote control over the mechanics of the matrix.

*Methodology: Magnetic hollow scaffolds were produced by mixing a polymer solution of 300PEOT55PBT45 (PolyVation) with a magnetic Fe₃O₄ nanoparticles (FeNPs) solution, followed by an electrospinning process. The mixture was fed to a charged needle between 12 - 18 kV at 3000 µL/h, and the mandrel with a diameter of 1.5 mm was charged with -1.5 kV. A biomimetic polyisocyanide² (PIC) hydrogel was formed by heating, non-covalently by a linker dopamine-DBCO³ (DD) or covalently crosslinked by a linker DBCO-PEG4-DBCO (DPD). Alginate hydrogels were ionically crosslinked with calcium ions. Magneto-rheology was used to study the magneto-rheological properties.

*Results: Hydrogels were injected into a hollow scaffold, named Hydrocups. For stress-stiffening PIC gels, we hypothesized that the magnetic hydrocups would apply external force to hydrogels under a magnetic field, inducing gel stiffening (Figure 1A). We used magneto-rheology to test magnetic hydrogels to quantify the stiffening effects. The absolute stiffness of covalently crosslinked PIC (0.2 wt%) with particles (15 wt%) increased up to 23.3 kPa, whereas particles alone was 6.3 kPa (Figure 1B). As a control, magnetic alginate (0.6 wt%) only increased up to 3.6 kPa. Preliminary data showed that hMSC cells significantly spread and formed cell network structures (Figure 1C).

Figure 1. (A) Scheme of the magnetic responsive Hydrocups. (B) Absolute increase of storage modulus of magnetic gels. (C) Human mesenchymal stem cells (hMSC) spreading after 14 days of culture in PIC hydrogels under bright field (i) and stained for nuclei (ii), cell skeleton (iii) and merged image (iv). Scale bar 100 µm.

*Conclusion/Significance: We developed a highly stiffening scaffold by switching “ON” a magnetic field, and cells had high affinity in the PIC hydrogels. We believe magnetic Hydrocups have great potential for the cellular mechanostransduction studies. Currently, we are investigating the potential of these new magnetoresponsive constructs on cell activities, such as proliferation and cell secretome.

183 - Nanoliposome Functionalized Colloidal Scaffolds For Tissue Engineering Applications

E. Omidvari¹, M. Samandari², D. Ghanbariamin², J. Quint², E. Mollocana², C. Kahn¹, A. Tamayol², E. Arab-Tehrany¹

¹ Université de Lorraine, Nancy, France,

² University of Connecticut Health Center, Farmington, CT

*Purpose/Objectives: Bioprinting technologies, specifically extrusion-based bioprinting, are regarded as promising methods for creating intricate 3D scaffolds that replicate the physical, chemical, and structural characteristics of natural tissues. Recently created GelMA scaffolds with multiscale porosity have been shown to effectively treat soft tissue injuries. The colloidal inks exhibited ease of printing, provided bioadhesion through in situ crosslinking, and notably improved cellular ingrowth, tissue remodeling, revascularization, and tissue regeneration in various mouse models. In order to increase their biological efficacy, we investigated the process of functionalizing them with nanoliposomes, which have been identified as one of the most promising methods for drug delivery.

*Methodology: This study examined the impact of varying doses of nanoliposome inclusion on the characteristics of colloidal inks and the resulting scaffolds. The inks consisted of solutions containing GelMA at concentrations of 10%, 15%, and 20% (w/v), together with a photoinitiator at a concentration of 0.3% and starch as a foam stabilizer at a concentration of 0.5%. The physical and electrostatic properties of nanoliposomes were analyzed, followed by an investigation into the physicochemical interactions between liposomes, stabilizer, and GelMA and the potential effect of incorporating nanoliposomes on the crosslinking of GelMA. Furthermore, we investigated the impact of nanoliposome concentration on foam porosity and mechanical properties. The rheology of the bioink was utilized to control the extrusion printing parameters of the 3D scaffold. An analysis was conducted to evaluate the biocompatibility and antioxidant properties of the nanofunctionalized scaffolds.

*Results: The findings demonstrated that increasing the concentration of anionic nanoliposomes with sizes of 111 ± 1 nm (Fig. 1A) resulted in an elevation in the magnitude of the ζ-potential (Fig. 1B) and viscosity of bioinks (Fig. 1C). The embedded foam bioinks showed shear-thinning behavior, hence improving printability. Furthermore, the incorporation of nanoliposomes allows for precise tuning of the macropore shape and mechanical properties without any chemical interaction or impact on complete crosslinking. By augmenting the concentration of nanoliposomes, the foam scaffold’s Young’s modulus (Fig. 1 D) and macropore diameters (Fig. 1 E, F) were increased. The nanofunctionalized foam bioinks made exclusively from natural components demonstrated significant antioxidant activity (Fig. 1G) due to the presence of α-tocopherol in phospholipid bilayers. These nanoliposomes had the ability to provide endogenous protection against oxidation, which enhanced their stability. Consequently, they can perform as anti-inflammatory and radical scavenger agents against the free radicals generated by macrophages and neutrophils during an inflammatory response. In addition, the nanofunctionalized foam bioinks were successfully bioprinted in different sizes and shapes. The foam-embedded nanoliposomes showed remarkable compatibility with C2C12 cells, even when present at a high concentration of 5%, which is an uncommon concentration. This rare characteristic has significant potential for various biological applications

*Conclusion/Significance: To summarize, nanoliposome multi-scale porous scaffolds have the potential to be utilized in biomedical applications that require precise delivery of therapeutic substances to cells enclosed within or at their interface, as well as tissue regeneration. This is due to their porous structure and ability to scavenge free radicals while protecting the bioactive materials from physiological conditions.

186 - Biofabrication Of Granular Hydrogel-based Bioinks For Dermal Regeneration

R. Shamasha^1,2, S. Kollenchery Ramanathan³, A. Zielinska¹, K. Oskarsdotter⁴, F. Rasti Boroojeni³, S. Naeimipour³, P. Lifwergren³, L. Roberts¹, N. Reustle³, A. Starkenberg¹, J. Rakar^1,2, P. Apelgren⁴, K. Säljö⁴, L. Kölby⁴, D. Aili³, J. P. Junker^1,2

¹ Department of Biomedical and Clinical Sciences, Linköping, Sweden,

² Center for Disaster Medicine and Traumatology, Linköping, Sweden,

³ Department of Physics, Chemistry and Biology, Linköping, Sweden,

⁴ University of Gothenburg, Gothenburg, Sweden

*Purpose/Objectives: Wounds imbue a tremendous burden on the health care system and cause pain and suffering for affected patients. Treatment using split-thickness skin grafts results in poor functional and aesthetic outcome, while cultured epidermal autografts fail to recapitulate the full diversity of cells and extracellular matrix components (ECM). We have previously shown that porous gelatin microcarriers (PGMs) can serve as a transplantation vehicle for cells in the treatment of wounds. Here, we investigate cell-laden PGMs in combination with 3D bioprinting to biofabricate tissue-engineered skin microtissue for transplantation.

*Methodology: Using bioorthogonal copper-free click chemistry, PGMs were functionalized to enable polyethylene glycol (PEG)-azide bridged crosslinking with hyaluronic acid (HA). Human dermal fibroblasts were seeded on the PGMs for three days to ensure proper attachment and expansion. Zombie Red^TM, immunofluorescent staining, and semiquantitative real-time PCR were used to investigate cell viability and phenotype, as well as ECM production over time. Constructs composed of cell laden PGMs, HA and gelatin were created using a Cellink Bio X bioprinter and transplanted into an in vivo mouse model and followed for 28 days. At set timepoints, samples were fixed, paraffin-embedded and stained using immunofluorescence to assess cell viability, proliferation, and protein expression.

*Results: Fibroblast viability on PGMs was 86.6 ± 5.3 % before printing. Cells per PGM increased from 13.6 ± 5.2 to 31.9 ± 14.2 in 96h (p > 0.001). Immunofluorescent staining showed cell production of ECM components. Complementary gene expression assays revealed significant upregulation of COL3A, LAMB3, LAMC2, FBN1 and ELN. The presence of myofibroblasts on the PGM was confirmed by a ten-fold increase of ACTA2 gene expression as well as α smooth muscle actin positive staining. Directly after printing, cell viability was 78.9 ± 6.0 %. After 24 and 74h, viability was 84.9 ± 5.0 % and 75.6 ± 15.9 % respectively. Ki67-positive staining revealed a subset of cells to be in a proliferative state (G1, S, G2 or mitosis). Ki67 staining also confirmed that cells in the constructs survive transplantation and continue proliferating over time in vivo. Furthermore, immunofluorescence staining revealed positive staining for Collagen I, III and IV, CD31 and Laminin V.

*Conclusion/Significance: The combination of functionalized PGMs and HA enabled 3D-bioprinting of granular skin hydrogel constructs. Following transplantation, cells in the constructs were shown to survive and proliferate over time, as well as produce ECM molecules relevant for tissue reconstitution. The proposed methodology is conducive to tissue regeneration and recapitulation of normal dermal architecture, as opposed to healing via scar formation.

187 - COMBINATION OF 3D PRINTING, PLASMA POLYMERIZATION, AND BIOACTIVE COATINGS TOWARDS FABRICATION OF EGGSHELL BIOWASTE/PCL COMPOSITE SCAFFOLDS FOR BONE REGENERATION

A. Jafari, H. Savoji

University of Montreal, Montreal, QC, Canada

*Purpose/Objectives: 3D printing is a robust and versatile technique that offers a unique opportunity for making tissue-engineered scaffolds for bone regeneration. Eggshell (ES) contains bone-like compositions, which makes this biowaste an exciting material for bone tissue engineering. In this study, we fabricated 3D printed scaffolds using polycaprolactone (PCL) and ES powder and investigated the effect of ES concentration on the printability, mechanical properties, and morphology of the scaffolds. Furthermore, to enhance the scaffolds’ bioactivity, we used plasma polymerization to deposit an oxygen-rich thin film coating to activate the scaffolds’ surface. Subsequently, gentamicin, an antibiotic, as a model bioactive agent, was grafted on the surface of the scaffolds.

*Methodology: To investigate the physicochemical surface properties of the 3D printed scaffolds, scanning electron microscopy, X-ray photoelectron spectroscopy, X-ray diffractometry, thermogravimetric analysis, and antibacterial activity were implemented. In addition, the potential application of these scaffolds for bone tissue engineering was studied by biocompatibility and mesenchymal stem cell proliferation/differentiation assessments.

*Results: The 3D printed scaffolds' performance and bioactivity were significantly improved by using plasma polymerization to modify the surface and grafting gentamicin. Sample characterizations demonstrated that we were successfully able to develop bioactive scaffolds in terms of printability, mechanical properties, morphology, and antibacterial activity. The scaffolds also exhibited excellent biocompatibility and supported cell adhesion, proliferation, and osteoblast differentiation, indicating its potential application for bone tissue engineering.

*Conclusion/Significance: We reported that 3D printing using ES powder and PCL was a promising technique for creating tissue-engineered scaffolds for bone regeneration. Overall, the results suggested that the 3D printed scaffold has shown great potential in the field of bone tissue engineering and could be a significant step toward developing effective treatments for bone regeneration.

188 - Topographical And Fucoidan Modification Of Polyvinyl Alcohol Hydrogel To Enhance Neuronal Neurite Outgrowth And Axon Guidance

Y. Yao, F. Feng, E. K. Yim

University of Waterloo, Waterloo, ON, Canada

*Purpose/Objectives: Peripheral nerve injuries are common clinical problems worldwide. Artificial nerve grafts that support axon growth hold promises in promoting nerve regeneration and function recovery. Nerve grafts made of collagen and synthetic materials, such as polyglycolic acid (PGA) and polyvinyl alcohol (PVA) have been approved by FDA and are clinically available for bridging short nerve defects. However, current artificial nerve grafts are insufficient to regenerate axons across long nerve gaps. Specific biochemical and biophysical cues are required to be incorporated to artificial nerve grafts to promote neural cell adhesion and guide neurite outgrowth. While PVA nerve conduit has been clinically approved, the applicability of PVA nerve conduits is limited to short injuries due to low cell binding. In this study, we explored the incorporation of biochemical cues and topographical cues for promoting neurite outgrowth and axon guidance.

*Methodology: PVA was conjugated with extracellular matrix proteins and a bioactive fucose-based sulfated polysaccharide fucoidan to improve cell adhesion. Micro-sized topographies, including 1.8 μm convex lenses, 2 μm gratings, and 10 μm gratings were successfully fabricated on PVA planar film and the lumen of 2mm internal diameter conduit by nanofabrication. The quality and fidelity of topographical patterns were examined with scanning electron microscopy (SEM) or laser scanning confocal microscopy. PC12 cell lines from American Type Culture Collection were cultured according to supplier’s instruction. PC12 cell were seeded onto PVA smaples at a density of 20,000 cells/cm². Cells were visualized by either using calcein AM or staining with Alexa Fluor 546 Phalloidin. Cells were imaged and analyzed using Fiji.

*Results: The synergistic effects of topography and biochemical molecules on pheochromocytoma 12 (PC12) neuritogenesis and neurite alignment were studied. Conjugated fucoidan promoted the percentage of PC12 with neurite outgrowth from 0 to 2.8% and further increased to 5% by presenting laminin on the surface. The incorporation of 2 μm gratings could double the percentage of PC12 with neurite outgrowth and neurite length, and guided the neurites to extend along the grating axis. For example, the neurite outgrowth percentage was significantly increased from 0% on blank PVA surface to 9.9 ± 4.0% on fucoidan modified PVA with laminin coating and 2 μm gratings. Additionally, fucoidan was able to bind nerve growth factor (NGF) on the surface and allow for PC12 to extend neurites in NGF-free media. Without NGF coating, PC12 cells has 0% of neurite outgrowth. PC12 cells on unpatterned fucoidan-modified PVA with NGF coating (PVA-G/NGF) showed a significantly higher 4.4 ± 0.8% neurite outgrowth, while fucoidan-modified PVA with NGF coating and 2 μm gratings (PVA-F2/NGF) induced the highest percentage of PC12 neurite outgrowth (7.0 ± 1.8%).

*Conclusion/Significance: Fucoidan could present both extracellular matrix protein, laminin, and NGF to induce and promote neurite outgrowth of PC12 cells. By combining topography with biochemical modification, we demonstrated that gratings and biochemical cues could synergistically promote neurite formation and guide neurite extension. The work presents a promising strategy to enhance neurite formation and axon guidance, presenting significant value in promoting nerve regeneration.

189 - Antimicrobial Peptide/Chlorin E6 Functionalized Gelma Hydrogels Combined With Photodynamic Therapy Show Strong Antibacterial And Antibiofilm Activity On Resistant Bacteria

E. Bilgiç, N. Çelebi, G. Pulat, N. Topaloğlu Avşar

Izmir Katip Celebi University, Izmir, Turkey

*Purpose/Objectives: Infected chronic wounds are considered one of the most serious health problem. Although pathogen-specific antibiotics are used, high concentration of antibiotics leads to systemic toxicity and antibiotic-resistance. Consequently, the development of new antimicrobial strategies is critical. Antimicrobial peptides (AMPs) are an essential part of the immune system that show broad-spectrum antimicrobial activity. Tryptophan-rich AMP, TetraF2W-RR, is effective against Gram-positive/Gram-negative bacteria in both planktonic and biofilm forms. AMPs can be combined with different antimicrobial approaches to obtain the optimum antimicrobial effect. Photodynamic therapy (PDT) is a promising method to treat infections caused by microorganisms. A second-generation photosensitizer widely used in PDT, Chlorin e6 (Ce6), has many advantages such as short photosensitization time and selective accumulation. Since antibacterial agents cannot show sufficient effect when used directly on wound due to characteristics of wound and environmental factors, hydrogels could be promising to deliver antimicrobial agents. GelMA hydrogel facilitates wound closure through its inherent bioactive properties and supportive matrix for proliferation and regeneration. Structure of GelMA can be considered an ideal platform for both AMP and Ce6 delivery for the management of infected chronic wounds. Herein, GelMA was functionalized with TetraF2W-RR and Ce6 to develop a novel wound dressing and combined with PDT to provide optimum antimicrobial activity.

*Methodology: TetraF2W-RR (WWWLRRIW) was synthesized on 4-Methylbenzhydrylamine resin. GelMA solution (10% w/v) was prepared and crosslinked with UV. To conjugate AMP and Ce6 (1mM:1mM) to the surface of GelMA, EDC/NHS chemistry was utilized. Characterization was carried out by examining FT-IR spectrum, swelling and degradation behaviors. A diode laser emitting light at 655 nm was used as the light source (output power: 500mW; fluence: 100-250 J/cm²). All antibacterial experiments were conducted with/without laser application of GelMA, GelMA/AMP and GelMA/AMP/Ce6 to obtain comparative results on the planktonic form of MRSA and MDR P. Aueriginosa with 10⁷ CFU/ml bacterial concentration. Biofilm viability and EPS was determined by MTT and safranin staining on 3-day-old biofilm, respectively. Effect of hydrogels on L929 viability was analyzed via MTT.

*Results: Succesfull conjugation of AMP/ Ce6 on GelMA was confirmed with the extention of the peaks around 1644 cm^-1 which represents C=O stretch and 3283 cm^-1 which represents N-H bending in FT-IR spectrum. GelMA/AMP and GelMA/AMP/Ce6 were more stable and degradation occurred slower than GelMA. Swelling ratio of GelMA/AMP and GelMA/AMP/Ce6 was lower compared to GelMA. Laser application did not cause significant difference in degradation and swelling. GelMA/AMP and GelMA/AMP/Ce6-250 J/cm² provided 3.45 and 7.85 log deaths, respectively, on MRSA; whereas, on MDR P. Aueriginosa, it has resulted in 1.13 and 2.14 log deaths. Biofilm viability and EPS amount of biofilm with GelMA/AMP/Ce6-250 J/cm² were reduced to 60.6% and 86% in MRSA; to 21.86% and 58.28% in MDR P. Aueriginosa, respectively. According to MTT analysis, no significant change was observed in L929 cell viability at the 24th hour.

*Conclusion/Significance: Antimicrobial activity was successfully increased by combining GelMA/AMP/Ce6 hydrogels with PDT compared to GelMA/AMP. Developed wound dressing has the potential to be an ideal wound dressing with its broad-spectrum antimicrobial activity and biocompatibility.

190 - Towards Next Generation Implants: Augmentation Of In Vitro Mineralization Via Novel Nanosecond Pulsed Laser Texturing Of Titanium Compared To Standard Sla-treatment

T. Stich¹, T. Křenek², M. Saller³, T. Kovářík², D. Docheva⁴

¹ University Regensburg Medical Centre, Regensburg, Germany,

² University of West Bohemia, Pilsen, Czech Republic,

³ Ludwig-Maximilians-University (LMU) Hospital, Munich, Germany,

⁴ Orthopaedic Hospital König-Ludwig-Haus and University of Würzburg, Würzburg, Germany

*Purpose/Objectives: Numerous hip and knee joint arthroplasties require revision due to early or late loosening of the implant. Moreover, a cementation is required for aged/osteoporotic patients, a procedure that is very adverse to the neighbouring bone tissue. Micro- and nano-features of implant surfaces greatly influence cell behaviour at the bone to implant interface and thereby, the subsequent osteointegration. The goals of this study were: i) to generate novel titanium (Ti) surfaces via shifted Laser Surface Texturing (sLST) using nanosecond pulsed laser, and ii) to compare the behaviour of bone forming cells onto the sLST-Ti versus clinically used standard roughening technique sandblasted acid-etched (SLA).

*Methodology: Ti grade 2 disks (d/h: 12mm/1.2mm) were subjected to sLST (SPI G3, beam 1064nm, pulse duration 200ns) to generate open pores (l/w: 500µm, h: 300µm) and micro-nano-texturing. SLA-Ti disks were prepared as a control. Surface roughness was examined via confocal microscopy (405 nm laser). Mesenchymal stromal cells (MSCs) from young/healthy (YH, n=6, 29 ± 6 years), aged/healthy (AH, n=5, 79±5 years) and aged/osteoporotic (OP, n=5, 76± 5 years) donors were investigated. Both disk types without and with MSCs were imaged via SEM. Cells on sLST-Ti and SLA-Ti were assessed by live/dead staining at day 1 and 5 and with Resazurin (metabolic/proliferative) assay on day 1, 3, 5 and 7. Osteogenic differentiation (21 days) and matrix mineralization was validated with Alizarin Red S (ARS) staining and quantification. Bulk RNA-seq (Eurofins, Germany) was performed at day 1 using a MSC-hTERT cell line (n=3 RNA preps/disk type) and analysed with R Studio software. Statistical testing was performed with unpaired t-test and one way ANOVA.

*Results: In contrast to the standard SLA surface, sLST-Ti disks exhibited stochastically distributed Ti droplets with different micro- and nanometer sizes that were formed by the laser-induced Ti plasma, a feature closely resembling the micro-nanotopography of bone tissue. Roughness measurements demonstrated higher peak to valley distances and 5-fold increased Ra and Rq values for sLST-Ti. SEM imaging showed that the cells adopt different morphologies, namely “parachute-like” on sLST-Ti while “kite-like” on SLA-Ti. Live/dead and Resazurin assays revealed for each of the MSC cohorts high biocompatibility as well as comparable survival rates and proliferative activity on both disk types. ARS quantification first detected significantly higher (p<0.0001) matrix mineralization by YH cells on sLST-Ti compared to SLA-Ti. Most intriguingly, sLST also significantly induced the matrix mineralization of AH as well as OP cells with 5-fold higher ARS values versus SLA-Ti (AH: p<0.0001; OP: p<0.001). To investigate the primary molecular response and to avoid the masking due to cell-produced matrix, RNA seq was carried out at day 1 and bioinformatic analysis identified a number of differentially expressed genes on sLST-Ti versus SLA-Ti; namely SULT1A4, TNFSF12-TNFSF13 and DUSP6 were upregulated.

*Conclusion/Significance: Our novel results demonstrate that the sLST-Ti surfaces are safe, highly biocompatible, can rescue patient cohort specific mineralization behaviour and thus represent a great potential for development into next-generation-implants suitable for elderly and bone compromised population.

191 - Synthetic Poly(ethylene Glycol) Hydrogel Supports Development And Hormone Production Of Dissociated, Reconstructed, And Implanted Human Ovarian Tissue

M. Wall, M. Brunette, D. Pavlidis, M. Tong, N. Hanby, A. Shikanov

University of Michigan, Ann Arbor, MI

*Purpose/Objectives: Ovarian tissue cryopreservation and transplantation (OTCT) is the sole fertility preservation option available to prepubertal girls facing gonadotoxic anticancer treatments. Existing OTCT methods rely on transplantation of large pieces of the ovarian cortex which are difficult to characterize, slow to revascularize, and may contain malignant cells. Isolation of ovarian follicles from the surrounding, potentially malignant, ovarian stroma cells before implantation would not only dramatically reduce the risk of cancer recurrence following OTCT, but also allow for characterization of each patient's follicle population and implementation of tissue engineering approaches to improve revascularization and reduce follicle death. However, primary cells survive poorly when transplanted without a support matrix. To date, isolated human follicles have been xenografted to immunocompromised mice encapsulated in several naturally derived matrices. Synthetic matrices, however, offer the advantage of being highly tunable and reproducible but, have not yet been explored for the transplantation of isolated human follicles. We have developed a proteolytically degradable poly(ethylene glycol) (PEG) hydrogel that supports the survival, growth, and hormone production of digested human ovarian tissue in a mouse model of primary ovarian insufficiency (POI) and hypothesize follicle survival and graft revascularization can be enhanced through sequestration of cell-secreted extracellular matrix (ECM) within our scaffolds.

*Methodology: Previously cryopreserved ovarian cortex from four donors 25 to 34 years old was enzymatically digested to obtain a cell suspension for encapsulation. We encapsulated this cell suspension in two different PEG formulations: one functionalized with 0.375mM of the cell adhesion ligand RGD served as a control, the other functionalized with 0.375mM each of RGD and basement membrane binding peptide (BMB) was designed to sequester cell secreted extracellular matrix. Constructs were implanted to the fat pad of ovariectomized immunocompromised mice and the function of the transplanted ovarian tissue was evaluated through daily vaginal cytology. Grafts were explanted and fixed for immunofluorescent staining to evaluate follicle development and cellular remodeling.

*Results: We first confirmed our digested tissue remained viable throughout the digestion and encapsulation process. We then evaluated the constructs prior to implantation (D0) to ensure grafts contained both ovarian follicles (essential for restoration of fertility and endocrine function) and endothelial cells (beneficial for follicle survival and revascularization). Hormone production of the implanted ovarian constructs was demonstrated by the presence of nucleated and/or cornified epithelial cells in the vaginal smears of over 75% of mice monitored for 10 or more weeks. Explanted grafts were markedly softer than D0 gels, indicating degradation and cellular remodeling of the hydrogel. Finally, fluorescent staining revealed that encapsulated ovarian endothelial cells persist within the graft following transplant.

*Conclusion/Significance: This work demonstrates for the first time that a synthetic PEG hydrogel can recapitulate the ovarian microenvironment, support the survival of digested human ovarian tissue in vivo, as well as the capacity for these constructs to restore ovarian endocrine function in a mouse model of POI. Future work will evaluate the quantity and composition sequestered ECM within our system, as well as the potential benefits of this retained ECM on follicle survival, development and graft revascularization.

192 - Scaffold-guided Breast Tissue Engineering - Preclinical Study Of 3D Printed Large Volume Medical-grade Polycaprolactone Scaffolds In A Pig Model

R. Finze^1,2, S. Saifzadeh^3,4, F. Medeiros Savi^1,4,5, U. Kneser², L. Klein¹, M. Cheng^1,6, O. Ung^7,8, M. Wagels⁷, D. W. Hutmacher^1,5,9

¹ School of Mechanical, Medical, and Process Engineering, Queensland University of Technology, Brisbane QLD, AUSTRALIA,

² Department of Hand, Plastic and Reconstructive Surgery, BG Trauma Center Ludwigshafen, Ludwigshafen, GERMANY,

³ Medical Engineering Research Facility, Queensland University of Technology, Brisbane QLD, AUSTRALIA,

⁴ ARC Industrial Transformation Training Centre for Multiscale 3D Imaging, Modelling and Manufacturing (M3D), Brisbane QLD, AUSTRALIA,

⁵ Max Planck Queensland Centre for the Materials Science of Extracellular Matrices, Brisbane QLD, AUSTRALIA,

⁶ Department of Plastic and Reconstructive Surgery, Princess Alexandra Hospital, Brisbane QLD, AUSTRALIA,

⁷ Herston Biofabrication Institute, Brisbane QLD, AUSTRALIA,

⁸ Comprehensive Breast Cancer Institute, Brisbane QLD, AUSTRALIA,

⁹ ARC Centre in Additive Biomanufacturing, Brisbane QLD, AUSTRALIA

*Purpose/Objectives: In 2022 more than 500.000 oncoplastic surgeries on breast cancer patients were performed worldwide. Breast implants were used in 87% of patients despite being associated with a variety of significant complications, such as infection, rupture, leakage, and capsular contracture. Current literature reports that 5 years after implant placement, 9.8% to 18.9% of patients require reoperation and implant replacement due to capsular contracture. In 2019, worldwide regulatory action to suspend, cancel, or recall some of these higher risk devices is supported by the increasing evidence of patients presented with breast implant-associated anaplastic large cell lymphoma. The eminent crisis for both patients and clinicians mandates the fundamental and applied research and ultimately the clinical translation of a radical innovation to address the problem of current breast implants. Hereby, we present first-time preclinical findings of large-volume soft tissue regeneration using a scaffold-guided breast tissue reconstruction (SGBTR) approach.

*Methodology: In this pilot study, eight biodegradable scaffolds made from medical-grade polycaprolactone (mPCL) were implanted into the flanks of two pigs (4 scaffolds per pig, 2 per side). Two weeks later, autologous fat graft (AFG) was harvested via liposuction from the pigs’ abdomen, and injected into the scaffolds (experimental group). In vivo longitudinal imaging via computed tomography was performed immediately, as well as 1-, 6- and 12 months post-surgery. At the 12-months endpoint, the SGBTR implants were harvested, and a comprehensive analysis was undertaken: ex vivo biomechanical testing, magnet resonance imaging, scaffold degradation analysis using gel permeation chromatography and differential scanning calorimetry, as well as histological- and immunohistochemical analyses were performed to investigate tissue quality and quantity, vascularization, and the immune response as well as in vivo degradation profile of 3D printed medical-grade polycaprolactone. Furthermore, macroscopic- and microscopic results were correlated.

*Results: There were no short-term or long-term complications associated with the implantation of the mPCL breast scaffolds. Soft tissue regeneration was observed within all scaffolds with qualitative and quantitative variations between the timepoints, but with no significant difference between the AFG-injected and non-injected group. No clinical signs of inflammation were seen at any timepoint, in alignment with the scaffold degradation profile, thus an immune response within the newly formed tissue in proximity to the scaffold material could be observed.

*Conclusion/Significance: These study results show and underline the importance of longitudinal in vivo analysis as part of any preclinical model. Moreover, based on these results, current boundaries and limitations in SGBTR could be identified for the refinement of the model and as well as the scaffold design and biodegradable polymer.

193 - Modifiable, Biomimetic Polyethylene Glycol (PEG) Hydrogel Enables Co-culture Of Early-stage Ovarian Follicles And Stromal Cells

T. T. Schissel¹, M. Wall¹, A. Shikanov²

¹ Biomedical Engineering, University of Michigan, Ann Arbor, MI,

² Biomedical Engineering, Cellular and Molecular Biology, Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI

*Purpose/Objectives: Prepubertal patients undergoing lifesaving yet gonadotoxic treatments have limited options for fertility preservation, especially those with bloodborne or metastatic cancers. Currently, the only available method is cryopreservation and auto-transplantation of ovarian tissue, which carries the risk of reintroducing malignant cells. In vitro culture of ovarian follicles can mitigate this problem; however, traditional materials like alginate prevent large-scale volumetric expansion and have limited functionality. In this report, we utilized a proteolytically degradable polyethylene glycol (PEG)-based hydrogel modified with extracellular matrix (ECM) sequestering bioactive peptides. This design created a 3D system that can be locally remodeled as a follicle expands and that promotes follicle-follicle, follicle-somatic cell, and follicle-matrix interactions.

*Methodology: Ovaries from 10-12-day old mice were enzymatically digested using Liberase to obtain primary stage follicles that were encapsulated in groups of ten. Hydrogels were formed by pre-reacting 5% wt 8-arm PEG-VS with ECM-sequestering (BMB) peptides prior to crosslinking with plasmin-degradable crosslinkers. Hydrogels were cultured in 150uL of alpha-MEM based media supplemented with fetuin, insulin, transferrin, selenium, bovine serum albumin, and follicle stimulating hormone for 12 days, with brightfield images taken and half of the media (75uL) refreshed every two days. In an attempt to translate our system for human follicle culture, we thawed and enzymatically digested eight 1mm x 10mm x 1mm strips of vitrified human ovarian tissue from a 23-year-old donor for four hours. 69 follicles were manually selected and evenly distributed for co-encapsulation with 10 million stromal cells/mL in 10uL gels. We performed the same encapsulation and culture protocols with the following adjustments: hydrogels were also pre-reacted with cell-adhesive (RGD) peptides; human serum albumin replaced bovine; and gels were cultured for 14 days. vinyl sulfone (VS); basement membrane binder peptide (BMB)

*Results: During culture, murine follicles formed multi-follicle, organoid-like aggregates that exhibited granulosa cell proliferation and antrum formation (Fig 1A). LIVE/DEAD analysis demonstrated high cell viability in human follicle culture after two weeks, and brightfield images also depicted the presence of activated primary and secondary stage follicles with a significant increase of 70.7% in diameter (p=0.0063) compared to day zero (Fig 1B). Collectively, these experiments highlight that bioactive synthetic hydrogels enable in vitro culture of mammalian ovarian follicles to study early-stage folliculogenesis. Future experiments will include hormone analysis to benchmark follicle development and immunofluorescent staining for theca and endothelial cell markers.

*Conclusion/Significance: Co-encapsulation of multiple follicles and support cells in a biomimetic hydrogel promoted murine and human folliculogenesis in vitro. Further development of this platform has the potential to move human follicle culture towards a more translatable method for fertility preservation and improved patient quality of life.

194 - Ovx-induced Bone Loss At Different Skeletal Sites In A Preclinical Model Of Osteoporosis

M. Uberti, C. Intini, A. Matheson, O. D. Kennedy

Department of Anatomy, Royal College of Surgeons in Ireland, Dublin, IRELAND

*Purpose/Objectives: Osteoporosis is a disease that affects more than 200 million women worldwide. It is characterised by a systemic impairment of bone mass and quality that results in fragility fractures. The ovariectomized (OVX) rodent model, is a well established experimental model for investigation of postmenopausal osteoporosis due to estrogen deficiency in women. The OVX mouse is an ideal candidate for studying of pathogenesis of postmenopausal osteoporosis, while the OVX rat model is largely used for the evaluation and development of new drug compounds for postmenopausal osteoporosis treatments. However, it remains unknown whether bone loss occurs in the same way at different skeletal sites in these models. Therefore, the aim of this study was to assess bone changes, in particular trabecular microarchitecture by microCT and histology at tibial, vertebral and patellar site in a rodent model of osteoporosis.

*Methodology: Female Sprague Dawley OVX rats (n=8) 26 weeks old were used for this study and had OVX surgery at 11-12 weeks old. Control rats (n=8) 26 weeks old were also used. Samples from different skeletal sites including lumbar vertebra, proximal tibia, and patella were harvested, then scanned using microCT. The scanning parameters that were used were as follows: - Energy/intensity: 70kVp, 114 µA, 8 W - Calibration: 7:70 kVp, BH: 1200mg HA/ccm. Imaging software CTAn version 1.16 was used to measure the bone parameters following OVX.

Histology Samples were decalcified, embedded in paraffin and sectioned to 10µm thickness, deparaffinised and then stained using Haematoxylin and Eosin (H<E) to differentially stain cell nuclei, extracellular matrix and bone. Masson's Trichome staining was also used to highlight purple cell collagen and bone tissue. Also, the Tartrate-resistant acid phosphatase (TRAP) staining was used to better investigate the cellular population of osteoclasts.

*Results: Overall, the results demonstrated that all OVX samples exhibited significantly lower mineralized bone content compared to controls. OVX tibia, vertebra, patella showed decreased BV/TV [%] (3.81, 28.86, 29.93), Tb.th [um] (87.73, 110.22, 107.77), Tb.N [1/mm] (0.44, 2.64, 2.78), and Tb.sp [um](1363.84, 321.49, 254.64) in comparison to their respective healthy counterparts tibia, vertebra, patella BV/TV [%] (21.41, 37.65, 36.99), Tb.th [um] (71.78, 117.39, 113.75), Tb.N [1/mm] (2.08, 3.21, 3.25), and Tb.sp [um] (396.88, 280.73, 231.47). Interestingly, OVX from tibia was demonstrated to possess significantly lower values of BV/TV compared to vertebra and patella OVX. Moreover, Tb.N measured from OVX samples exhibited a significant decrease compared to healthy samples. On the other hand, Tb.sp showed an increase in OVX samples only. Although the difference in BV/TV between OVX and the healthy patella is 7% (and in tibia is 17.60%), these observations align with the trends seen in other skeletal sites. Furthermore, histological assessment confirmed the trends observed in BV/TV analysis.

*Conclusion/Significance: This study underscores the unique impact of osteoporosis on trabecular bone microarchitecture from various skeletal locations in rat models. The findings from our analysis, which were validated by histological evaluation, indicate that bone loss is most pronounced in tibia-isolated samples, but the patella bone is also affected.

195 - Naturally-Derived Electrospun Scaffolds As An Alternative To Surgical Mesh In Women's Health Applications

R. Thomson¹, C. Basham¹, J. Shaw², K. Hixon¹, D. Van Citters¹

¹ Engineering, Dartmouth College, Hanover, NH,

² Obstetrics and Gynecology, Trinity Health, Grand Rapids, MI

*Purpose/Objectives: Over 25% of adult women experience pelvic floor disorders, including stress urinary incontinence (SUI) which can greatly affect their quality of life. The most common surgical intervention for SUI is implantation of a mid-urethral sling composed of synthetic, polypropylene mesh. Of the 300,000 patients who receive midurethral sling surgeries in the US every year, 2-3% experience mesh erosion and up to 20% remain incontinent. These patients may require revision surgeries where more synthetic mesh is placed, compounding the problem. Mesh erosion and exposure can cause an increased risk of infection, bleeding, and chronic pain. The objective of this study is to characterize composite electrospun scaffolds for use as an alternative to synthetic mesh for pelvic floor reconstructive surgery. We hypothesize that our naturally-derived scaffolds will promote adhesion and proliferation of fibroblasts, based on the material composition, while promoting the aligned extracellular matrix necessary to support the urethra in vivo.

*Methodology: All scaffolds were fabricated using an electrospinner (Nanoscience Instruments®) to create a nanofibrous mesh out of naturally-derived polymers: silk fibroin (SF) and polyhydroxybutyrate (PHB). A high voltage is applied to the polymer solution to polarize the liquid, and the solvent evaporates as the thin polymer fiber is drawn toward the collecting drum by electrostatic forces. We combined these polymers at 3 and 5% wt/v with the ratios of PHB/SF: 100/0, 75/25, 50/50, 25/75, and 0/100. Outcome measures include imaging, tensile testing, and in vitro culture. Imaging samples were cut into 1cm² sections and sputter coated for scanning electron microscopy (SEM). Tensile samples were cut into 4x2.5cm sections along the length of the scaffolds and tested at a rate of 10mm/min on an Instron 68SC. In vitro samples were cut into 2cm² pieces, seeded with donor human vaginal fibroblasts (hVFs), and cultured for 7 and 14 days. Cultured samples were then imaged in the SEM and stained with immunofluorescence markers for confocal microscopy.

*Results: Our results show increased mechanical properties in scaffolds with higher amounts of PHB (Fig 1a). There were no differences between tensile properties of scaffolds before and after cell culture. Fibroblast attachment and proliferation on SF (Fig 1c) was increased compared to PHB (Fig 1b) after 7 days of culture. However, it should be noted that no cellular toxicity was observed with any of the scaffolds. Confocal imaging showed alignment of ECM, specifically nuclei (blue) within mechanically aligned collagen fibers (red staining), indicating growth of fibrous tissue to resist the in vivo forces that will be applied to the scaffold (Fig 1d-f).

*Conclusion/Significance: Maintaining high mechanical properties to support the urethra is important during fibrous tissue generation. We were able to characterize the mechanical and cellular properties of the composite nanofibrous scaffold with promising results for the generation of aligned fibrous tissue to support the urethra. This work demonstrates the tradeoffs between strength and hVF-specific biocompatibility associated with PHB/SF ratios in a novel electrospun scaffold. Future work will investigate coaxial configuration of the materials with an SF exterior and PHB core to optimize cellular attachment and proliferation.

196 - In Vivo Performance Of Angiogenic/antibacterial Tissue Interposition Flap Substitute For Use In Vesicovaginal Fistulae Repair

H. Serincay¹, N. Mangır², N. Zeybek³, O. Bozdemir⁴, S. Ozturk⁵, C. Ozkul⁶, M. Ayva², I. Eroglu⁵, H. Eroglu⁷, K. Ulubayram⁵

¹ Department of Bioengineering, Hacettepe University, Ankara, TURKEY,

² Department of Urology, Hacettepe University, Ankara, TURKEY,

³ Department of Histology and Embryology, Hacettepe University, Ankara, TURKEY,

⁴ Department of Stem Cell Sciences, Hacettepe University, Ankara, TURKEY,

⁵ Department of Basic Pharmaceutical Sciences, Hacettepe University, Ankara, TURKEY,

⁶ Department of Pharmaceutical Microbiology, Hacettepe University, Ankara, TURKEY,

⁷ Department of Pharmaceutical Technology, Hacettepe University, Ankara, TURKEY

*Purpose/Objectives: Vesicovaginal fistulae (VVF) is an abnormal opening between the bladder and the vagina that causes continuous urinary leakage. Surgical repair of VVF often necessitates the use of a tissue interposition flap (TIF) to promote healing and prevent recurrences. So far, autologous TIFs have been used however they are not always available for use, harvesting of autologous tissues are associated with comorbidities at the donor site and also autologous tissues may have limited capacity to promote tissue regeneration. We designed a novel TIF with pro-angiogenic and antibacterial properties to improve the outcomes of VVF repair surgeries.

*Methodology: The blend of silk fibroin (Fib) and polycaprolactone (PCL) was electrospun into two layers where estradiol (E2) was added to the first layer of the fibers to promote angiogenesis and silver nitrate (AgNO₃) was added to the second layer to prevent potential infections around the bladder and vagina. Morphological characterizations, thermogravimetric and structural analysis of fibers were conducted by SEM, TGA and FTIR, respectively. The amount of E2 and AgNO₃ released from the fibers was determined by the in vitro release tests. The cytotoxicity was determined by MTT and angiogenic properties of TIF was evaluated via the ex ovo chorioallantoic membrane (CAM) assay. The disc diffusion test was performed by using E. coli, S. aureus, L. acidophilus and C. albicans strains to investigate antibacterial effects of TIFs. Finally, constructed TIFs were implanted in the vesico-vaginal space (mimicking the actual clinical application) of the female rats (n=8) for 28 days after which tissue samples were collected and examined macroscopically and histologically to investigate tissue response.

*Results: Fib/PCL fibers exhibiting a homogeneous structure without bead formation and it was verified through SEM analysis. The FT-IR spectra exhibited characteristic peaks consistent with silk fibroin and PCL. TGA thermograms exhibited a characteristic behavior of silk fibroin and PCL containing fibers. After three weeks, it was measured that a total of 0.021 mg E2 (17%) and 3.19 ppm (81.8%) Ag was released from E2Fib/PCL-Ag fibers. The non-cytotoxic fibers, incorporating Ag (E2Fib/PCL-Ag), demonstrated antibacterial effect against all tested strains but, among the tested strains, they were more detrimental to L. acidophilus (p <0.0001). CAM assay showed an increased vascular density in the E2Fib/PCL and E2Fib/PCL-Ag groups by 36% and 10%, respectively compared to the control (p<0.05). Gross examination of the vesicovaginal complexes containing the TIFs did not show any material complications. Histologic analysis revealed that adding E2 and AgNO₃ into fibers significantly decreased infiltrated PMN amount (p<0.05) and connective tissue organization was similar in Fib/PCL and E2Fib/PCL-Ag groups (p>0.05). The incorporation of E2 to the fibers clearly increased blood vessel formation at in vivo conditions (p<0.05).

*Conclusion/Significance: A novel bilayered mesh exhibiting angiogenic and antibacterial properties have been designed and tested in vivo. This translational approach where pre-defined clinical needs constitute the core design parameters has the potential to lead to real life clinical benefits for patients undergoing surgery for VVF repair. Further tests in appropriate in vivo model are necessary to complete pre- clinical testing of these materials.

197 - Engineered Extracellular Vesicles For Targeting And Activation Of Lymphatic Vegfr-3

J. Oesterreicher^1,2,3, L. Narendja^1,2,3, M. R. Bobbili^1,2,3, M. Jeltsch^4,5,6, J. Grillari^1,2,3, D. Hercher^1,3, W. Holnthoner^1,3

¹ LBG, Ludwig Boltzmann Institute for Traumatology, The Research Centre in Cooperation with AUVA, Vienna, Austria,

² University of Natural Resources and Life Sciences, Institute of Molecular Biotechnology, Vienna, Austria,

³ Austrian Cluster for Tissue Regeneration, Vienna, Austria,

⁴ Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland,

⁵ Individualized Drug Therapy Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland,

⁶ Wihuri Research Institute, Helsinki, Finland

*Purpose/Objectives: The lymphatic vasculature has not received overwhelming scientific attention for many years, although it is fundamental for the development and homeostasis of higher organisms. To re-establish true tissue homeostasis after traumatic injuries and disease, therapeutic strategies need to consider the regeneration of the lymphatic system. Vascular endothelial growth factor C (VEGF-C) is a key mediator of lymphangiogenesis upon binding and activation with the lymph-specific vascular endothelial growth receptor 3 (VEGFR-3). It has mainly been investigated in settings such as lymphedema and cancer. Nevertheless, most tested applications of this ligand did not consider its highly complex biosynthesis process. Proteolytic processing gives rise to different variants showing varying levels of receptor specificity and affinity which influences all of its applications.

*Methodology: To enhance the pro-lymphatic effects of VEGF-C and its systemic availability, the possibility of linking it to proteins such as tetraspanins (e.g. CD9, 63, 81), which are enriched in extracellular vesicles (EVs), has been proposed recently. In this project, we aimed to establish telomerase immortalized adipose-derived stroma cell (ASC) lines which express different variants of VEGF-C genetically fused to CD81, thus enriching VEGF-C on the EV membrane, facilitating a bimodal function of receptor activation and tissue-specific homing of ASC-derived EVs.

*Results: Expression and subcellular localization of the fusion protein were confirmed by western blotting and immunofluorescence microscopy. Using a BaF3 cell line expressing a VEGFR3/EpoR chimeric protein, we showed that different variants of CD81-VEGFC show significant differences in their VEGFR3 activation. Pro-lymphangiogenic function was confirmed via network formation and 3D spheroid assays using primary lymphatic endothelial cells.

*Conclusion/Significance: In this study, we provide proof of principle that we can generate different ligand variants of fusion proteins on EV surfaces, test the bioactivity of engineered EVs in subsequent receptor activation and angiogenesis assays, and thus pave the way for testing their therapeutic potential for enhancing lymphangiogenesis in various in vivo models of traumatic injuries as a next step.

198 - Extracellular Vesicles Derived From Induced Msc (imsc) And Chondrocytes (icho): Evaluation Of Anti-inflammatory Effects Using An In Vitro Osteoarthritis Model

M. Balbi¹, M. Rovere¹, E. Quarto², M. Formica², S. Pontara¹, G. Shaw³, S. Ravera¹, S. Coco², V. Rotter Sopasakis⁴, K. Vukusic⁴, A. Lindah⁴, M. Mastrogiacomo¹, M. Murphy³, C. Gentili¹

¹ University of Genoa, Genova, Italy,

² IRCCS Ospedale Policlinico San Martino, Genova, Italy,

³ National University of Ireland Galway, Galway, Ireland,

⁴ University of Gothenburg, Gothenburg, Sweden

*Purpose/Objectives: Osteoarthritis (OA) is a complex, multi-factorial chronic joint disease with few treatment options with the pathogenesis still uncertain. Currently, new therapeutic approaches are being studied with mesenchymal stromal cells (MSC) and derived extracellular vesicles (EVs) considered promising therapeutic approaches. EVs, containing proteins and different genetic information including microRNA, are involved in cell-to-cell communication and enter target cells to deliver this information.Since, the use of adult MSC has some limitations like heterogeneity linked to the different sources of origin and donors, we aimed to generate MSC and chondrocyte cells differentiated from induced pluripotent stem cells (iMSC and iCHO), which could reduce this heterogeneity and allow a longer cell expansion. The objectives of the study were to characterize iMSC- and iCHO-derived EVs, validate the biological effects of these vesicles on an in vitro OA model with particular attention to their anti-inflammatory properties, their potential to influence the redox state and their miRNA content.

*Methodology: Three different iMSC and iCHO lines were differentiated from a commercial iPSC line and maintained in culture in a serum-free medium through extensive passaging (P5 to P16). Cells were evaluated during the culture period based on morphology, proliferation, and differentiation. EVs were isolated from iMSC and iCHO conditioned medium at each culture passage following a differential ultracentrifugation protocol with iEVs characterized through quantification of specific EVs proteins, nanoparticle tracking analysis (NTA), western blot, non-conventional flow cytometry and the miRNA content was assessed (Agilent technologies).The in vitro OA model consisted of primary culture of human articular chondrocytes, primed with IL-1α to mimic the chronic inflammation of the disease. The OA model was exposed to EVs for 24h and anti-inflammatory effects were investigated using Nanostring technologies. ROS damage (malondialdehyde, 4-hydroxynonenal, nitro-tyrosine, 8-hydroxy-2’-deoxyguanosine) and redox balance (GSH, GSSG, glutathione reductase and catalase activity) were studied by luminometric and enzymatic assays.

*Results: iMSC maintain the classical mesenchymal phenotype and differentiation potential until P16. The differentiation of human iPSCs towards iCHO using specific growth factors enabled through an epithelial-to-mesenchymal transition (EMT) followed by chondrogenic differentiation.During the progressive culture passages EV secretion was increased and the expression of the classical markers such as CD63, CD81, Flotillin-1 and Synthenin-1 was detected. EVs isolated from different iMSC lines demonstrated an anti-inflammatory effect on the in vitro OA model with COX-2, IL-6 and IL-8 expression analyzed by qRT-PCR. Data on ROS damage and redox balance demonstrated that iMSC-derived EV treatment can significantly reduce ROS damage marker levels in chondrocytes primed with IL-1α, through reactivation of an antioxidant response.Similar studies are underway to validate the biological activity of iCHO-derived EV.

*Conclusion/Significance: Isolation of EVs from iMSCs and iCHOs could represent an alternative therapeutic strategy compared to EVs isolated from bone marrow derived MSCs or terminally differentiated chondrocytes, possessing the advantage of maintaining their potential in culture for a longer period. Treatment with EVs derived from iPSCs could represent an innovative approach in the treatment of osteoarthritis. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 874671.

199 - Stress-Induced Premature Senescence And Senolytic Intervention For Therapeutic Implications In The Adipose Stromal Vascular Niche

M. Wahlmueller¹, M. S. Narzt¹, K. Missfeldt¹, I. Lämmermann², B. Schaedl¹, M. Mairhofer³, N. Hashemi¹, S. Doettl⁴, A. Rohwedder⁵, S. Suessner⁶, E. Priglinger⁴, S. Wolbank¹

¹ Ludwig Boltzmann Institute for Traumatology, Vienna, Austria,

² Rockfish Bio AG, Vienna, Austria,

³ Johannes Kepler University, Medical Faculty, Department of Hematology and Internal Oncology, Linz, Austria,

⁴ Johannes Kepler University, Medical Faculty, Department for Orthopedics and Traumatology, Linz, Austria,

⁵ Johannes Kepler University, Medical Faculty, Linz, Austria,

⁶ Austrian Red Cross Blood Transfusion Service for Upper Austria, Linz, Austria

*Purpose/Objectives: Adipose tissue has emerged as important source of therapeutic cells, since it is abundantly available and easily accessible. In an autologous setting, the derived Stromal Vascular Fraction (SVF) has established as popular treatment option for several conditions including osteoarticular, neurological and cardiovascular diseases. The therapeutic efficacy of autologous cell therapies might be impacted by the presence of senescent cells especially in elder or diseased patients. Multiple cell types are affected by the senescent fate, and involved in the generation of a pro-inflammatory environment, the establishment of chronic diseases, and progression of age-related metabolic dysfunction. Targeting and eliminating senescent cells with senolytic compounds represents a key novel strategy to enhance the efficacy of autologous cell therapies and to ameliorate pathological states.

*Methodology: We have investigated the impact of senolytic treatment on the senescence status and therapeutic potential of adipose derived SVF. We generated stress-induced premature senescence (SIPS) in vitro models using Doxorubicine, representing the human adipose stromal vascular niche, including 3D microtissue-stromal vascular fraction (MT-SVF), where cells are embedded within their native extracellular matrix. We characterized the models by multiple hallmarks of senescence on different cellular levels (morphology, cell cycle, senescence-associated β-galactosidase (SA-βgal) activity, mRNA and histology). We determined the abundance of senescent cells in adipose tissue and MT-SVF, with respect to aged and lipedema patients. To eliminate senescent cells, we applied inhibitors of arachidonic acid converters (RFB01016, MK-886, Diclofenac), for which we optimized the senolytic treatment in the respective models. Senolytic activity was determined by histological analysis of apoptosis and senescence markers and by live fluorescence apoptosis monitoring using caspase staining. The therapeutic effect of MT-SVF was evaluated by characterizing the pro/anti-inflammatory secretome and in an osteoarthritis (OA) in vitro model based on human cartilage explants.

*Results: SIPS in vitro models were most efficiently generated using two subsequent exposures with 200 nM Doxorubicine for six days. Senescence was confirmed in 2D through SA-βgal activity, at mRNA level (LMNB1, CDK1, p21), G2/M cell cycle arrest and significant differences in Lamin B1 and p21 protein expression in the MT-SVF 3D-model. We have demonstrated that human adipose tissue and the derived MT-SVF are afflicted by a senescence cell burden in a donor-dependent manner. Senolytic treatment of SIPS ASC and endogenous MT-SVF induced apoptosis measured by caspase activity in senescent cells, and concomitant decrease in p16 positive cells, which was confirmed by Lamin B1 and p21. Secretome analysis of the pro/anti-inflammatory balance and the OA in vitro assay showed the proof of principle suitability of senolytic treatment in this setting.

*Conclusion/Significance: As multiple cell types cause heterogeneity and complexity in adipose tissue senescence, our established models representing the perivascular niche are highly relevant for studies of adipose tissue pathologies linked to senescence and facilitate analysis of the endogenous senescent state. In addition, since adipose tissue is an attractive source of regenerative cells, reducing the senescent cell burden, which is commonly observed in obesity and aging might improve the outcome of future autologous cell therapies for patients with an insufficient endogenous cell material.

200 - Enhancing Targeted Regeneration Of Peripheral Nerves Via Laminin-binding Extracellular Vesicles Derived From Adipose Tissue-derived Stem Cells

M. Q. Nguyen^1,2,3, J. Oesterreicher^1,2,4, M. R. Bobbili^2,4,5, J. Jacak^2,6,7, R. Grillari^4,8, S. Couillard-Després⁹, J. Grillari^2,4,7, D. Hercher^1,2

¹ Neuroregeneration, Ludwig Boltzmann Institute for traumatology, Vienna, AUSTRIA,

² Austrian cluster for Tissue Regeneration, Vienna, AUSTRIA,

³ Paracelsus Medical University, Salzburg, AUSTRIA,

⁴ University of Natural Resources and Life Sciences Vienna, Vienna, AUSTRIA,

⁵ Ageing, Ludwig Boltzmann Institute for traumatology, Vienna, AUSTRIA,

⁶ Center of Excellence Medizintechnik/TIMed Center, Research Center Linz, Linz, AUSTRIA,

⁷ Ludwig Boltzmann Institute for traumatology, Vienna, AUSTRIA,

⁸ Evercyte GmbH, Vienna, AUSTRIA,

⁹ Institute of Experimental Neuroregeneration, Paracelsus Medical University, Vienna, AUSTRIA

*Purpose/Objectives: Peripheral nerve injuries result in impaired sensory and motor function, with current regeneration methods often providing suboptimal recovery. Schwann cells (SCs) play a crucial role in facilitating the regeneration process, by providing guidance cues for regrowing axons via their basement membrane - primarily consisting of laminin. Extracellular Vesicles (EVs) are vital for intercellular communication and the transfer of biological information. Recently, adipose derived stem cell- EVs (ASC-EVs) have emerged as a promising drug delivery system. However, the precise targeting of EVs to the desired location upon intravenous administration remains a significant challenge. To overcome this issue, overexpressed EV surface marker protein CD81, from the tetraspanin protein family, has been biologically engineered toward preferential binding of laminin subsequently allowing preferential homing to the target site. This study focuses on producing laminin-binding EVs in ASCs to enable specific targeting to injured peripheral nerve sites.

*Methodology: To allow evaluation of uptake and biodistribution, we primarily cloned specific CD81-LEL sequences into lentiviral vectors encoding the expression cassette for full-length CD81 proteins fused with eGFP or Akaluciferase under the control of human cytomegalovirus (CMV) promoter. Stable cell lines were obtained upon transformation of adipose derived Stem cells (ASCs) and selected by sorting for high expressers. ASC-EVs were further enriched by using tangential flow filtration (TFF) and characterized by nanoparticle tracking analysis and flow cytometry. To assess their binding affinity to laminin, we coated thin glass plates with either bovine serum albumin (BSA) to detect nonspecific binding or human laminin and subsequently allowed the EVs to adhere to the coated glass plates. Images were taken directly after addition of EVs at 30-second intervals over a 15-minute time range via a high-resolution fluorescence microscope (FM). The experiment was repeated on rat laminin coated glass plates to confirm consistent behaviour of the EVs on both human and rat laminin substrates. A similar method was used to visualize the uptake of the EVs by primary SCs. Furthermore, qRT-PCR was performed to detect the pro-regenerative capacity of the laminin-binding EVs on primary SCs.

*Results: The characterization of ASC-EVs revealed a size distribution ranging from 100-180 nm and a substantial positivity for GFP signals of up to 75%. Performed fluorescence imaging showed an enhanced binding affinity of the produced engineered ASC-EVs on both human and rat laminin. Additionally, the uptake of laminin-binding EVs by SCs was detectable within a time span of up to three hours and subsequent qRT-PCR analysis revealed increased expression of the pro-regenerative genes p75, BDNF and C-Fos.

*Conclusion/Significance: The results demonstrate that targeted delivery following biological engineered EV membrane proteins is achievable in vitro. Our next steps involve their translation into an in vivo median nerve defect model to investigate its impact on peripheral nerve regeneration processes. This study represents the basis for both EV-based therapies and their potential homing capabilities, not only applicable to peripheral nerve injuries but also in other medical fields where local administration is challenging, and targeted delivery after application offers a non-invasive therapeutic option.

202 - Using 3d Tumoroids At The Air-liquid Interface To Find A New Tool For Monitoring Treatment Outcomes In Non-small-cell Lung Cancer Patients

D. Movia¹, S. Mullen²

¹ Maynooth University, Maynooth, Ireland,

² Trinity College Dublin, Dublin, Ireland

*Purpose/Objectives: Lung cancer is classified as a priority disease by the World Health Organisation. Non-small-cell lung cancer (NSCLC), the most common type of lung cancer accounting for 80-85% of all lung cancer diagnoses, is a devastating disease and the leading cause of cancer-related mortality in Europe and worldwide. The average five-year survival rate (17.8%) is much lower than other leading cancers. Worryingly, 70% of patients show stable or progressive disease even after targeted therapy, due to acquisition of drug resistance. Our ongoing study aims to determine how to tackle the current issue of acquired resistance that inevitably develops after a first response to targeted therapies in NSCLC patients. In detail, our study investigates whether resistance acquisition translates into changes in the surface biomarkers expressed on extracellular vesicles (EVs), nanosized particles that are naturally released from cancer cells. Here, we will present our most recent experimental findings.

*Methodology: To date, due to the limited availability of patient samples, intrinsic inter-species differences between human and animal EV biology, and the complex nature of EV interactions in vivo, where several events occur simultaneously, the use of conventional preclinical and clinical models has hindered reaching conclusive results on EVs’ potential as a diagnostic tool. To overcome this, our study uses a proprietary in vitro new approach methodology (NAM) as the test model. In detail, a three-dimensional multi-layered cell culture (MCC) of human NSCLC cells grown, alone or in co-culture with healthy human lung fibroblasts, in Air-Liquid Interface (ALI) conditions, is employed (Figure 1). Our data show that the tumoroids formed reproduce cancer cell-fibroblast crosstalk, multidrug resistance mechanisms, and feedback activation loops seen in NSCLC patients. The tumoroids allow for sustained culture (> 14-day viability) and long-term (> 2 weeks) experiments. Also, they reflect the apicobasal polarity of the human alveoli, where NSCLC most often originates.

*Results: The results of our study clearly demonstrate the presence of EV-associated surface epitopes in samples harvested at different time-points from the apical side of the tumoroids. Furthermore, cancer-specific markers can also be detected in the EV-containing samples. The latter are harvested from the tumoroids by using an in vitro technique we developed to mimic the bronchoalveolar lavage (BAL) procedure used in the clinic. Surface epitopes and cancer-specific biomarkers are characterised by means of the state-of-the-art MACSPlex Exosome Kit and the Proteome Profiler™ Human XL Oncology Array Kit, respectively.

*Conclusion/Significance: Our preliminary results clearly support the hypothesis that surface biomarkers of EVs isolated from the BAL fluid of NSCLC patients, could be used as a tool for monitoring treatment outcome in the future. Our study pushes the frontiers of the cancer field, by addressing a clinical challenge through the use of NAMs - The pursuit of fundamental biological understanding of how to use EVs to monitor in real-time the development of acquired resistance to targeted therapies in NSCLC patients, something that currently cannot be achieved. The ultimate ambition is to make sure that targeted drugs can translate in lasting and transformative treatment solutions against this disease in the future.

204 - Engineering 3-dimensional Assembloid Platforms To Investigate Fibroblast-cancer Cell Crosstalk And Its Role In Colorectal Cancer Metastasis

E. Sim, A.-M. Finger, M. Gbenedio, J. Roose

University of California, San Francisco, San Francisco, CA

*Purpose/Objectives: Metastatic colorectal cancer (CRC) remains a lethal malignancy, with a 5-year survival rate of only 14%. Historically, mouse models and monoclonal cancer cell lines have been the driving force behind cancer research. Yet, these preclinical models have limited human physiological relevance or do not adequately represent the tumor microenvironment (TME). Classical (epithelial cell) organoids are 3-dimensional (3D) in vitro cultures — generated from patient samples, expanded, characterized, and cryobanked — that better preserve native tissue morphology and microarchitecture. However, classical organoids still lack crucial TME elements (e.g., fibroblasts, immune cells, or other non-malignant cells), and to understand metastasis, the field requires novel, patient-centric platforms that incorporates the TME. Here, we investigate the generation of human CRC ‘assembloids’ — malleable, 3D in vitro culture systems that capture cancer-TME interactions by combining human classical organoids, fibroblasts, and extracellular matrix (ECM) units in droplet-based suspension cultures. We anticipate this system will improve the field’s understanding of fibroblast-cancer cell crosstalk, its role in the progression of mCRC, and provide a novel human platform to eventually improve clinical outcome.

*Methodology: To establish assembloids, we first generated Matrigel-supported CRC- and colon- organoids via our clinical sample pipeline. Using scRNAseq- and spectral flow-based characterization, we demonstrated sustained stem cell function of epithelial lineages in organoids that remain stable over passages and can be indefinitely propagated. Secondly, patient-derived, tissue-matched, primary fibroblasts are combined with these organoids at an approximate 1:5000 ratio, suspended in Matrigel, and assembled into 4 ul droplets. Droplets are polymerized at 37 degrees Celsius, transferred to full 3D organoid medium, and cultured under rotational force. Assembloids are optimized using dye- and fluorescent imaging-based approaches, and additional co-culture technologies and platforms are explored to promote scaling up and standardization. Assembloid characterization will include immunofluorescence targeting epithelial and stem cell markers indicative of self-organization; spectral flow cytometry with scRNAseq and computational analysis to identify markers implicated in disease-driving cellular networks; and subsequent comparison of 3D assembloids with 2D co-culture platforms.

*Results: The assembloid generation pipeline has produced preliminary live-cell images that show colon organoids embedded in a dense fibroblast network, cultured for 5 days in full 3D organoid medium (Figure 1). Fluorescent dyes were used to visualize approximate organoid and fibroblast localization within the assembloid. Paraffin sectioning and immunofluorescence will be used to evaluate assembloid microarchitecture and organoid self-organization. Spectral flow cytometry and scRNAseq will be used to obtain single-cell resolution insights into cancer cell-TME interactions.

*Conclusion/Significance: We anticipate that our assembloid platform will elucidate fibroblast-cancer cell crosstalk and its role in the progression of mCRC. By providing more mechanistic insights into specific fibroblast-ECM units and formulating hypotheses on receptor-receptor and ligand-receptor interactions, we hope to identify meaningful biomarkers and cellular networks implicated in mCRC progression. Ultimately, we anticipate that our assembloid system will result in a novel human platform to test experimental cancer therapies and drive new innovations in translational cancer research.

206 - Advanced 3D Model Of The Neurovascular Unit And Blood-brain Barrier Using Ex Vivo Human Brain Tissue To Model Neurovascular Pathologies

B. O’Grady¹, M. Schrag², E. Lippmann¹

¹ Vanderbilt University, Nashville, TN,

² Vanderbilt University Medical Center, Nashville, TN

*Purpose/Objectives: The human brain has complex physiology that poses a substantial challenge for developing anatomically and physiologically relevant models of human brain pathologies in animals and in vitro. Consequently, animal models used to screen potential therapeutics in vivo are not able to provide essential information regarding the interactions between the therapeutic and the human-specific physiology. This knowledge gap contributes to widespread clinical trial failures. These failures and the lack of in vitro platforms that accurately represent the human in vivo niche highlight an urgent need for technologies mimicking complex brain anatomy, physiology, and functions to study mechanistic interactions in drug discovery. To address these limitations, we have developed a perfusable, fully vascularized and anatomically correct 3D model of the NVU in a perfusable microfluidic chip using ex vivo human tissue embedded in a biomimetic hydrogel containing the N-Cadherin adhesion motif. We verified the structural hierarchy of all cells required to create a perfusable vascular network.

*Methodology: Microfluidic devices were fabricated using Parylene C coated stereolithographic 3D printed mold. Fresh human brain tissue was collected and minced in a 1mL conical tube filled with complete media for 5 minutes, and centrifuged at 2500 rpm. Then, 100µL of minced ex vivo tissue was transferred to 1mL of 10% hydrogel and microbial transglutaminase was added to the hydrogel for crosslinking, mixed and injected into the microfluidic device. The hydrogel was cured for 30 minutes at 37°C. Vascular growth in the hydrogels was imaged with confocal immunofluorescence 7 days following tissue embedding.

*Results: We established a neurovascular in vitro model using human brain. The newly formed arterioles and capillaries formed a patent lumen with correct concentric spatial organization. Additionally, newly formed vasculature anastomosed with the hydrogel wall to form a perfusable vascular network. We established a biomimetic hydrogel that supports iPSC-derived glutamatergic neurons, glial and mural cells in 3D. Additionally, our biomimetic hydrogel promotes arteriogenesis of ex vivo human brain tissue (Figure 1), and we established a neurovascular in vitro model with the human brain tissue. The newly formed arterioles and capillaries formed a patent lumen with correct concentric spatial organization. Highlighting a key feature of this novel ex vivo model, the newly grown capillary network demonstrated a robust and impermeable BBB integrity. Diffusion and permeability coefficients were quantified, revealing barrier properties two orders of magnitude greater than models using immortalized cells.

*Conclusion/Significance: We developed a neurovascular unit containing endothelial cells, pericytes and smooth muscle cells. Using ex vivo human brain tissue in our biomimetic hydrogel induces continuous vascular growth with anatomically correct hierarchy and expression of characteristic markers of vascular cell phenotypes. Finally, we demonstrated lumen-perfusion of this newly formed vascular network using fluorescent microbeads and demonstrated this ex vivo model develops a robust blood-brain barrier. We believe this platform represents a realistic biomimetic microenvironment that will allow for unprecedented access to not only gain a deeper understanding of disease progression and pathology, but gain the ability to spatially and temporally observe the interactions of multiple cell types involved in neuropathologies.

209 - Characterization Of Micro-vascularization And Hepatic Function In FRESH 3D Bioprinted Liver Lobule-mimicking Scaffolds

A. Asghari Adib¹, M. Cruz¹, B. D. Coffin², S. P. Moss¹, J. M. Bliley¹, A. W. Feinberg¹

¹ Carnegie Mellon University, Pittsburgh, PA,

² University of Pittsburgh, Pittsburgh, PA

*Purpose/Objectives: Chronic liver disease affects over 1.5 billion individuals worldwide. While liver transplantation is the most effective treatment, its potential is constrained by the limited supply of donor organs and the need for chronic immunosuppression. Tissue engineering (TE) has emerged as an alternative therapy promising to eliminate the need for donor supply. However, current TE liver constructs are far from transplantation grade. One reason is that the TE constructs fail to recapitulate the multi-scale structure, multi-cellular composition, and vascular complexity of native tissue. Recently, 3D bioprinting approaches such as freeform reversible embedding of suspended hydrogels (FRESH) have emerged, enabling scaffold fabrication with <100 µm resolution. Here, we utilize FRESH bioprinting to (i) fabricate liver lobule-mimicking constructs and characterize their functionality, and (ii) investigate the in vitro self-assembly of endothelial cells into micro-vasculature in the bioprinted multi-cellular scaffolds.

*Methodology: Square lattice scaffolds were FRESH bioprinted in thrombin-doped support with a cell-laden bioink consisting of 72 mg/mL fibrinogen (F) combined with 1% (w/v) xanthan gum (X). The cells used were HepG2 cell line to model hepatocytes together with human umbilical vein endothelial cells (ECs) and mesenchymal stem cells (MSCs) as pericyte-like cells. The bioink had HepG2 (H) cells (FX-H bioink) for functionality testing, and a combination of HepG2s (H), MSCs (M), and ECs (E) with varying ratio (FX-HME) for vascularization studies. Muti-material FRESH was used to print lobule-mimicking scaffolds with a 23 mg/mL collagen bioink and the FX-H bioink. Post-print cell viability was assessed via LIVE/DEAD assay. Post-print hepatic function, cellular reorganization, and proliferation were evaluated at several time points over 28 days in culture via human albumin ELISA assay and immunofluorescence (IF) staining for albumin and Ki-67. The organization of ECs in the FX-HME prints and the formation of microvasculature-like constructs were monitored over 28 days via IF.

*Results: Lattice and lobule-mimicking scaffolds were bioprinted successfully. Post-print viability was ∼80%. IF revealed that the HepG2s proliferate (Ki-67 positive) and grow in 3D clusters over time in culture. Albumin secretion, as an indicator of hepatic function, was increased from 1084.7 ± 128.4 ng/mL on day 1 to 2371.6 ± 201.9 ng/mL on day 28. IF results also demonstrated that the cells continue to express intracellular albumin over time in culture. These results show that hepatic function in the FRESH printed scaffolds is achieved and is enhanced over time in culture as the cells proliferate. While the MSCs changed morphology and showed an interconnected network in FX-HME constructs with an 8:1:1 cell ratio in 28-day static culture, the ECs did not show capillary-like vessel formation. The ECs in FX-HME constructs with a 6:1:3 cell ratio formed some interconnected vessel-like structures highlighting the effect of ECs concentration and the EC/MSC ratio in the scaffold vascularization.

*Conclusion/Significance: In conclusion, we demonstrated the successful fabrication of functional hepatic constructs and studied the behavior of endothelial cells in static co-culture with HepG2s and MSCs towards vascularization of FRESH bioprinted hepatic constructs. Further work will focus on studying the formation of microvasculature-like constructs in dynamic perfusion culture.

210 - Hydrogel Optimisation For Evaluation Of Angiogenic Responses In 3d Tissue Models Containing Perfusable Microvascular Networks

R. Dai¹, H. Genc¹, M. Ryma², S. Heilig², F. Oliver³, C. Alexiou¹, S. Härteis⁴, J. Groll², I. Cicha¹

¹ University Hospital Erlangen, Erlangen, Germany,

² University of Würzburg, Würzburg, Germany,

³ Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany,

⁴ University of Regensburg, Regensburg, Germany

*Purpose/Objectives: 3D tissue models enable biomedical research in more physiologically-like conditions, but the fabrication of such structures remains challenging due to size limits in the absence of vascularisation. In this study, a 3D printed bioreactor is used to perfuse hydrogel-embedded, bifurcated endothelialised microchannels. Our aim is to investigate and select a suitable matrix that enables sprouting from the main vessel while retaining the stability of the already established channel and the construct over the extended time of construct maturation.

*Methodology: To create perfusable channels, sacrificial micro-filament scaffolds were fabricated using a thermoresponsive poly(2-oxazoline) (POx) and embedded into gelatine-methacryloyl (GelMA) at different concentrations using dedicated perfusion bioreactors. Lowering the temperature allowed dissolution of the scaffold to create perfusable bifurcating channels which were subsequently seeded with primary human umbilical vein endothelial cells (HUVECs).

To mimic cellular interactions and further drive angiogenesis, primary human dermal fibroblasts (fibroblasts) and pericytes were embedded into the hydrogel surrounding the endothelialized channels. Cell migration was compared between different GelMA concentrations.

Furthermore, to assess the angiogenic potential of the hydrogels in more native-like conditions, the samples were incubated on the chick chorio-allantoic membrane (CAM) for 7 days.

*Results: Using a concentration of 7% GelMA warrants good long-term stability of the perfusable channel and the 3D construct, but the resulting hydrogel is too stiff to allow for cell migration. Therefore, the polymer concentrations lowered to 5%, 4.5% and 4% were tested. The 4.5% and 4% gels showed a high variability in their stability and a large variance in the diameter of the channel due to the POx scaffolds’ swelling. The 5% GelMA proved to be more promising, enabling better cell elongation while preserving the channel size and construct stability.

Incubation of the 5% GelMA samples containing endothelialized channels on the chick CAM showed a high degree of reabsorption into the CAM membrane, in addition to interactions of the CAM blood vessels with the hydrogel sample. Further histological evaluation is currently ongoing.

*Conclusion/Significance: The developed vascularized 3D models with a functional endothelial monolayer can, in the future, serve as a tool to investigate cell activation and angiogenesis, macromolecular, nanoparticulate or drug transport, as well as serve as a building block for generation of larger tissue constructs. However, further optimisation of the hydrogel matrix is necessary in order to enable these investigations and other research regarding interactions of perfusable microchannels with tumour spheroids and organoids.

211 - Enhanced Vascularization Of Brain Organoids: Towards A User-convenient And Reproducible Protocol

J. Fumadó Navarro^1,2, W. Chan³, J. Mason³, A. Pandit², M. Lomora^1,2

¹ University of Galway, Galway, Ireland,

² Science Foundation Ireland (SFI) Research Centre for medical devices (CÚRAM), Galway, Ireland,

³ The University of Edinburgh, Edinburgh, United Kingdom

*Purpose/Objectives: Brain organoids (BOs) are emerging bioengineering tools used in neuroscience as a more relevant alternative to animal experimentation. These in vitro 3D models are derived from stem cells through a specific patterning process and resemble the premature morphological and physiological features of the human brain. BOs are multicellular, self-organised, and attractive models for developmental biology and drug discovery. However, the lack of vascularity limits their use for drug delivery screening or for studying brain tissue-vascularity interactions in healthy and diseased contexts (i.e. ischaemic stroke). The process of in vitro vascularization of BOs poses several technical challenges, including the simultaneous differentiation of two different tissue origins, with insufficient support for both the organoid and vascular components, limited penetration of the vascular networks within the organoid, and the absence of luminal perfusion. This project aims to mitigate some of the challenges associated with cerebrovascular interfaces by developing relevant models for investigation.

*Methodology: To address some of these challenges, we devised an encapsulation approach in which endothelial cells (ECs) were delivered to developing organoids from a progressively degrading and surrounding biomaterial. Human brain microvascular endothelial cells (HBMVECs) were selected for their specialized characteristics, which may enhance interactions with neural tissue more effectively than other endothelial cell types. The composition of the media and the concentration of the hydrogel have been optimized to promote both neurodevelopment and vascular network formation.

*Results: Using the proposed encapsulation strategy, a higher density of vascular-like networks was noted compared with control standard organoids when whole-organoid staining was performed. These vascular organisations appear to expand within the organoid tissue, surrounded by collagen depositions and co-existing with different neural populations, as revealed by immuno- and histological staining of the organoid cryosections (Figure 1). Vascularised BOs from the most efficient and reproducible set of conditions will be further characterised using gene expression and functional assays (i.e. perfusability). Moreover, to build on these encouraging results, investigations were carried out to understand the degree to which these vascular-like networks originate: i) from vascular endothelial growth factor (VEGF)-induced endogenous differentiation, ii) from externally incorporated HBMVECs; or iii) from both VEGF and HBMVECs.

*Conclusion/Significance: Hydrogel delivery of encapsulated HBMVECs enables the creation of enhanced vascular-like networks within the brain organoids. However, further mechanistic investigations are required to confirm the sustainment of both functional vascularity and neurodevelopment. Overall, this project will allow for the establishment of an informed and user-convenient protocol for enhanced vascularized BOs, as the basis for generating suitable platforms for screening drug delivery devices and human cerebrovascular modelling.

213 - A Digital Twin To Determine The Pericellular Biochemical Microenvironment Seen By The Spheroids Cultured In A Perfusion Bioreactor

M. BANKOUE NTATE, S. EL HAJJ, M. DUPUY, B. DAVID, H. DUVAL

CentraleSupelec, Gif-sur-Yvette, France

*Purpose/Objectives: Spheroids are increasingly used in tissue engineering (TE) applications. A TE strategy relies on the direct formation of spheroids within scaffolds, followed by their maturation in a perfusion bioreactor. The perfusion flow mechanically stimulates the spheroids and enhances the solute transport (i.e., nutrient supply and waste elimination). However, the stress and solute concentration fields seen by the cells are generally unknown and not uniform in a bioreactor. Thus, a digital twin might be an efficient tool to determine the cell's chemical and mechanical microenvironment. It would help to understand the cell behavior within the bioreactor better. This approach was applied to the dynamic coculture of mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) in hydrogel scaffolds for bone tissue engineering applications.

*Methodology: The device was a fixed-bed reactor with a cylindrical chamber hosting perforated disk-shaped scaffolds. The scaffolds, comprised of polysaccharide porous hydrogel supplemented with hydroxyapatite, were seeded with MSCs and HUVECs (ratio 1:1). Then, the constructs were cultured in the bioreactor for 21 days under perfusion flow. The cell viability was tested using a LIVE/DEAD kit. The osteogenesis was assessed by alkaline phosphatase (ALP) assay. The digital twin solved the hydrodynamics and the transport of three metabolites (dissolved oxygen, glucose, and lactate) within the bioreactor using lattice-Boltzmann methods. The microgeometry of the constructs was acquired at micrometer spatial resolution using optical coherence tomography (OCT). The metabolism model was adapted from a model initially developed for multicellular tumor spheroids since energy carrier molecules in MSCs and HUVECs are mainly produced by aerobic glycolysis as in tumor cells.

*Results: The size of the embedded MSCs/HUVECs spheroids measured at day 1 ranged between 30 and 300 μm, with a mean size of 95 μm. Their viability remained high (> 94%) throughout the 21 days of culture in the bioreactor. After seven days, the spheroids rearranged, and the HUVECs clustered preferentially at the center. The mechanical and chemical conditions seen by the spheroids were determined from numerical simulations carried out on one-day-old constructs. The wall shear stress within the scaffolds was within the 0.01-0.1 mPa interval, significantly lower than the stress level found in the literature to ensure an optimal cellular response (1-10 mPa). Yet, ALP activity peaked on day 7, signaling the start of MSCs osteogenic differentiation. The pericellular chemical microenvironment was physiological: the glucose and dissolved oxygen concentrations were significantly higher than the corresponding Michaelis-Menten constants, and the pericellular pH ranged between 6.8 and 7.4. However, the glucose and dissolved oxygen concentrations decreased by 40% between the periphery and the core of the spheroids. We propose that the oxygen gradient is one of the factors explaining cell rearrangement: HUVECs migrate following the oxygen gradient to induce angiogenesis.

*Conclusion/Significance: By computing the fluid velocity field, the wall shear stress, and the metabolite concentration maps at the pore scale within the bioreactor, the digital twin elucidates the chemical and mechanical microenvironment experienced by the spheroids and provides valuable insights for interpreting biological outcomes.

214 - Artificial Intelligence For Quality Control And Parameter Optimization In Extrusion Based Bioprinting

A. Bonatti¹, G. Vozzi², C. De Maria²

¹ Research Center “E. Piaggio”, University of Pisa, Pisa, Italy,

² Department of Information Engineering, University of Pisa, Pisa, Italy

*Purpose/Objectives: Extrusion-based bioprinting (EBB) represents the most widespread approach in Bioprinting, thanks to its simple implementation and wide variety of processable materials. The integration of quality control solutions in the EBB workflow ranks among one of the current main technological challenges for the technique, and its implementation is expected to: i) reduce the trial-and-error process and associated material consumption, ii) achieve standard results across different set-ups, laboratories and batches, and iii) comply with relevant standards and regulations, thus accelerating the translation of EBB products to more impactful clinical applications. Recently, Machine Learning algorithms have been proposed as advanced tools to implement quality control in the bioprinting workflow, thanks to their ability to learn complex input-output relationships from training data. In this context, we propose a Deep Learning model able to monitor the EBB process online and predict the quality of the print result.

*Methodology: A comprehensive dataset was created, consisting of videos recorded from a front view using a high-resolution webcam (Logitech C920). Each video recorded the printing process of a woodpile scaffold (10 mm sides and 5 mm height) using Pluronic F127 at 25% w/v as test material. Variability in the dataset was introduced by changing, for each video, multiple printing parameters (i.e., layer height, flow, infill percentage, printing speed, needle internal diameter and shape) and by using different EBB set-ups (i.e., piston-driven and pneumatic extrusion-based bioprinters, uniform background, material color). The printed scaffolds were classified after printing in three categories based on the overall quality, namely “ok prints”, “under-extrusion” (i.e., not enough material is being extruded) and “over-extrusion” (i.e., an excess of material is being extruded). Frames were extracted from each video and pre-processed to reduce the computational cost of the Deep Learning model. Then, the resulting dataset was used to train and test a Convolutional Neural Network (CNN) with the task of classifying the quality of the scaffold during the printing process starting from frames of the video feed. The model was defined in Python using the Tensorflow library. Different model architectures were tested in order to: i) maximize the classification performance (measured in terms of accuracy, precision, and recall considering a multi-class classification set-up), and ii) minimize the time to get a prediction in compliance with the online monitoring application.

*Results: Results from the training and testing of the CNN highlight that the optimized model can effectively classify the video frames with a high accuracy score (above 90%). Furthermore, using the optimized architecture, the model showed a fast response time (≈ 180 ms) even when tested on non-optimized hardware using CPU only.

*Conclusion/Significance: In this work, we have shown that a CNN model can be effectively used for the task of monitoring online the EBB printing process. These promising results pave the way for a more widespread and effective implementation of quality control in the bioprinting workflow, with the final aim of accelerating the translation of these techniques to the clinics.

215 - Vip Cells: Screening Key Players And Calculating Their Contributions In Cardiovascular Tissue Remodeling And Regeneration With Supervised Learning

J. Han¹, F. Takaesu¹, E. L. Schwarz², J. D. Humphrey², M. E. Davis¹

¹ Emory University and Georgia Tech, Atlanta, GA,

² Yale School of Engineering and Applied Science, New Haven, CT

*Purpose/Objectives: Identifying functional driver genes and cell populations in heterogeneous tissues is crucial for understanding tissue remodeling and regeneration. Cutting-edge single-cell transcriptomics technology has provided us a detailed road map of the cellular landscape within tissue, but calculating their specific contributions to tissue function still remains a challenge. To address these unmet needs, we developed a supervised learning-based computational pipeline that integrates single-cell and bulk RNA-Seq data to rank functional driver genes and populations (VIP genes/cells).

*Methodology: The pipeline consists of two parts: (1) screening the most important VIP gene clusters involved in tissue function using Partial Least Squares (PLS) regression, and (2) matching VIP genes to clustered populations in scSeq data to discover VIP cell clusters. We applied this pipeline to decipher VIP genes and cells involved in elastin formation during mouse pulmonary artery development.

*Results: This approach identified three highly influential genes (VIP genes): Acan, Ptn, and Igf2. Interestingly, mapping these genes onto the cellular landscape of the developing mouse pulmonary artery pinpointed a previously undefined subpopulation of smooth muscle cells in neonates as potential VIP cell candidates for elastin formation. This finding suggests that these VIP cells and their associated genes warrant further investigation as potential therapeutic targets for pulmonary arterial hypertension.

*Conclusion/Significance: Our results demonstrate the pipeline's possibility to provide quantitative and statistical evidence for functional drivers in tissue remodeling and regeneration, offering key clues for precise tissue engineering and regenerative medicine.

216 - Scalable Spheroid Biofabrication Platforms For Musculoskeletal Regenerative Medicine And Disease Modelling

T. B. Woodfield¹, K. Lim², G. Lindberg¹, S. Cui³, L. Veenendaal¹, G. Major¹, G. Hooper¹

¹ University of Otago Christchurch, Christchurch, New Zealand,

² University of Sydney, Sydney, Australia,

³ The Chinese University of Hong Kong, Shenzen, China

*Purpose/Objectives: Biofabrication technologies, including 3D bioprinting, bioassembly and volumetric, enable generation of engineered constructs that replicate the complex 3D organization of native tissues via automated hierarchical placement of cell-laden bioinks, tissue modules, and/or bioactive factors. Photo-initiated radical polymerization combining light and photo-initiators to generate radicals for crosslinking photo-polymerizable macromers, has been widely employed in 3D bioprinting of cell-laden hydrogels. Despite rapid advances in biofabrication technologies, development of individual bioinks for each biofabrication technique and specific tissue niche. Our overall goals are to address major bottlenecks that still remain in designing materials that are both cell-instructive and compatible with high resolution additive manufacturing and 3D biofabrication techniques. Bottom-up spheroid biofabrication strategies offer flexibility to precisely arrange cellular modules to mimic the zonal structure of tissues as well as uncouple the reliance on narrow biofabrication windows and manipulate cell signaling through spatial presentation of different biomolecules.

*Methodology: This talk discusses alternative strategies to engineer highly tuneable hydrogel platforms that 1) promote a specific cell-instructive niche using light-activated crosslinking in gelatin-based micro-gels, bioresins and high-throughput modular spheroids, and 2) are printable across multiple biofabrication technologies, including extrusion-bioprinting, lithography-bioprinting and microfluidic-based bioprinting.

*Results: We describe the design of versatile photoinitiator system (Ru/SPS) and photo-clickable, cell-instructive gelatin-based bioinks and bioresins for biofabrication of 3D in vitro models. Tailoring macromolecular chemistry offered by the platform (primarily in allylated-gelatin hydrogels; GelAGE), we modulated the cell-instructive tissue niche for multiple cell types via: covalent incorporation of thiolated bioactives (e.g. heparinSH), nanocomposites (e.g. strontium, Laponite); tailored stiffness gradients in cell-laden microgel filaments fabricated using a diffusion-based microfluidics approach, as well as di-tyrosine cross-linking of decellularized extracellular matrix (ECM) bioinks. Examples discussed include yielding enhanced chondrogenic/osteogenic differentiation and vascular network formation.We further discuss experience in developing hybrid tissue constructs and convergence with 3D spheroid bioassembly platforms for probing multicellular spheroid fusion, extracellular matrix (ECM) formation and stem-cell niche, offering new paradigms for high-throughput screening and disease modelling in healthy and osteoarthritic sphroid fusion models. Examples include a 3D spheroid bioassembly platform combining allogeneic umbilical cord blood derived MSCs in chondrogenic (GelMA-HepSH) and osteogenic (GelMA-strontium) cell-instructive microgels bioassembled within mechanically stable thermoplastic scaffold (PEGT/PBT) as a potential off-the-shelf osteochondral tissue repair strategy.

*Conclusion/Significance: This work demonstrates advances in development of cell instructive universal bioink platforms for 3D bioprinting and regenerative medicine, and advances scalable, modular approaches for bioassembly of functional tissues towards clinical translation.

219 - Upscaling Manufacture Of Osteogenically Differentiated Mesenchymal Stem Cells Using Nanoscale Vibration And Microcarrier Suspension Culture

J. Williams¹, M. Dalby², M. Salmeron-Sanchez², P. Childs¹, S. Reid¹

¹ Centre for the Cellular Microenvironment, Department of Biomedical Engineering, University of Strathclyde, Glasgow, United Kingdom,

² Centre for the Cellular Microenvironment, University of Glasgow, Glasgow, United Kingdom

*Purpose/Objectives: Bone is the second most transplanted tissue following blood, and over 2 million procedures are performed each year involving bone grafts, which poses issues of supply and efficacy. Nano-amplitude sinusoidal vibration (1 kHz, 30 nm) delivered continuously in a pistonic manner - by our bespoke nano-amplitude vibrational bioreactor - drives the differentiation of human mesenchymal stem cells (hMSCs) towards an osteoblast lineage, in both 2D and 3D environments, further maturation has been confirmed by the presence of mineralisation. Upscaling the nanovibrational cell stimulation process is key for supplying the large quantities of phenotypically controlled nanovibrated hMSCs needed for cellular therapies and orthopaedic use, e.g. for repair of critical-sized or non-union bone defects. The vibrational bioreactor can currently deliver precise vibration to T150 flasks and multi-layer CellSTACKs. However, microcarrier suspension culture systems offer almost unbound upscaling potential to provide sufficient numbers of cells for clinical and commercial viability of a new therapy.

*Methodology: In this present work an experimental setup designed to deliver nanoscale vibration to hMSCs adhered to polystyrene microcarriers, suspended in a 250 mL spinner flask, was developed, optimised, characterised, and tested (Figure 1A). The spinner flask was adapted such that the impeller was driven with an overhead motor, to enable application of nanovibration from its base. This osteogenic vibration is within a precise range, laser interferometry was used to quantify, shift and reduce as far as possible motor-induced vibration. Further, scanning laser vibrometry was used to confirm that suitable nanovibration was delivered by measuring the motion of the fluid’s top surface. With this validated setup, the potential to upscale the nanovibrational cell production process was assessed. Specifically, hMSCs were seeded onto microcarriers, 24 hours later the spinner flask was continuously nanovibrated and stimulation was applied for 3 weeks. Seeding efficiency, proliferation, stemness and osteogenic markers were assessed and compared to non-vibrated control spinner flasks, using a variety of techniques including RT-qPCR, immunofluorescence, alizarin red, micro-computed tomography (microCT) and flow cytometry.

*Results: Vibration analysis enabled fine-tuning of the setup to ensure that only lower frequency (<400 Hz) and lower amplitude (5 nm) vibration was introduced to setup due to motor-driven impeller rotation. Further, the fluid vibrated pistonically at 1 kHz (replicating prior results of 2D multiwell plate nanovibration). Proliferation, assessed via MTT assay showed slower growth in the vibrated flask. Osteogenesis and mineralisation on microcarriers were assessed with mRNA expression of relevant osteogenic markers (osteocalcin, osteopontin, RUNX2), osteocalcin immunofluorescence, alizarin red and quantifiable amounts of mineralisation detected with microCT (Figure 1B).

*Conclusion/Significance: The integration of nanoscale vibration and microcarrier suspension culture represents one of the first attempt to exploit mechanotransduction for cell therapy manufacture. Microcarrier culture is readily exploitable within good manufacturing practice (GMP) for advanced therapy medicinal products (ATMPs) and offers the potential to provide large quantities (billions) of nanovibrated “osteoprimed” hMSCs per batch. This development will be crucial to make a commercially viable and clinically relevant cell therapy for the repair of critical-sized (non-healing) bone defects.

220 - Injectable Hydrogel Comprising MicroRNAs Promotes Nerve Regeneration Following Brain Stroke

R. Singh¹, V. S Ramanujan^1,2, C. Huang¹, B. Chen³, K. Zhang⁴, L. Bian⁴, L. Ping³, S. Chew^1,5,6

¹ Nanyang Technological University, Singapore, Singapore,

² CNRS@create, Singapore, Singapore,

³ National Neuroscience Institute, Singapore, Singapore,

⁴ South China University of Technology, Guangzhou, China,

⁵ Lee Kong Chian School of Medicine, Singapore, Singapore,

⁶ School of Materials Science and Engineering,, Singapore, Singapore

*Purpose/Objectives: Stroke is a common disease globally with high mortality rates. The formation of ischemic cavities, inflammation, cell apoptosis, and scarring often restricts neural regeneration post-stroke. Current treatments target the inhibitory microenvironment that impedes nerve recovery. However, mature neurons have a reduced intrinsic ability to regenerate, which is a leading cause of regeneration failure. This study aims to find ways to encourage the intrinsic growth capability of damaged neurons. Injectable hydrogels that mimic the extracellular matrix and fill the damaged area show potential in the treatment of brain tissue injuries.

*Methodology: In our study, we have encapsulated microRNAs (miRs) which are known to play important roles in regulating axon-localized protein synthesis. We aimed to improve the intrinsic growth ability of neurons by administering these miRs via injectable hydrogel directly to the stroke cavity in the rat model with middle cerebral artery occlusion (MCAo). We used a combination of gelatin and acrylated beta cyclodextrin (β-CD) to form injectable hydrogels through host-guest complexation, followed by controlled chemical cross-linking.

*Results: The injectable hydrogel has a 3D porous structure, high swelling ratio, biodegradation, and tunable mechanical strength. An in vitro release test demonstrated at least 3 weeks of sustained miR release, and the hydrogel supported high cell viability for cortical neurons. Differentiation of cortical neurons into mature neurons with axonal projections was observed after 7 days of culture. After being implanted in the stroke cavity for two weeks, the hydrogels helped to promote the growth of neurons into the lesion site. It is important to note that the hydrogel integrated well with the host tissue and did not cause inflammation or glial scarring around the injection site. After the treatment of animals with axon miRs, it was observed that neurofilament penetration was denser and longer at the interface and into the scaffold (about three times more) as compared to animals treated with negative miRs. Moreover, new blood vessels were also visible at the interface and their numbers were comparatively higher in the miRs treated animal than the animals with negative miRs.

*Conclusion/Significance: The results indicate that Gelatin and beta-cyclodextrin Injectable hydrogels are effective in promoting an endogenous regenerative response that restores tissue in the brain's ischemic cavity. Therefore, the use of axon miRs encapsulated injectable hydrogel for stroke treatment is a highly promising therapeutic approach for nerve tissue regeneration.

221 - Co-delivery Of A Thermostabilized Enzyme And Human Neural Progenitor Cells Using Tailored Hydrogels Enhances Recovery After Stroke

N. Letko Khait, D. Li, C. Morshead, M. Shoichet

University of Toronto, Toronto, ON, Canada

*Purpose/Objectives: Stroke is one of the leading causes of morbidity and long-term disability worldwide, with no clinically available treatments to promote tissue regeneration. While transplanting neural cells is a promising approach, demonstrating improved functional behavior in pre-clinical studies, cell survival poses a significant challenge. One of the causes is the accumulation of inhibitory chondroitin sulfate proteoglycans (CSPGs) that are secreted post-injury to the central nervous system and are believed to limit the regenerative capacity of the tissue. CSPG degradation was therefore suggested as a therapeutic strategy after stroke to improve local plasticity and promote axonal regrowth. The bacterial enzyme chondroitinase ABC (ChASE) can degrade CSPGs, yet its inherent thermal instability limits its therapeutic potential. Moreover, continuous delivery is needed to obtain significant improvements after stroke.

*Methodology: To overcome these obstacles, we designed an injectable hyaluronan-based hydrogel that mimics the brain’s extracellular matrix and supports human induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs). We studied their survival and neuronal differentiation within the hydrogel using methods of gene expression and confocal microscopy. Independently, we engineered ChASE to enhance thermostability by introducing 37-point mutations (ChASE37). We expressed the enzyme as a fusion protein with a Src homology 3 (SH3) domain (SH3-ChASE37) and modified an injectable cross-linked methylcellulose hydrogel with SH3 binding peptides to enable affinity-controlled release. We studied the controlled release and bioactivity using ELISA and cell-based assays. Lastly, we co-delivered iPSC-NPCs and affinity release SH3-ChASE37 directly to the brain of endothelin-1-induced stroke injured rats, where we assessed improvements in motor function using behavioral studies, and tissue regeneration through immunohistochemistry.

*Results: Hydrogel-encapsulated iPSC-NPCs survived injection through a fine needle, and showed high cell viability and neuronal differentiation ability for at least 14 days in vitro. SH3-ChASE37 demonstrated a 6.5 times higher half-life than the native enzyme, with a higher melting temperature, and increased activity for its substrate. The hydrogel attenuated the release of bioactive SH3-ChASE37 in vitro and in vivo, and the efficacy of the released enzyme was demonstrated with degradation of the inhibitory CSPGs. We co-delivered encapsulated iPSC-NPCs and SH3-ChASE37 directly to the stroke-injured rodent brains, and observed improved motor function relative to vehicle controls. We are currently examining the fate of the transplanted cells in terms of promoting tissue repair and the impact of SH3-ChASE37 release on tissue plasticity.

*Conclusion/Significance: We demonstrated efficacy of a new thermostable mutated variant of the catalytic enzyme chondroitinase ABC, SH3-ChASE37, for the first time using both cell culture and animal models of stroke. We utilized specialized hydrogels to enable cell survival and the sustained affinity release of bioactive SH3-ChASE37. Importantly, in vivo bioactivity was achieved, as evidenced by the degradation of CSPGs in the stroke-injured rat brain. This multifaceted approach holds immense potential for advancing stroke therapeutics by addressing key challenges in cell survival and promoting neuroplasticity. This work was supported by the Stem Cell Network, Heart & Stroke Foundation, CFREF through Mend the Gap, and PRiME clinical catalyst fellowship.

222 - Electrostimulation Via A 3D-printed, Biomimetic, Neurotrophic, Electroconductive Scaffold For The Promotion Of Axonal Regrowth After Spinal Cord Injury

L. M. Leahy^1,2, I. Woods^1,2, A. Dervan^1,2, M. G. Monaghan^3,2, F. J. O’Brien^1,2,3

¹ Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland,

² RCSI and Trinity College Dublin (TCD), Dublin, Ireland,

³ TCD, Dublin, Ireland

*Purpose/Objectives: Spinal cord injury (SCI) is a devastating neurotrauma, affecting up to 500,000 people annually, and typically results in lifelong paralysis. Electrostimulation can promote neuronal growth, but the formation of a lesion cavity post-SCI inhibits regrowth, limiting its efficacy. Bridging the lesion with a structured, electroactive substrate to direct electrostimulation to growing axons could support and drive regrowth to enable functional recovery. Through mimicking the architecture of the tissue, regrowing neurons could also be guided to meet their distal targets. However, to date, no such platform exists. This study describes the application of electrostimulation via an electroconductive 3D-printed scaffold, comprising an electroconductive polypyrrole/polycaprolactone framework filled with biomimetic & neurotrophic extracellular matrix, to induce significantly longer process extension in neurons.

*Methodology: Biomimetic polycaprolactone (PCL) scaffolds were 3D-printed (Allevi 2) to produce uniaxially aligned, interlocking cylindrical architectures, utilizing several channel sizes to match the diameters of human lateral corticospinal cord axonal tracts. Polypyrrole (PPy) was polymerized in situ to form an electroconductive nanoparticle coating on the scaffolds, verified using FTIR and SEM imaging. Electroconductivity was measured via the 4-point probe method. A hyaluronic acid-based extracellular matrix (ECM) was directionally freeze-dried within scaffold channels to provide a neurotrophic environment, and a physical guide to direct neuronal growth. Biocompatibility was assessed by growing neurons and astrocytes on 2D PPy/PCL films and measuring metabolic activity, cellular DNA and morphology. Neurons were electrically stimulated for 7 days on ECM-functionalised PPy/PCL and PCL scaffolds using an Ionoptix bioreactor and imaged to assess the effect of electrostimulation-induced neurite extension.

*Results: PCL scaffolds with anatomically relevant channel sizes were 3D-printed and functionalised with PPy and ECM (Fig 1A). The conductivity of the PPy coating was measured at 15±5S/m, 30 times higher than native spinal cord tissue. PPy/PCL substrates displayed excellent biocompatibility - with both neurons and astrocytes colonising the surface with typical morphologies and no difference in metabolic activity or cellular DNA on PPy/PCL in comparison to PCL controls. The freeze-dried neurotrophic ECM formed aligned pore structures within scaffold channels, to guide neuronal neurite outgrowth. Scaffolds of varying channel sizes exhibited no difference in electrical properties or cell activity, showing scaffolds can be scaled to match native cord tracts without losing functionality. Electrostimulation induced significantly longer neurite outgrowth (Fig 1C) and average neurite length was significantly increased on stimulated PPy/PCL scaffolds compared to all other groups (Fig 1D).

*Conclusion/Significance: This work represents the development of a novel, biphasic electroconductive scaffold, which can direct electrostimulation to promote neurite growth through its longitudinal, ECM-filled channels. The data indicates that PPy/PCL is an excellent substrate for neural tissue engineering and can be developed into ordered structures that can be tailored to match the native tracts to direct neurite growth. Applying electrical stimulation via this electroconductive scaffold results in significantly longer neurite length, demonstrating the potential of this system to direct and guide axonal growth post-SCI to restore connection and promote functional recovery. FUNDING ACKNOWLEDGEMENTS: Irish Rugby Football Union Charitable Trust and SFI Advanced Materials and Bioengineering Research (AMBER) Centre (SFI/12/RC/2278_P2).

223 - Motor Function Evaluation Of A Prevascularized Cell-based Tissue-engineered Nerve Tube In Rabbit Nerve Defect Regeneration

A. Thibodeau, O. Hayouni, T. Galbraith, H. Khuong, F. Berthod

Département de Chirurgie, Faculté de médecine, Université Laval, Québec (Québec), Canada, LOEX, centre de recherche du CHU de Québec-Université Laval, Québec, QC, CANADA

*Purpose/Objectives: Peripheral nerve damage remains a challenge even in the era of cutting-edge technologies. Nerve injuries not only result in a severe loss of sensibility but, more gravely, can lead to paralysis, significantly impacting patients' quality of life. While the gold standard in clinical practice involves the use of autologous nerve transplantation, it has major limitations. This approach induces a deficit at the donor site and results in poor functional recovery in most cases (a 50% success rate of functional recovery). An alternative avenue is to use a nerve conduit made of biomaterials to guide axonal migration through the distal nerve stump. However, this strategy faces several clinical constraints, particularly in achieving prompt and adequate blood circulation - a critical component of nerve regeneration for lesions more than 3 cm long. In our research group, we hypothesize that a prevascularized cell-based tissue-engineered nerve grafts (TENGs) could enhance a rapid and efficient vascularization. A viable conduit pre-seeded with both endothelial (ECs) and Schwann cells (SCs) could allow a faster recovery for patients undergoing major peripheral nerve transections.

*Methodology: TENGs are composed of human fibroblast sheets seeded with endothelial cells (ECs) and Schwann cells. This cellular sheet is then rolled to form a tubular structure. In a one-year study, TENGs were implanted in immunosuppressed New Zealand rabbits to address a 4 cm fibular nerve defect. Monthly assessments, including electromyography and nerve conduction studies, were conducted under light anesthesia. The Toe Spread Index (TSI) served as a reliable metric for monitoring functional outcomes. At the end of the study, graft innervation was observed by immunofluorescence and toluidine blue staining.

*Results: Nerve conduction analysis conducted on the tibialis anterior muscle indicated the onset of nerve conduction recovery around the fourth month. The electromyogram revealed a return of resting electrical activity with multiple polyphasic activities (I.e., reinnervation in progress) around the fifth month. After 6 months, an improvement in the TSI showed a partial motor function recovery in rabbit with autograft, a phenomenon also observed in the Schwann cell enriched TENG group.

*Conclusion/Significance: Our team previously demonstrated in a rat model that pre-vascularized TENGs allows for a better motor function recovery following a 15mm nerve defect. The fast establishment of vascularization post-grafting ensures a well-oxygenated environment, a critical factor supporting axonal migration over extended distances. Primary SCs have the potential to release nerve growth factors that will enhance nerve regeneration. Our approach to develop a prevascularized living autologous nerve tube, promoting a rapid vascularization of the graft, is crucial to support axonal migration over long distances. This strategy holds promise as a novel clinical tool to bridge large lesions (>3cm).

224 - Automated Production Of Nerve Repair Constructs Containing Endothelial Cells Derived From Human Induced Pluripotent Stem Cells

P. O. Smith¹, G. Huang², K. Devries², P. Jat¹, S. N. Nazhat², J. B. Phillips¹

¹ University College London (UCL), London, United Kingdom,

² McGill University, Montreal, QC, Canada

*Purpose/Objectives: Traumatic peripheral nerve injury has a sudden, debilitating effect on millions of people every year, resulting in loss of sensation and movement, significantly reducing quality of life. The current clinical standard autograft has multiple limitations including donor tissue availability and donor site morbidity. Tissue engineering offers a promising alternative, artificial nerve repair constructs produced from aligned cells and biomaterials. Cell source and production time can limit the clinical translatability of nerve repair constructs. Whilst Schwann cells are the traditional therapeutic cell type for nerve repair, constructs containing endothelial cells have been shown to further improve regeneration. Here, a novel source of endothelial cells was derived and incorporated into nerve repair constructs in a newly developed automated, scalable fashion.

*Methodology: Human induced pluripotent stem cells, derived by reprogramming research equivalents of a clinically-approved conditionally-immortalised neural stem cell line (ReNeuron Ltd, UK), were differentiated to endothelial cells using an established monolayer culture protocol, sorted by FACS, and characterised by RT-qPCR, immunofluorescence and tubulogenesis assays. Collagen hydrogels, seeded with endothelial cells, were stabilised by controlled aspiration under negative pressure using the Gel Aspiration-Ejection (GAE) technique which was advanced to automation by integrating a syringe pump with a robotic positioning system, using software coded in Python. Viability, alignment and construct maturation were assessed. Co-culture of endothelial cell constructs with primary sensory neurons was used to assess support of aligned neurite growth in vitro.

*Results: Endothelial cells were successfully differentiated from hiPSCs with a differentiation efficiency of 26% as determined by expression of endothelial markers CD31 and CD144. A decrease in pluripotency markers and an increase in endothelial markers were detected as differentiation progressed (Fig. 1b), with differentiated cells capable of tubulogenesis. Through combination of a syringe pump and a 3D printer adapted to act as a robotic arm, the GAE technique was automated, producing nerve repair constructs from cellular collagen hydrogels in ∼1 minute (Fig. 1c). The GAE technique generated 48 constructs per batch (<1 h), with the option to customise construct length and diameter. The resulting nerve repair constructs exhibited ∼80% cell viability with significantly improved cell alignment compared with the starting hydrogels (p<0.0038) with the median cell deviating 17° from the longitudinal axis (Fig 1d). Endothelial cell constructs significantly improved in vitro neurite alignment compared to acellular constructs by 2.25-fold (p<0.0169) (Fig. 1e). Culture of endothelial cell constructs for 2 to 4 days resulted in a tight cobblestone cell layer on the outside and a network of tube-like structures inside the constructs (Fig. 1ciii).

*Conclusion/Significance: Human endothelial cells were successfully derived from hiPSCs (for which direct GMP equivalents are available) and used to produce aligned and viable nerve repair constructs capable of supporting neurite growth in vitro. When cultured for 2-4 days, these constructs exhibited angiogenic behaviour permitting the benefit of endothelial cells in nerve repair constructs to be studied. In addition, the GAE technique was advanced to automation accelerating and scaling the production of nerve repair constructs, improving clinical translatability of the technique.

225 - A Cell-derived Extracellular Matrix-based Biologic To Reverse Axonal Degeneration In Diabetic Neuropathy

Z. Zhirenova^1,2, K. Lit^1,2, Z. Wang¹, A. M. Blocki^1,2,3

¹ School of Biomedical Sciences, Hong Kong, Hong Kong,

² Center for Neuromusculoskeletal Restorative Medicine, Hong Kong, Hong Kong,

³ Department of Orthopaedics & Traumatology, Hong Kong, Hong Kong

*Purpose/Objectives: Diabetic neuropathy (DN), a complication of diabetes mellitus, leads to peripheral sensation loss due to axonal degeneration, increasing the risk of limb amputation. DN contributes to the formation of chronic diabetic wounds characterized by ischemia, chronic inflammation, and an extracellular matrix (ECM) deficient in pro-reparative factors. The degeneration of sensory neurons is detrimental to tissue repair due to diminished release of neuromodulators. Current therapeutic approaches for DN are insufficient, necessitating novel approaches to promote peripheral nerve regeneration and facilitate diabetic wound healing. This project aims to develop a composite biologic based on mesenchymal stem cells (MSC)-derived and decellularized pro-reparative ECM to reverse axonal degeneration in DN. Previously, we synthesized Microparticles of Solidified Secretome (MIPSOS) from bone-marrow MSC-derived and decellularized ECM supplemented with dextran sulfate (DxS), a heparan sulfate mimetic. MIPSOS enhanced skin wound healing and revascularization, surpassing the therapeutic potential of ECM derived from naïve MSCs.

*Methodology: An in vivo model for DN was validated by immunohistochemical staining of tail skin tissue sections from wildtype (C57BL/6) and diabetic (db/db) mice for neuron-specific markers. The bioactivity of MIPSOS on neuropathic skin re-innervation was evaluated by immunohistochemical staining for the neuron-specific markers of diabetic wound sites treated with MIPSOS wound fillers. To enable delivery into uninjured neuropathic skin, MIPSOS was required to be solubilized by urea extraction. To assess the in vitro bioactivity of the solubilized biologic through various functional assays, sensory neuron-like cells were differentiated from rat pheochromocytoma cells (PC-12), whilst Schwann cells (SC) and their precursors (SCP) were differentiated from induced pluripotent stem cells (iPSCs).

*Results: Diminished expression of neuron-specific markers in diabetic murine skin compared to healthy counterparts underscores the peripheral nerve degeneration in DN. Proteomic analysis revealed the enrichment in various pro-neurogenic factors in MIPSOS versus ECM-derived from naïve MSCs, suggesting it promising for reversing axonal degeneration in DN. Applying MIPSOS to diabetic skin wounds enhanced overall healing, surpassing the empty delivery vehicle and showing comparable results to tissue-ECM, commercially known as GraftJacket. Moreover, an improved recovery of neuron-specific proteins after MIPSOS treatment suggests in-situ regeneration of peripheral nerves. MIPSOS were successfully solubilized to render them suitable for the application into uninjured skin. SCPs and sensory neuron-like cells were successfully established from iPSCs and PC12 cells, respectively, to enable further functional evaluation of solubilized biologic’s effects on SCP proliferation, migration and neurotrophic factors secretion, as well as neuronal outgrowth in vitro.

*Conclusion/Significance: MIPSOS and their soluble format have the potential to reverse axonal degeneration in DN. The increase in neuronal markers after MIPSOS treatment suggests the promotion of nerve regeneration during diabetic skin wound healing, making it a promising biologic for treating DN in uninjured skin. The proposed approach offers an innovative off-the-shelf therapy for DN, potentially preventing axonal degeneration and improving the quality of life of diabetic patients.

226 - Age-associated Functional Healing Of Musculoskeletal Trauma Through Regenerative Engineering And Rehabilitation

K. Habing¹, V. Duke¹, Y. Tan¹, N. Willett², K. H. Nakayama¹

¹ Oregon Health & Science University, Portland, OR,

² University of Oregon, Eugene, OR

*Purpose/Objectives: Traumatic musculoskeletal injuries that lead to volumetric muscle loss (VML) are challenged by irreparable soft tissue damage, impaired regenerative ability, and reduced muscle function. Regenerative rehabilitation strategies involving the pairing of engineered therapeutics with physical exercise have guided considerable advances in the functional repair of skeletal muscle following VML. However, few studies exist that evaluate the efficacy of regenerative rehabilitation across age-diverse populations.

*Methodology: In the current study, the combined treatment of an engineered muscle was paired with running exercise following induction of a tibialis anterior VML injury and evaluated in both young (8-week) and aged (80-week) C57BL/6 mice. Engineered muscle was generated from nanofibrillar aligned collagen scaffolds laden with differentiated primary mouse myogenic cells. Healing and recovery from injury were assessed in the following three groups: 1) sham; 2) voluntary wheel running; 3) voluntary wheel running combined with engineered muscle [Fig. 1a-e].

*Results: The size of the VML injury was verified to be similar between both young and aged cohorts [Fig. 1f]. Differences in self-selected activity were demonstrated by a 3-fold greater running distance of young compared to aged mice [Fig. 1g]. Four weeks post-injury, young mice that received exercise in combination with engineered muscle, demonstrated significantly greater muscle mass (1.31-fold, p<0.05), in situ contractile force (2.45-fold, p<0.001), and vascular density (1.91-fold, p<0.01) compared to age-matched controls [Fig. 1h-j]. However, the aged animals did not experience a regenerative healing response regardless of treatment, suggesting a diminished efficacy of regenerative rehabilitation in aged populations. Additionally, aged mice exhibited chronic upregulation of circulating inflammatory cytokines including IL-7, IL-15, and MIG compared to young mice.

*Conclusion/Significance: Taken together, these findings highlight the restorative potential of regenerative rehabilitation for the treatment of traumatic musculoskeletal injuries in young populations and the complimentary need for age-specific evaluations to serve broader patient demographics.

228 - Electrical Stimulation Via Conducting Polymers For Neural Repair

S. Song

University of Arizona, Tucson, AZ

*Purpose/Objectives: Electrical signals are essential for the communication of neurons to other cell types^1,2 and play an important role in neuronal survival, differentiation, and functional expression^3,4. Previous research has shown that exogenous electrical stimulation is effective in promoting cell mobilization and differentiation in a voltage- and time-dependent manner^5-10. We developed implantable conductive platforms with varying physical shapes and properties to apply electrical stimulation to cells and tissues for peripheral nerve injury¹¹, stroke¹², and epilepsy neuromodulation¹³.

*Methodology: We created 2D and 3D conductive platforms made of polypyrrole (PPy) based on electrochemistry to understand the electrical effects on human neural progenitor cells (NPCs). We investigated the physical effect (2D vs 3D) as well as the effect of electrical stimulation on the viability and gene expression of encased hNPCs¹⁴. We subsequently implanted the 2D and 3D conductive platform containing hNPCs to treat stroke¹² and peripheral nerve injury¹¹ using small animal models, respectively. All animal procedures were approved by the Institution’s Administrative Panel on Laboratory Animal Care. For the stroke study, adult nude rats (NIH-RNU) underwent distal middle cerebral artery (dMCA) occlusion model with occlusion of both common carotid arteries lasting 30 min as described previously¹⁵. One week of stroke, animals were randomized by vibrissae-whisker paw score, and implantation surgeries performed by a blinded individual. A craniectomy was created between the neuroanatomical lambda and bregma markers, and the dural layer was excised. The 2D conductive platform with hNPCs were implanted over the exposed brain tissue primarily on the penumbral cortex medial to the lesion. For the peripheral nerve injury study, adult nude rats (NIH-RNU) received a critical nerve defect using a sciatic nerve transection model as described previously¹⁶. The 3D conductive platform with hNPCs under applied electrical stimulation were implanted to repair the critical nerve defect. In vivo electrical stimulation was applied post-operatively based on parameters identified from in vitro hNPC study. For both animal studies, a battery of animal behavior and molecular and cellular techniques were used to assess the level of neural repair and functional recovery.

*Results: The physical dimension (2D vs 3D) of stimulating platforms alone changed the hNPCs gene expression related to cell proliferation and metabolic pathways¹⁴. The addition of electrical stimulation was critical in upregulating gene expression of neurotrophic factors that are important in regulating cell survival, synaptic remodeling, and nerve regeneration^11,12,14. The 2D conductive platform containing hNPCs improved functional stroke recovery in rodents¹². Transcriptome analysis identified stanniocalcin 2 (STC2) as one of the genes most significantly upregulated by electrical stimulation. Intraventricular administration of recombinant STC2 post-stroke conferred functional benefits. The 2D conductive platform containing hNPCs promoted peripheral nerve repair from paracrine secretion of neurotrophic factors¹¹. Increased myelination and nerve conduction speed demonstrated the synergistic potential of using hNPC and electrical stimulation as a novel regenerative rehabilitation approach for neural repair.

*Conclusion/Significance: Our conductive polymer systems enable electrical modulation of transplanted stem cells as a new regenerative rehabilitation approach to identify important therapeutic targets and improve neural recovery.

230 - De Novo Organs For Drug Discovery, Cell Therapy Development And Transplantation

I. M. Rogers^1,2, J. Castillo Prado^1,2, C. Tat Lui^1,2, A. Bhadwal^1,2, Y. Ping^1,2, M. Sadat^1,2

¹ Lunenfeld Tanenbaum Research Insitute, Sinai Health System, Toronto, ON, Canada,

² University of Toronto, Toronto, ON, Canada

*Purpose/Objectives: The extracellular matrix (ECM) is an element crucial to organogenesis and regeneration. Here we demonstrate that the ECM can direct the differentiation of definitive endoderm to pancreas, and mesoderm to kidney, without the need for exogenous growth factors. The ability of the ECM to direct differentiation to specific cell types was associated with the growth factors sequestered in adult ECM. To achieve proper organ development a decellularization method that maintains ECM proteins was critical. Also, for decellularization /recellularization and organ growth a suitable ex vivo perfusion system is required. Combining properly decellularized organ ECM from mouse or porcine with human stem-cell derived progenitor cells allowed for the production of human organs and tissues that are suitable for drug discovery, cell therapy development and eventually transplantable organs.

*Methodology: 1) Development of an ex vivo perfusion organ system for mouse and pig organs. 2) Perfusion decellularization of mouse and porcine whole organs and tissue sections to remove endogenous cells and maintain ECM proteins. Different protocols were required for each different organ. 3) ES cells and iPS cells differentiated to endoderm or mesoderm were then applied to the acellular ECM and grown in air:liquid or ex vivo perfusion culture. 4) analysis of the new organ or tissue.

*Results: Native mouse organs were used to establish an ex vivo organ perfusion system for the production of humanized organs. We achieved perfusion times of days to weeks for the mouse kidney and pancreas after optimization of the media, flow rates and oxygenation. A porcine organ ex vivo perfusion system was also developed and tested successfully with pig kidneys. We demonstrated successful preservation of the ECM, importantly the glycosaminoglycans and associated growth factors. Recellularized mouse kidney and pancreas with endoderm or mesoderm resulted in organized tissue similar to endogenous organs. Importantly we demonstrated that the ECM provides a unique biological address that determines the fate of the progenitor cell at each specific cell location within the organ. In contrast, recellularization of the acellular kidney with mature kidney cells resulted in healthy kidney cells but they lacked the organization we observed when progenitor cells were used for recellularization. Using RNA-seq our study also indicated the presence of the ECM increased the rate of maturation of the new organ.

*Conclusion/Significance: The production of a new organized organ is dependent on the optimal decellularization of the ECM. The retention of ECM proteins, especially the glycosaminoglycans, allows for the ECM to direct the differentiation of stem-cell derived organ progenitor cells resulting in an organized organ. Although the acellular ECM supports adult kidney cells, they remained disorganized with no obvious arrangement. The establishment of an ex vivo organ perfusion system was important to maintain vascular flow which is critical for nutrition and oxygen delivery.

231 - Decellularization Of Whole Rat Kidneys: Application Of Physical And Chemical Methods

H. Salti¹, S.-C. Nelz¹, C. Pohl², J. Hofrichter¹, A. Breitrück¹, S. Mitzner^3,1, R. Wasserkort^1,3

¹ Fraunhofer Institute for Cell Therapy and Immunology (IZI), Rostock, Germany,

² Greifswald University Medical Center, Rostock, Germany,

³ Department of Internal Medicine, Rostock University Medical Center, Rostock, Germany

*Purpose/Objectives: The increasing prevalence of chronic kidney disease highlights the urgent need for innovative interventions in end-stage renal disease (ESRD) beyond conventional transplantation and dialysis. Tissue engineering, particularly the development of bioartificial kidneys, emerges as a potential alternative. Perfusion decellularization of whole kidneys yields scaffolds with the extracellular matrix (ECM) featuring the natural architecture of the target organ. Considering the detrimental effects of chemical reagents on the ECM, the exploration of physical treatments aims to enhance the efficiency of perfusion decellularization. Building on insights from a previous study, which used precision-cut kidney slices (PCKS) for investigating physical treatments during immersion decellularization, this research assesses how these physical pretreatments impact the success of perfusion decellularization in whole rat kidneys.

*Methodology: Physical methods included pretreatment of whole rat kidneys with high hydrostatic pressure (HHP) or freezing-thawing cycles (FTC), followed by perfusion decellularization using the (CHEM-Prf) protocol, which involved several chemical reagents (Trypsin, Triton X-100, sodium deoxycholate, sodium dodecyl sulfate). The evaluation of decellularization encompassed assessing the preservation of ECM components and removal of residual DNA, along with histological examination of the scaffolds and perfusate analysis. To evaluate the ability of decellularized rat scaffolds to support recellularization, PCKS were prepared from whole decellularized kidneys and seeded with human immortalized renal proximal tubular epithelial cells (RPTEC/TERT1). Assessing recellularized scaffolds involved evaluating cell viability and examining the expression of the tight junction protein zonula occludens-1 (ZO-1). Additionally, artificial neural networks (ANN) were utilized for a location-specific assessment of the seeded cells, automating the identification of Bowman capsules (BC) in scaffolds stained against collagen type IV (Col-IV). Subsequently, the number of seeded cells within the BCs relative to the total number of seeded cells was counted, serving as an indicator of specific recellularization.

*Results: FTC-Prf demonstrated the most substantial reduction in DNA but also a significant decrease in glycosaminoglycans (GAG). Conversely, HHP200-Prf exhibited the least effectiveness in DNA removal, primarily attributed to HHP-induced tissue compression. In perfusate analysis across all tested methods, SDS resulted in the highest release of GAGs (70%), followed by SDC (20%). Conversely, SDC caused the most significant loss of collagen (60%), followed by Triton X-100 (16%). For further comparison, PCKS prepared from whole decellularized kidneys by perfusion (PCKS-Prf) were recellularized with RPTEC/TERT1 cells. The viability of seeded cells in CHEM-Prf and FTC-Prf scaffolds exhibited consistent growth over the 7-day incubation period, while HHP200-Prf scaffolds displayed the lowest viability. Interestingly, similar to the approach of PCKS decellularization by immersion, PCKS obtained from whole kidneys decellularized by FTC-Prf demonstrated superior results concerning the expression of the tight junction protein ZO-1.

*Conclusion/Significance: This study not only affirms the effectiveness of FTC as a pretreatment in whole kidney decellularization but also reinforces the role of the ECM in imparting instructive memory, guiding the migration of immortalized cells and preventing non-specific attachment. The research highlights the significance and suitability of utilizing PCKS as a model for the initial exploration of de- and recellularization methods, thus contributing to a reduction in the need for animal sacrifice.

232 - New Engines For Polybiosensing And Analysis Of Growing Cells: From Disease Models To Therapeutic Strategies

M. Veiseh

Polybiomics, Inc. / Lawrence Berkeley National Lab (LBNL), Kirkland, WA

*Purpose/Objectives: Advancements in hybrid bioelectronics, cell-based devices, and 3D organotypic disease models have enabled us to detect cellular bioelectricity, metabolism, and phenotypes at multiple scales. However, the heterogeneous changes in living cells across spatiotemporal dimensions pose an engineering challenge, necessitating dynamic measurement within the same organotypic culture. To catalyze a paradigm shift from current sequential cellular measurements in separate culture wells and equipment or at different times, we developed an integrated system—polybiosensing—for continuous and simultaneous polysensing and imaging of multiple phenotypic and functional properties in a single living culture. We hypothesize that spatiotemporally linked datasets from polybiosensing offer new actionable intelligence for better disease modeling and therapy development.

*Methodology: Biocompatible and optically thin gold substrates and polysensor-incorporated culture wells (PICW) were developed for 2D and 3D culture. Breast cancer (BCa) cells and human mammary-derived cell types (HMT-3522 BCa progression series) were seeded on ultrathin Matrigel-coated gold substrates and PICWs. After establishing phenotypes and structural signatures with a known biology in response to an abundant ligand in the tumor microenvironment of aggressive cancers, hyaluronan (HA), the polybiosensing system enabled temporal quantitative cellular analysis of a) morphology and motility by standard brightfield and RGB fluorescence imaging, b) growth, attachment, and detection of membrane integrity by electrical impedance sensing, and c) metabolism by pH, oxygen, and glucose levels per PICW. AI-driven data analytics connected and interpreted spatiotemporal datasets, providing deep insights for predicting outcomes.

*Results: Initial studies on optically thin gold substrates hosting BCa cells revealed distinct 3D phenotypes via high-resolution scanning electron microscopy (Figure 1A). Aggressive BCa cells exhibited nano-scale surface domains with distinct architectures and distribution, as well as tendrils extending >2mm from the central 3D colony mass. Dynamic polybiosensing enabled non-invasive orthogonal testing and streamlining of rich matrix information (currently up to 16 readouts) from a single living culture. Spatiotemporal monitoring up to 3 weeks did not inhibit cell growth. Real-time imaging and impedance sensing at 30-minute intervals showed stable differences between 2D/3D structures and the growth of the malignant MDA-MB-231 and T4-2, and the non-malignant S1 cells. Electrical impedance detected and quantified cell growth and death (induced by cytotoxic agents) faster than real-time imaging. Multi-frequency impedance scans revealed cell type dependencies with improved detection at higher frequencies. Clear distinctions were made between non-malignant and malignant cell types in terms of pH, oxygen and glucose consumption, and orthogonal electrical impedance assessments over time (Figure 1B). Continuous datasets (orthogonal, phenotypic and functional) were fed into our AI model for generating deep insights and actionable intelligence.

*Conclusion/Significance: Polybiosensing introduces a transformative cell analysis approach and the PICW acts as a discovery and modeling platform in pre-clinical and clinical R&D of regenerative medicine. This comprehensive and AI-based cell analysis engine not only saves time, cost, and scarce patient samples but also allows for one experiment with “n” sensors instead of “n” separate experiments on different samples.

233 - Hydrogel Delivery Of MicroRNA:nanoparticle Complexes Enhances Osteogenesis In MSC-laden 3D Hydrogels

S. Shrestha, T. Tieu, M. Wojnilowicz, N. Voelcker, J. Forsythe, J. E. Frith

Monash University, Melbourne, Australia

*Purpose/Objectives: MicroRNAs are small non-coding RNAs that regulate gene expression and which have powerful effects on cell proliferation, differentiation and migration. This makes them attractive candidates to improve the repair and restoration of damaged tissues, including for bone tissue engineering where osteoinductive signals can be used to promote osteogenesis of mesenchymal stromal cells (MSCs). Commonly used osteoinductive signals, such as growth factors, are hampered by their high cost, low stability and potential immunogenicity leading to growing interest in the delivery of microRNAs as a simple, inexpensive and powerful alternative to modulate MSC osteogenesis. However, advances in the materials used for miRNA transfection and an efficient way to deliver them to cells in a tissue engineering scaffold is required to provide efficient and reliable outcomes. This study aimed to develop a simple and efficient way to deliver pro-osteogenic miRNAs to MSCs encapsulated within a hydrogel to promote osteogenesis for bone tissue-engineering.

*Methodology: We have identified miR-125b-5p as strong pro-osteogenic miRNA which acts upon the fatty acid biosynthesis pathway to modulate MSC osteogenesis. A system was developed using porous silicon nanoparticles (pSiNP) modified with polyamidoamine (PAMAM) dendrimers (PAMAM-pSiNP) to deliver miR-125b-5p to human bone marrow-derived MSCs encapsulated within a photo-crosslinkable gelatin-polytheylene glycol (PEG) hydrogel. Loading and release of miRNAs from the PAMAM-pSiNPs, and release of PAMAM-pSiNPs from the hydrogel was optimized by monitoring miRNA levels in solution. Transfection efficiency was evaluated by flow cytometric analysis of MSCs transfected with fluorescently-tagged miRNA with internalization of the NPs evident from confocal imaging. A functional response was shown by using siTOX and via Western blotting of miRNA targets. Cell viability was determined by Live/Dead staining and osteogenesis measured via qPCR for RUNX2 and Osteoimage staining of mineral deposition.

*Results: Our results show that PAMAM-pSiNPs can be encapsulated within the hydrogel matrix and released gradually over 96 hrs. MSCs encapsulated within the hydrogels maintained high viability and were reliably transfected, with a higher efficiency than MSCs transfected via standard 2D methods. When evaluating MSCs in gelatin-PEG hydrogels, osteogenesis was increased when delivering PAMAM-pSiNPs and miR-125b-5p, showing greater expression of osteogenic markers and mineralization than in untransfected controls or those transfected with a scrambled miRNA sequence. Interestingly, a dual-effect on osteogenesis was observed, whereby the miRNAs increased expression of the osteogenic genes alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2) whilst the pSiNP themselves substantially enhanced mineralization. Measurements of pSiNP degradation confirmed that this was likely via their degradation into silicic acid, which can also enhance osteogenesis.

*Conclusion/Significance: Overall, this work presents insights into to the role of miR-125b-5p and fatty acid signalling in the regulation of osteogenesis. It also presents a simple yet effective system to enhance osteogenesis of MSCs by delivering miRNAs directly from a hydrogel. Together the work provides future targets to improve bone formation and a promising system for MSC-based bone tissue engineering.

234 - Electrical Stimulation Via Temporal Interference For CNS Repair

S. Merlo-Nikpay, S. Peressotti, A. Lee, R. Green

Imperial College London, London, United Kingdom

*Purpose/Objectives: Many pathological central nervous system (CNS) conditions are driven by dysregulated neuroinflammation, which can lead to loss of function and impaired regenerative capacity. Electrical stimulation (ES) has been proposed as a method to promote endogenous repair mechanisms in the CNS by influencing both neural precursor cells (NPCs) fate and inflammatory cells phenotype. Temporal interference (TI) has emerged as a new promising approach to electrically activate deep structures in the body without the need for invasive procedures. Specific TI stimulation frequencies can be leveraged to modulate neural tissue regeneration and inflammation via glial cells.

*Methodology: To examine the influence of TI on NPCs, bio-inspired stimulation frequencies of 2, 8, and 40 Hz were applied on two in vitro cell models for one hour a day. The first model consisted of NPCs cultured as neurospheres (NS) derived from the spinal cord of E14 Sprague-Dawley rats (NS-only model). As neuroinflammation can exacerbate damage and impair tissue repair, a second in vitro model was developed to recapitulate the injury microenvironment. This consisted of a monolayer of mixed glial population (MGP) from the hippocampus of P4 Sprague-Dawley pups, with NPC NS introduced on top of them (MGP-NS). This MGP-NS model was then subjected to the same bio-inspired frequencies to evaluate the interaction between NPC and inflammatory phenotypes. Proliferation, differentiation, and functionality of NPCs were compared to unstimulated controls using fluorescence confocal microscopy. Population percentage expression of Sox2 and Nestin were quantified to evaluate electrical stimulation-induced proliferation of NPCs at 7, 14 and 21 days in culture. Relative expression of glial fibrillary acidic protein (GFAP), β-III tubulin and oligodendrocyte factor 2 (Olig2) was used to evaluate TI-induced NPC differentiation into astrocytes, neurons and oligodendrocytes, respectively. TI-induced proliferation of the MGP monolayer was characterized by immunofluorescence confocal microscopy using GFAP, Olig2 and ionized calcium-binding adaptor molecule 1 (Iba1) as markers for astrocytes, oligodendrocytes and microglia, respectively. Moreover, morphological analysis of microglia and astrocytes was performed using GliaTrace (MATLAB), as cytoskeletal rearrangement has been implicated in indicating inflammatory status.

*Results: 8 Hz TI stimulation induced NSC proliferation and differentiation into neurons, as well as enhancing neuronal maturity. However, the behaviour of relevant glial populations remains unknown. It is expected that 8 Hz ES will promote branching of microglia and reduction of pro-inflammatory signalling, which can partly explain improved neural function and tissue regeneration.

*Conclusion/Significance: ES is a promising strategy to enhance neural repair for the treatment of CNS pathologies. TI stimulation at 8 Hz was shown to promote NSC proliferation and differentiation, and to influence inflammatory glial cells phenotypes. Thus, TI stimulation may provide therapeutic benefits in CNS conditions where neuroinflammation is an important component of the pathology, such as Alzheimer’s disease or spinal cord injury.

235 - Surface Functionalization Of Polidimetilxilosane For Organ-on-a-chip

M. P. Pérez Calixto¹, A. Shapiro¹, C. Peto-Gutierrez¹, L. Huerta¹, M. Macias-Silva¹, D. Pérez-Calixto², G. Vázquez-Victorio¹

¹ National Autonomous University of Mexico, Mexico City, Mexico,

² National Institute of Genomic Medicine (INMEGEN), Mexico City, Mexico

*Purpose/Objectives: An organ-on-a-chip is a microfluidic device for culturing cells in continuously perfused micrometer-sized chambers, in order to recapitulate model physiological functions of tissue or organs. PDMS is the most common polymer used to fabricate microfluidic devices, due to the relatively low-cost and easy fabrication procedures, non toxicity and biocompatibility¹. Even though it is the most common, the hydrophobic nature of PDMS makes a cell attachment challenging². Currently, the surface modification can be carried out by physical or chemical methods. One of the methods is using UV light; however, the wavelength emitted by a UV lamp (254 nm) is not energetic enough to turn into a hydrophilic surface, as a result the modification is temporary and does not allow more than 1 or 2 days of culture³. To overcome this problem, the surface modification can be carried out by ozone using a heterobifunctional molecule, since it does not require instrumentation or expensive and complex processes.

*Methodology: In this work, a liver organ-on-a-chip was fabricated, taking into account the physicochemical characteristics of a liver lobule using PDMS applying a cast-replica process from a CNC-molded PMMA mold. Simulation of fluid flow in the microfluidic device was performed by finite element analysis using Comsol Multiphysics 5.5 © software. To allow for adequate adhesion of extracellular matrix protein and cell attachment, the surface was functionalized with sulfo-SANPAH/O3 and Sulfo-SANPAH/UV lamp as a comparative method. Then, it was deposited in a combination of extracellular matrix protein (collagen and fibronectin) which promoted cell monolayer formation⁴.

*Results: In order to try to elucidate the functional group to which Sulfo-SANPAH molecule binds to the PDMS and the protein mixture the modified surface was characterized physicochemically by X-ray spectroscopy (XPS), Raman spectroscopy, contact angle, SEM and AFM, besides biological characterization was carried out. Interestingly, the XPS spectrum of PDMS ozone-oxidized showed a surface oxidation with OH groups, which allows the bond reaction with some functional groups of a Sulfo-SANPAH; however, surface oxidation by UV lamp did not show a surface oxidation. In all stages of surface modification,contact angle was measured. PDMS modified with sulfo-SANPAH/O3 - protein mixture decreased to 24° from 94°C of the pristine PDMS; meanwhile modification with only UV lamp a slight change is observed. For biological characterization, human liver endothelial cells were used and culture medium was perfused into the device at a volumetric flow rate of 2 µL/min by peristaltic pump assembly for 7 days. The cultured cells exhibited a high percentage of cell viability in static and flow conditions. Additionally, a different channel with hepatocyte was cultivated for 5 days showed a high viability and functionality showed canaliculi structure.

*Conclusion/Significance: Finally, the physicochemical and biological characterization of the channel microfluidic device demonstrated that it can recapitulate the adhesion and flow conditions necessary for liver endothelial cell functionality, making the modified surface with ozone and sulfoSANPAH molecule a useful technique for research use as an organ-on-a-chip in vitro model.

236 - Harnessing DNA Hybridization To Engineer A Novel Dynamic Biointerface With Tunable RGD Kinetics At Nanoscale To Influence Stem Cell Fate

X. Zhang, S. v. Rijt

Maastricht University, Maastricht, Netherlands

*Purpose/Objectives: The native extracellular matrix (ECM) undergoes constant remodeling, where adhesive ligand presentation changes over time and space to influence stem cell fate. Studying this dynamic ECM ligand-cell interaction is a crucial step towards the fabrication of advanced ECM-mimicking biomaterials with dynamic complexity associated with complex cell processes. To recapitulate the ECM dynamicity, different approaches and strategies have been explored to create dynamic platforms with smart surface properties capable of controlled display of bioactive ligands. Most current dynamic platforms are based on stimuli-responsive systems. Next to that, current approaches largely ignore the importance of ligand clustering, which is an essential feature in the ECM. In contrast to the previous reports, we propose a unique and novel strategy based on DNA hybridization, which allows us to study the importance of (clustered) ligand presenting kinetics in regulating stem cell fate.

*Methodology: In our approach, first, mesoporous silica nanoparticles (MSN) functionalized with a single-strand DNA (MSN-ssDNA) were developed to create 2D bioengineered MSN films. Using MSN to engineer the platform offers possibilities to control the ligand clustering at the nanoscale, essential feature of native ECM biology. Then, an adhesion ligand RGD was covalently conjugated to a complementary DNA strand (csDNA), which can specifically interact with a tether strand DNA (ssDNA) immobilized on the MSN films. DNA constructs with different nucleotides in length were employed for RGD conjugation to vary the ligand kinetics on surfaces. Finally, we study stem cell adhesion and migration in response to RGD presenting kinetics on the prepared MSN films.

*Results: Our results showed that low RGD dissociation kinetics led to enhanced stem cell adhesion, and stem cell spreading resulting in elongated cell morphology. Cells on surfaces with low RGD dissociation kinetics also exhibited higher motility with migration in multiple directions compared to cells on surfaces with fast RGD dissociation kinetics.

*Conclusion/Significance: This study contributes to the existing body of knowledge on dynamic ligand-stem cell interaction. Importantly, it aids in unraveling the complexity of cell adhesion and migration processes, contributing to a deeper understanding of stem cell fate in complex environments. Understanding the dynamic interaction between stem cells and biochemical cues has been a topic of broad interest, especially in the field of regenerative medicine but the platform as well as the biological results are translatable to other biomedical fields.

237 - Multi-responsive Hydrogels Act As Smart Sweeper Of Pathological Microenvironment To Promote Diabetic Wound Healing

M. Bao

Shanghai Jiaotong University, Shanghai, CHINA

*Purpose/Objectives: Wounds in Type II diabetes mellitus (T2DM) are characterized by complex pathogenesis and difficulty in wound healing. Overcoming the inhibitory effects of high glucose and inflammatory environment on tissue regeneration in T2DM remains challenging in T2DM-related tissue engineering. Traditional biomaterials suffer from issues such as mismatch between drug dosage and demand, inadequate efficacy, and the need for repeated administration. To address these problems, we designed a multi-responsive composite hydrogel enabling spontaneous and intelligent release of the load to promote diabetic wound healing. This provides new insights and strategies for diabetic tissue regeneration.

*Methodology: We synthesized a smart composite hydrogel based on a PBA-PGA system, loaded with metformin, to create a microenvironment-responsive hydrogel. Chemical T2DM models were established on C57BL/6 mice and full-thickness skin defects were made. Smart hydrogels were then applied to the wounds. After 14 days, the mice were sacrificed and the tissue around the defects were collected to evaluate the effects. When applied to the defects, this material can detect characteristic pathological factors of the diabetic microenvironment, such as high glucose and low pH, in a multi-responsive way. When the level of any pathological factors reaches a critical threshold, it triggers the degradation of the material and enables the real-time dynamic release of the loaded microenvironment regulator, metformin. This helps maintain a stable regenerative microenvironment by regulating blood glucose and pH levels, thereby promoting tissue regeneration. Metformin, a traditional antidiabetic modulator, as the core component of the composite hydrogel, acts an AGE-breaker and ROS sweeper- a brand new role to reverse the detrimental microenvironment.

*Results: The smart hydrogel demonstrates mechanical adaptability to wounds and exhibits efficacy in reducing inflammation and promoting collagen formation. It possesses the ability to eliminate advanced glycation end-products and relieve oxidant stress-associated effects, thus effectively enhancing wound healing.

*Conclusion/Significance: Our study indicates the potential of this smart hydrogel in promoting diabetic wound healing, especially in 1) its targeted elimination of diabetic pathological factors as a microenvironmental regulator; 2) the novel application of the ‘old star’ metformin. The easy availability and practicability of the smart hydrogel might provide us with a new perspective of diabetic wound treatment.

238 - Development Of Bifunctionalized Microcarriers For The Isolation, Culture And Ex Vivo Expansion Of Endothelial Progenitor Cells

M.-A. Campeau, H. Level, C. Hoesli

McGill University, Montreal, QC, Canada

*Purpose/Objectives: Through their role in vascular regeneration, endothelial progenitor cells (EPCs) have emerged as promising therapeutic candidates to treat cardiovascular diseases. Recent clinical studies demonstrated accelerated revascularization of ischemic tissue after localized EPC delivery, holding great promise as a minimally invasive treatment for myocardial infarction or critical limb ischemia. Yet, our ability to effectively isolate, culture and expand EPCs remains limited and prohibitive to the development of EPC-based therapeutic products. While typical EPC culture involves a multi-step process resulting in reduced cell yield, functionalized material surfaces can be used to drive specific cell responses and to provide a scaffold for growth as in the case of microcarriers. By consolidating the isolation and culture steps, our goal is to reduce process complexity and to minimize manipulations with a potential for higher yield, which represent critical hurdles in biomanufacturing. We hypothesize that the use of functionalized microcarriers with a dual function of cell capture and growth will accelerate the ex vivo expansion of EPCs.

*Methodology: Antibodies and biomimetic peptides were immobilized on microcarriers after adapting our patented surface functionalization method, enabling the selective capture and subsequent culture of EPCs. Aminomethyl polystyrene beads (200 μm) were first functionalized with a combination of Fc-binding RRGW peptides and cell-adhesive RGD peptides though maleimide-thiol conjugation, followed by immobilization of anti-CD309 antibodies through interaction with RRGW. The resulting beads present oriented anti-CD309 antibodies to selectively capture EPC while RGD can mediate clonal expansion of ECFCs (Fig 1A). RDG peptide was used as a negative control. Affinity immobilization of antibodies was tested by ELISA with peptide concentrations ranging from 1 to 1000 µM and various combinations of RRGW and RGD. Human EPCs from adult human peripheral blood were expanded in standard conditions (collagen-coated flasks) up to passage 2 to 4, after which their response to functionalized surfaces was tested in static or stirred conditions. Captured EPCs were enumerated and their cytoskeletal organization as well as proliferation were evaluated. The efficiency of capture on microcarriers under agitation was assessed through metabolic assays and fluorescent microscopy.

*Results: As expected, increasing the RRGW:RGD ratio led to a linear increase in surface antibody density. Increasing RRGW concentration resulted in an increase in the number of EPCs captured on microcarriers after 1h-incubation under agitation (Fig 1B). Compared to control RDG peptides, RGD-presenting surfaces improved cell spreading and retention which synergized with the effect of the antibody alone (Fig 1C). RGD also accelerated the initial attachment and spreading in serum-free conditions (Fig 1D-G), suggesting an enhanced adhesion to microcarriers for subsequent expansion (Fig 1H-K).

*Conclusion/Significance: Future work will test the compatibility of the antibody+RGD modified microcarriers on EPC expansion in scale-up bioreactor systems, as well as selective capture from mixed populations. Ultimately, this validation study could support the development of therapies for the treatment of ischemic tissues as well as the application of in situ endothelialization strategies for cardiovascular implants.

239 - Towards Seamless, Biomimetic, Bioactive Multi-channel Peripheral Nerve Guidance Conduits Made Of Silk Fibroin Nanofibres

D. Torres¹, S. Cartmell¹, J. Knowles², S. Rawson¹, J. Blaker¹

¹ The University of Manchester, Manchester, UNITED KINGDOM,

² University College London, London, UNITED KINGDOM

*Purpose/Objectives: Peripheral nerves can, under certain conditions, regenerate and reinnervate tissue after an injury. This natural capacity can be enhanced by guiding axonal growth from the proximal to the distal end of the injury with biochemical or morphological cues. However, producing devices with both types of cues is a complicated challenge that has no definite solution yet. In this work, seamless, nanofibrillar multi-channel silk fibroin conduits have been designed with tuned topography (nanofibrillar alignment) and biochemical composition (silk fibroin functionalisation with norbornene). This was obtained by overcoating dissolvable phosphate glass macrofibres fibres with silk nanofibers exploiting an electrospinning nanofibre yarn module. To produce biochemical cues, silk was chemically modified to promote UV-catalysed functionalisation that could generate bioactive patterns on nanofibrillar surfaces.

*Methodology: Silk fibroin cast films were dissolved in HFIP to obtain 6% w/v solutions. These solutions were electrospun into nanoyarns using a custom-built yarn module to overcoat sacrificial phosphate glass fibre (PGF) bundles. Multiple overcoated PGF bundles were then put together and further overcoated, then the PGF bundles were dissolved out to produce seamless multi-channel structures of silk nanofibres. This was achieved by submerging the constructs in DI H₂O to dissolve the PGF, followed by freeze drying. The morphology of the multichannel conduits was elucidated and quantified using X-ray computer tomography. Wet-state mechanical properties were also measured on hollow conduits. To functionalise silk fibroin (SF) with norbornene, a 1% w/v SF, 5% w/v carbic anhydride solution was kept at pH 8-9 for 24 hours, then dialysed for 2 days. C-NMR was used to determine the presence of norbornene groups. Electrochemical Impedance Spectroscopy was used to measure electrochemical properties of the materials.

*Results: The nanofibrillar alignment of the surfaces can be successfully tuned by changing process variables, and a minimum alignment of 10° against the construct axis was achieved. The same applies for lumen diameter, which can be modified by changing the diameter of the sacrificial substrate before the nanofibrillar coating. Multichannel conduits were obtained by two-step electrospinning and the dissolution of PGF and freeze-drying allowed for the production of hollow channel structures with aligned inner surfaces and no seam section across the mantle. The wet-state mechanical properties of these devices are very similar to those reported for peripheral nerve tissues. Silk functionalisation with norbornene was confirmed through C-NMR spectrums and fluorescent patterns were obtained. A change in the electrochemical behaviour of the material was observed, from proton conductivity on SF and reflectin, to anionic conductivity on SF-NB.

*Conclusion/Significance: Nanofibrillar silk constructs appear to have good potential for nerve tissue regeneration applications. The tuneable size and scalable length inherent to the process can produce different micro or macro-organisations that mimic the architecture of specific native nerves, and this is easily achieved by modifying process variables. The chemical modification of silk and further functionalisation with UV-catalysed patterns also shows the potential of solid surface functionalisation for biochemical patterning on surfaces for cellular growth. The addition of norbornene does not show cytotoxicity or reduced biological behaviour in vitro.

240 - In Vitro Immunomodulation Of An Inflammatory Osteoarthritic Model With Sustained Release Of Curcumin From A Dual Crosslinked Photocurable Injectable Polymeric Nanoparticulate Composite System

A. Mukherjee, B. Das

Indian Institute of Technology Ropar, Rupnagar, India

*Purpose/Objectives: Osteoarthritis (OA) is one of the most common musculoskeletal diseases that afflicts 654.1 million people worldwide. Globally, osteoarthritis is the 4th most significant cause of incapability in women and 8th in men. In India, OA is the second most common rheumatologic problem, and it is the most frequent joint disease, with a prevalence of 22% to 39% in India. A hundred million people worldwide have disability due to osteoarthritis. The development of OA is closely related to the joint's decreased ability to lubricate, where articular cartilage loss is cumulative and permanent, and subsequent inflammatory responses are the main mechanisms.

*Methodology: In this study, we have fabricated a nanoparticle (NP) formulation of curcumin encapsulated in PLGA using a novel vehicle such as GelMA & sulphated polysaccharide-based injectable hydrogel crosslinked with potassium chloride and investigated the sustained release of curcumin for in vitro immunomodulation of inflammatory osteoarthritic model. Cur/PLGA-NPs encapsulated photocurable injectable hydrogel was formulated using photoinitiated radical polymerization to support human mesenchymal stem cells (hMSCs) adhesion by creating a microenvironment that promotes cell growth.

*Results: The dynamic light scattering (DLS) technique was used to determine the particle size, size distribution, and morphology of the effectively generated NPs, which were verified using the electron microscope. Different conjugation processes of functional groups were confirmed using the FTIR study. Detailed physiochemical properties such as swelling behaviour and degradation analysis, rheological properties, surface wettability, in vitro cytotoxicity, cytocompatibility and stem cell differentiation of NP-encapsulated hydrogel were investigated using cellular assay and PCR analysis. Conventional in vitro cell studies were also carried out to evaluate the viability of Cur/PLGA-NPs and the cellular uptake of Cur/PLGA-NPs into hMSCs in vitro. In vitro ROS scavenging activity was found to be favourable for the intended application. Further co-culture study was performed using chondrocytes and human THP-1, differentiated into M1 phenotype to determine the anti-inflammatory potential.

*Conclusion/Significance: In this way, we were able to provide a curative method for facilitating cartilage regeneration in osteoarthritic patients.

243 - Contact Drawn Biosynthetic Collagen Fiber Scaffolds Drive Anisotropic Cellular Organization For In Vitro Models

K. Pasmarthi¹, D. Bhattacharyya², A. Mishra¹, J. P. Frampton²

¹ Department of Pharmacology, Dalhousie University, Halifax, NS, CANADA,

² 3DBioFibR Inc., Halifax, NS, CANADA

*Purpose/Objectives: Anisotropic cellular organization is critical to the function of a variety of human tissues, including skeletal muscles and myocardial tissues. Surface patterns, such as microfabricated grooves or patterned coatings, are often used to model anisotropic cellular organization in vitro. However, surface patterning is limited by an inability to propagate the alignment cues into three dimensions. To create more robust 3D anisotropic cellular models, contact drawn collagen fibers have been used to create intricately aligned collagen scaffolds.

*Methodology: Collagen fibers are produced by contacting pins to the surface of a type I collagen/templating polymer solution, and then, rapidly pulling away the pins to nucleate liquid bridges that elongate into filaments through extensional flow. The filaments rapidly dry and can be collected on a substrate for assembly into an anisotropic collagen fiber scaffold. C2C12 myoblasts were seeded onto the scaffolds at 250,000 cells/mL and cultured for 2 days in growth medium and 7 days in differentiation medium. Immunolabelling for myosin and Hoechst staining were used to calculate the fusion index (# of nuclei in multinucleated structures/# of nuclei in the field of view). C2C12 cells cultured on glass served as a control. In addition, day 11.5 embryonic cardiac cells were seeded onto the scaffold at 200,000 cells/mL and cultured for three days. Immunolabeling was used to detect sarcomeric myosin heavy chain and connexin 40. Cell morphology (area, perimeter, circularity index, and aspect ratio) was also examined. Cardiac cells cultured on glass served as a control.

*Results: Relatively few myotubes formed for the glass control in the C2C12 experiments, and they were poorly aligned. In contrast, C2C12 cells cultured on the collagen fibers were well aligned and formed multinucleated structures indicative of myotube formation. The templating of myotubes by the collagen microfibers is clearly seen at higher magnification, where strong myosin expression closely follows the collagen microfibers. There is higher intensity of myosin and lower intensity of collagen in areas with stronger myosin expression, which may suggest that mature myotubes remodel the collagen fibers, which would resemble the natural process for muscle regeneration in which collagen fibers initially direct the growth and alignment of muscle fibers, followed by degradation/remodeling of the collagen fibers to form the mature muscle tissue. When cultured on the aligned collagen fibers, over two thirds of the cells formed myotubes (2X higher than the control). Similarly, cardiac cells were elongated on the collagen fibers as compared to the glass only control. Compared to the 2D glass control, when grown on the collagen fiber scaffolds, cardiomyocytes had 60% lower circularity index and 300% higher aspect ratio. The aspect ratio of 7.4 achieved on collagen fiber scaffolds falls within the normal range of adult cardiomyocytes, creating an efficient substrate to drive physiologically relevant cell morphology for in vitro modelling.

*Conclusion/Significance: Collagen fibers produced by contract drawing can be used to create 3D anisotropic cellular organizations that more closely match the physiological cellular morphologies, as demonstrated with skeletal muscle and embryonic cardiac cells.

244 - Nucleic Acid-Collagen Complexes: Unveiling Hierarchical Assembly And Physicochemical Characteristics Towards Advanced Tissue Engineering

N. Pipis, K. A. Stewart, S. Tabataei, J. B. Allen

University of Florida, Gainesville, FL

*Purpose/Objectives: In tissue engineering technologies, achieving biomimicry, biocompatibility, simplicity, and tunability, while facilitating targeted bioactivity poses a significant challenge. This study delves into Nucleic Acid-Collagen Complexes (NACCs) to explore their potential as a system with wide-ranging applicability. Single-stranded DNA (ssDNA) has been shown to complex with collagen type I to form fibrous structures that resemble native extracellular matrix architecture, without the need for external stimuli or complex chemistries. The presence of short ssDNA in the form of aptamers, exhibits versatile molecular recognition capabilities and can induce bioactivity towards numerous applications. The bioactive potential of NACCs has been demonstrated, and existing work suggests that DNA-collagen fibrous complexes utilizing function-specific aptamers can be used towards bone tissue engineering and angiogenic-like endothelial cell phenotypes. However, the detailed mechanisms behind this fibrillogenesis remain unknown. Here, we attempt to unravel the role of NACCs in shaping conducive microenvironments, paving the way for developing advanced biomaterial matrices for organoid growth and 3D cell culture with precise control over their properties and functionalities.

*Methodology: Our research investigates the hierarchical assembly dynamics of triple-helix collagen type I polypeptides and ssDNA aptamers. Microscopy techniques such as Confocal Reflectance, Transmission Electron (TEM), and Atomic Force Microscopy (AFM), revealed the topography of NACC fibers across multiple length scales. These structural characterizations were complemented by precise analysis using Infrared Spectroscopy (FTIR) to uncover DNA-protein interactions, as well as rheology to measure shear elastic moduli. Additionally, Isothermal Titration Calorimetry (ITC) shed light on the thermodynamic principles by which ssDNA triggers collagen aggregation, giving us general mechanistic conclusions regarding the ssDNA-collagen interaction.

*Results: Our results indicate that ssDNA serves as a linker and agglomerating agent that promotes the complexation of individual collagen fibrils (∼1μm wide) into bundles of around 10μm in width. This was confirmed both by CRM and TEM (Fig.1a-c). We also captured high-quality topographical AFM images of intertwined NACC fibrils down to nanoscale resolution. Collection of the IR spectrum revealed amplified amide band signals in NACCs compared to collagen alone, but no peak shifts, indicating a purely supramolecular interaction and unchanged chemical compositions of both ssDNA and collagen after complexation (Fig.1d). Alterations in the structural organization of collagen (i.e. filament size) due to the addition of ssDNA significantly increases the bulk shear elastic modulus, as measured by rheology (Fig.1e) and quantitative AFM. ITC suggests that the thermodynamic contribution of the aggregation decreases as the molar ratio of collagen to ssDNA increases, rendering ssDNA as the limiting reagent in this interaction. ssDNA directs the assembly of collagen, acting almost as a “molecular glue”, controlling and promoting the complexation of individual fibrils into large aggregates.

*Conclusion/Significance: NACCs emerge as a versatile, next-generation biomaterial platform, offering tunable composition and structure. Our understanding of their hierarchical assembly and molecular interactions positions NACCs as promising candidates for diverse applications. These biomaterials exhibit potential as osteoconductive coatings, implants, and 3D tissue models, showcasing adaptability across varied tissue engineering domains. Their simplicity and versatility suggest broader utility as coatings and scaffolds, hinting their transformative potential in advanced biomaterial design.

245 - An Open-source Approach To Accelerate Emerging Biofabrication Technologies

I. von Briesen¹, I. Liashenko¹, S. Luposchainsky², H. Xu², A. Reizabal³, P. Dalton¹

¹ University of Oregon, Eugene, OR,

² Kyoto Institute of Technology, Kyoto, Japan,

³ BC Materials, Leioa, Spain

*Purpose/Objectives: The high cost of commercial printers acts as a barrier to accessing biofabrication technologies, and innovation is impeded by the accompanying “no modification” warranties. The purpose of this project is to democratize access to emerging technologies by transforming a $700 open-source fused-filament fabrication (FFF) Voron printer into a system capable of performing functions well beyond its original design. This is exemplified here for an emerging technology, Melt Electrowriting (MEW), where commercial systems generally exceed $40,000 and can use only a limited set of materials. A Voron FFF printer is therefore converted into a high-performance system with expanded MEW capabilities, processing an important clinically relevant polymer, polydioxanone (PDO). This polymer degrades within six months and is widely used in the clinic as a bioresorbable suture. This accelerates both the accessibility and capabilities of this technology with a grassroots approach to building and modifying new innovative systems.

*Methodology: After assembling and calibrating a Voron 0.1 FFF printer, the default extrusion motor is replaced with a high-resolution stepper motor, facilitating the low flow rates essential for microfiber production (Figure 1). PDO, a biodegradable polymer that is difficult to melt-process due to its thermosensitivity and long print times, was chosen to challenge the MEWron system. PDO filament, sold as Dioxaprene (Poly-Med, USA) and made under Good Manufacturing Practice (GMP) conditions, was processed with a spectrum of printhead temperatures (125-150°C), extrusion rates (6-30 μL/h), distance between the nozzle and collector (1.5-3 mm), and corresponding voltages of 1.5 kV/mm were explored to demonstrate the breadth of outcomes resulting from different printing parameters. Poly-ε-caprolactone (PCL) made under GMP conditions was used as a control.

*Results: The fidelity of filament MEWron scaffolds was initially verified by printing identical validation scaffolds from a well-defined material control, poly-ε-caprolactone (PCL), on both filament and syringe systems. The resultant PCL scaffolds had homogeneous microfibers, precise vertical stacking, and very similar overall geometries compared to those made with commercial MEW printers. With a filament printhead containing a “hot zone” of only 55µL, the PDO filament was exposed to reduced melt times and allowed processing of microfibers. While extreme thermal degradation can be seen within two hours of printing PDO filament at higher temperatures (Figure 1), closer to the melting point, the filament MEWron can print well-organized and high-quality PDO microfibers (∼20μm) for over two hours (Figure 1). The continuous replenishment of filament enables stable printing of PDO for over 18 hours, demonstrating sustainable MEW printing of PDO from filament for the first time.

*Conclusion/Significance: Converting open-access printers accelerates the innovation of emerging biofabrication techniques, significantly reducing the overall cost while increasing performance. None of the existing commercial MEW printers are capable of processing PDO as they lack filament feed and a heated collector. Our experience demonstrates that the open-source Voron 0.1 and all possible variations can revolutionize university-based research. The enhanced accessibility of this system empowers researchers to delve into the exploration of new biomaterials and biofabrication processes to tackle diverse challenges in tissue engineering and regenerative medicine.

248 - Robotic-based In Situ Bioprinting Of Photo-curable Biomaterials Onto Complex Anatomical Defects

G. Vozzi, G. Fortunato, C. De Maria

University of Pisa, Pisa, Italy

*Purpose/Objectives: In situ bioprinting has emerged as a promising technology in the regenerative medicine field. It consists of the direct deposition of biological material into the patient, following the non-planar geometry of the anatomical defect. This study proposes an innovative method to generate objects with non-planar surfaces on highly geometrically complex substrates, by combining traditional planar layers with upper and lower non-planar ones. The algorithm was designed to be used on IMAGObot, a 5-axis robotic bioprinter equipped with a pneumatic extrusion-based bioprinting unit, which includes a photo-crosslinking unit focused on the just-deposited biomaterial.

*Methodology: The non-planar slicing algorithm was implemented in Matlab® and a graphical user interface was created to allow an intuitive interaction also by non-expert users. The algorithm can combine planar and non-planar layers (Fig. 1A-C). For generating the planar toolpath, the 3D model is cut horizontally by as many planes as the number of layers, defined by the layer height. Then, the top surface is generated: the 2D surface (as seen from the top view) is projected onto the model to obtain the Z-coordinates and generate the final 3D non-planar surface. Considering the lower surfaces, the geometry is rotated by 180° around the X or Y-axis, and all the steps previously needed are performed followed by an inverse rotation of the obtained points. The final geometry is thus composed of two non-planar portions, and an internal planar region.The path-planning algorithm enables the management of a purposely developed photo-crosslinking device installed on the IMAGObot pneumatic extruder. The system consists of a support adjustable in height with respect to the printing needle (to modulate the substrate exposure) which allows the direct accommodation of 8 LEDs (λ=405 nm) (Fig. 1D). To minimise the light exposure of the needle, and avoid its clogging, only the LEDs opposite to the printing direction are switched on to irradiate the newly deposited filament. The algorithm was validated performing different in situ bioprinting tests onto purposely designed phantoms using a 5% w/v GelMa+0.5% w/v LAP hydrogel. Different photo-polymerization parameters (e.g., speed, distance) were tested, assessing the swelling after 24 h in deionized water at 37°C and the mechanical properties (uniaxial compression).

*Results: IMAGObot was able to follow the non-planar trajectories during the printing phase with high accuracy (lower than 0.2 mm) and no collisions with the substrates, thus proving the robustness of the developed method. The photo-crosslinking device was able to polymerise the deposited hydrogel for all the tested fabrication parameters (Fig. 1E). The swelling behavior resulted not dependent both from the printing speed and distance from the exposure system. No differences were also highlighted in the mechanical properties (elastic modulus ∼50 kPa).

*Conclusion/Significance: In this work, a novel non-planar slicing algorithm also managing the photo-polymerisation during the deposition process was presented and validated for in situ bioprinting applications. The developed method can be used for the slicing and fabrication of geometries with any level of complexity (typical of the anatomical defects) thus representing a great improvement over approaches previously developed.

250 - Development Of A Novel Extrusion-based 3d-printing Method: Dragging Technique And Its Application To Tubular Tissue Engineering

H.-J. Jeong¹, H. Nam², J.-S. Kim^3,4, S. Cho², S.-J. Gwak^3,4, H.-H. Park^3,4, Y.-S. Cho^3,4, H. Jeon⁵, J. Jang^2,6, S.-J. Lee^3,4

¹ Columbia University, New York, NY,

² Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea, Republic of,

³ Wonkwang University, Iksan, Jeonbuk, Korea, Republic of,

⁴ Wonkwang University, Iksan, Korea, Republic of,

⁵ Seoul National University of Science and Technology, Seoul, Korea, Republic of,

⁶ Pohang University of Science and Technology, Pohang, Korea, Republic of

*Purpose/Objectives: Tubular tissue and organ, such as vascular system and gastrointestinal (GI) track, exhibit a hierarchical multi-cellular structure. Due to the inherent physiological role of these multi-layered cell structure, numerous endeavors have been attempted to replicate such configurations in tissue engineering and regenerative medicine. Although promising outcome has been reported through 3D fabrication methods, 3D bioprinting, electro-spinning, cell membrane roll-up, significant limitation still exists in the realization of tubular construct. The successfully create transplantable tubular construct needs to essential requirements, including simplified fabrication processes, multi-cellularity, and comprehensive target tissue-specific mechanical functionality. In this study, we established the new extrusion-based 3D printing technique, called by dragging printing, it allows to enable sequentially print synthetic polymer and cell-laden tissue-specific decellularized extracellular matrix (dECM) bioink to create porous multi-cells layered construct in one-step processing.

*Methodology: Dragging technique utilizes the elongation properties of synthetic polymers (Polycaprolactone, FDA approved) to create porous structures by moving quickly and continuously between the columns (Fig. 1A). This technique allows to create various tubular construct (Fig. 1B-D). Through dragging technique, we applied to development small diameter vascular construct and esophageal tissue engineering. To fabricate an artificial small-diameter vasculature (SDV), we prepared Human Umbilical Vein Endothelial Cell (HUVEC) and Human Aortic Smooth Muscle Cell (AoSMC) encapsulated collagen bioink (cell concentration rate 4:1) and printed bioink in dragging printed tubular construct to produce the SDV construct (inner diameter 5mm) (Fig. 2A). To build engineered esophageal tubular structure, we designed a multi-layered free-form tubular construct (MFT) with a wrinkle structure and bellows pattern to mimic histomorphology feature of native tissue. Decellularized extracellular bioinks derived from esophageal mucosa and muscle tissue were fabricated and printed within each compartment to construct a multi-cellular tubular structure. (Fig. 2B).

*Results: We confirmed that adjusting dragging parameter enable the pore size control on the tubular construct. Additinally, mechanical test revealed control the stiffness of the structure depending on pore size. In the engineered esophageal tissue study, immunohistology presented distinct separately loacted cell-layer, epithelial and smooth muscle, in each compartment for 2wks culture. This founding suggests that engineered esophageal structure not only sustains multi cellular configuration but also facilitate crosstalk between each compartment’s cell component during maturation. found that We speculated that it could hypoxia effect of HUVEC’s. In the oxygen concentration study, group with gradually decrease oxygen concentration exhibited notable HUVEC migration toward the inner later than consistent concentration group. This finding suggest that oxygen concentration could eggect to the simultaneously HUVEC migration. Finally, we were be able to conformed the endothelium functionality through a platelet attache test conducted with human blood flow.

*Conclusion/Significance: The dragging technique developed in this study is a unique technology that can easily fabricate a free-form porous multi-layered structure in a one-step process without sacrificial materials or post-processes. By combine with dragging technique and bioprinting technology, we successfully develop the multi-cellular tubular structure with physiological characteristics of native tissue. In the future, the dragging printed tubular structure could be applied to a variety of various tubular tissue engineering as promising approach.

251

252 - Patterning And Controlled Compaction Of Cells Embedded Within Thermosensitive Hydrogels For Anisotropic Tissue Engineering

A. Pylostomou^1,2,3, J. K. Wychowaniec², R. Tognato², C. J. Edwards-Gayle⁴, J. R. Weiser⁵, D. Loca^1,3, M. D’ Este², T. Serra², A. J. Vernengo^2,6

¹ Faculty of Materials Science and Applied Chemistry, Rudolfs Cimdins Riga Biomaterials Innovations and Development Centre of RTU, Institute of General Chemical Engineering, Riga Technical University, Riga, LATVIA,

² AO Research Institute Davos, Davos, SWITZERLAND,

³ Baltic Biomaterials Centre of Excellence, Headquarters at Riga Technical University, Riga, LV, Riga, LATVIA,

⁴ Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UNITED KINGDOM,

⁵ Department of Chemical Engineering, The Cooper Union for the Advancement of Science and Art, New York, NY,

⁶ Department of Chemical Engineering, Henry M. Rowan College of Engineering, Rowan University, Glassboro, NJ

*Purpose/Objectives: Anisotropic tissues are prevalent in the musculoskeletal system, presenting challenges in replicating cell-scale complexities during tissue regeneration. Traditional tissue engineering often focuses on scaffolds with spatially aligned cell patterns, but cell migration in random directions occurs due to the absence of dynamic signals maintaining alignment. To address this, we introduce EXPECT (EXtrusion Patterned Embedded ConstruCT), a thermosensitive hydrogel based on poly(N-isopropylacrylamide) grafted with chondroitin sulfate (pNIPAAm-CS). EXPECT exhibits shear thinning, yield stress, and self-recovery, allowing the creation of embedded mesenchymal stem cell (MSC)-laden cylindrical channels via 3D printing. The hydrogel undergoes a transition from a hydrophilic liquid to a hydrophobic solid state at its critical solution temperature of 32°C. We hypothesized that by periodically cooling the 3D printed constructs below the LCST for short intervals throughout the culture period, the formation of closely associated and oriented cellular patterns could be stimulated.

*Methodology: Human bone marrow single cells or pre-aggregated cells in pellets were suspended in fugitive bioink comprised of 6% w/v porcine gelatin at 4x10⁶ cells/ml. Bioinks were microextruded through a 300 µm (inner diameter) needle into 1 mL of EXPECT hydrogel in an embedded ring pattern (10mm diameter, figure 1a) at 25°C composed of 3 w/v % pNIPAAm-CS, 0.8 w/v % CP and 1 w/v % gelatin. Cells were cultured for 36 days under temperature-actuated (37°C to 25°C for 15 min every 5 days) or static (37°C) conditions. Using confocal microscopy with phalloidin and DAPI staining, coupled with semi-quantitative analysis using ImageJ, we measured width of the printed cell patterns at days 0, 7 and 36 to understand differences in alignment among the groups.

*Results: For single cells under temperature-actuated conditions, pattern width reduced from 390 ± 113 μm at day 0 to 213 ± 69 μm by day 36 (p<0.001). In static conditions, we measured a significant increase relative to day 0 to 536 ± 72 μm (p<0.001), indicating possible migration beyond their seeded channel placement. Pellets under static conditions maintained their shape, while under temperature-actuated conditions, their width decreased from 180 ± 32 µm to 119 ± 22 µm (p<0.01), forming column-like structures by 36 days (Figure 1b).

*Conclusion/Significance: In conclusion, exploiting the thermosensitive nature of pNIPAAm holds promise for enhancing the alignment and organization of engineered tissues, both in single cells and aggregated pellets.

254 - Biofabrication Of A Biomimetic Vocal Fold Microtissue

L. M. Medina, E. Dogan, H. Goodarzi, A. K. Miri

New Jersey Institute of Technology, Newark, NJ

*Purpose/Objectives: Understanding vocal fold (VF) pathologies and their mechanisms would require in vitro models that mimic the biophysical properties of VF tissue. The VF tissue is a multilayered structure with a soft avascular lamina propria and a vocalis muscle. During voice disorders, any change in the stiffness or biophysical properties of the extracellular matrix (ECM) impairs phonation parameters. Most of the current approaches used bioreactor models or excised larynges. They need more control over microscale phenomena and relevant repeatability. There is a need for an engineered biomimetic VF model that allows studying cell responses at microscales. We presented a microfluidic-based model of the VF tissue via hydrogel engineering and 3D printing. The tissue was modeled using a gelatin scaffold and a glycosaminoglycan matrix of hyaluronic acid (HA).

*Methodology: We introduced a hydrogel-based microfluidic model established in our lab, which consisted of two major layers. The first layer comprises a well-engineered form of gelatin as gelatin methacrylate (GelMA) and HA as hyaluronic acid Methacrylate (HAMA). The second layer will be acellular and be made of a synthetic hydrogel for the muscle: polyethylene glycol diacrylate (PEGDA; Sigma) at a fixed concentration (35% v/v). We followed our standard protocol to make GelMA in different mass concentrations: 2.5%, 5%, 7.5%, and 10% w/v. We developed a protocol for extrusion printing of HAMA at various mass concentrations: 0%, 0.5%, 1%, and 2% w/v. The photoinitiator was Irgacure (Sigma, 0.5% w/v). We tuned the mechanical properties of the target lamina propria using compressive modulus, storage/loss shear moduli, ECM porosity, as well as other physical properties of VFs. We defined three statuses of VF stiffness: healthy VF (10-100 kPa), inflammation-associated VF (1-10 kPa), and chronically scarred VF (250-750 kPa). We then studied the role of stiffness on VF fibroblast behavior at static (no vibrations) and oscillatory conditions (periodic vibrations at 100 Hz). We used an immortalized VF fibroblast cell line (HVOX) and immunostaining of cells to confirm the production of αSMA and Collagen I.

*Results: We showed the stability of our hydrogel scaffolds via swelling ratio and degradation rates up to two weeks. We evaluated the cell biocompatibility of our scaffolds using a quantitative essay over one week to assess cell proliferation at various cell densities. The model showed high cell proliferation in the lamina propria for all groups. We used the tunable properties of GelMA and HAMA to show how cells behave under oscillations and stiffness variations. We then showed how stiffer gel led to more generation of α-SMA and Collagen I proteins in the ECM model. The vibrations observed no variations among those markers, but the proliferation rate of HVOX was increased on Day 3. The results pave the way for more biological analysis of the model.

*Conclusion/Significance: Our objective is to optimize our material systems to create a biomimetic environment while maintaining stability of our microfluidic device for the long term. The next step is to add immune-response cells to the model to include the impact of macrophages in the development of scarring.

255 - Quadruple Culture Of Primary Human Osteoblasts, Osteocytes, Osteoclasts And Endothelial Cells - An Advanced 3D Bone Model

K. Wirsig, N. Bürger, R. F. Richter, A. Bernhardt

TU Dresden Faculty of Medicine Carl Gustav Carus, Dresden, Germany

*Purpose/Objectives: With an ageing population worldwide, research into bone metabolism and novel therapies for damaged and diseased bone is essential. Therefore, in vitro bone models that mimic the tissue as closely as possible are desirable. Bone is a vascularised, dynamic tissue that undergoes a remodelling process mediated by osteoblasts, osteoclasts and osteocytes. In addition, osteogenesis and angiogenesis are mutually dependent. We have developed an in vitro human bone model comprising the three main bone cell species - osteoblasts, osteoclasts and osteocytes as well as endothelial cells, isolated from primary human tissue. The established model is suitable for analysing the effect of versatile factors on bone cell differentiation, endothelial cell tube formation and crosstalk between these four cell types.

*Methodology: Osteoblasts were isolated from human femoral heads. Osteocytes were differentiated from osteoblasts in collagen gels and osteoclasts were differentiated from peripheral blood mononuclear cells (PBMC). All three bone cell types were separated from each other to allow exchange of soluble signalling molecules and separate analysis of the cell-specific markers. Commercially available human umbilical vein endothelial cells (HUVEC) were seeded on top of osteoblasts. Quadruple cultures as transwell insert constructs (figure 1) were conducted for 14 days under hypoxic conditions. Microscopic investigations, gene expression analysis as well as quantification of cell type-specific enzyme activities and protein secretion were performed.

*Results: Comprehensive tests were performed to find conditions allowing the simultaneous differentiation of osteocytes from osteoblasts, osteoclast formation from PBMC and tube formation of HUVECs. In the quadruple culture model, morphology, gene expression and marker enzyme activities of the three bone cell species like alkaline phosphatase for osteoblasts, tartrate-resistant acid phosphatase and cathepsin K for osteoclasts as well as CD31 for endothelial cells were detected proving the successful differentiation of all involved cell species in the 3D model.

*Conclusion/Significance: We demonstrated that the external addition of cytokines such as macrophage colony-stimulating factor (MCSF) and receptor activator of nuclear factor-κB ligand (RANKL) for the differentiation of multinucleated osteoclasts from PBMC or vascular endothelial growth factor (VEGF) for tube formation of HUVECs is not necessary in the quadruple culture. Therefore, the in vitro bone model represents a physiologically relevant culture system and might help to study and understand the complex crosstalk of bone cells, their precursors and endothelial cells during remodelling and vascularisation of bone tissue. Moreover, it enables preclinical testing of various parameters like the impact of bioactive factors, biomaterial extracts or drugs on the differentiation and behaviour of bone cells and endothelial cells, for translation into clinical practice without in vivo animal studies.

256 - Fabrication Of A Giant Spheroid With Pumping System

S. Ishimaru, H. Nakamura, N. Kojima

Yokohama City University, Yokohama, Japan

*Purpose/Objectives: Due to limitations in oxygen and nutrients exchange, spheroids must be formed smaller than several hundred microns in diameter. “Giant spheroids” with a diameter of millimeter must have necrotic core. However, if it is possible to create such giant spheroids without necrosis, it would be critical to develop new procedures for tissue engineering. This is because millimeter-sized objects can be easily handled as building blocks using existing technologies like microsurgery. The objective of this study is to create spheroids embedded with a pump that prevent necrosis, in order to achieve a millimeter-sized giant spheroids.

*Methodology: A hepatoma cell line, HuH-7 was used in this study. To prepare the pump, a hollow bead made of plastic material, a silicone tube with an inner diameter of 1 mm and an outer diameter of 1.5 mm was cut into a length of 1.5 mm. The tubes were then sealed on both sides with poly-dimethylsiloxane (PDMS) and baked at 100°C for 35 minutes. In order to embed the pump into spheroids, we applied a method utilizing 3% methylcellulose medium [1, 2]. Approximately 500,000 cells were used to from a single spheroid with or without a hollow balloon-like pump. Time-lapse observation using a phase contrast microscope was used to observe changes in the shape of the spheroids.

*Results: The pump that is embedded in spheroids needs to be small enough. It should also be resistant to moisture and be driven without wiring for power supply. To meet these requirements, we focused on the principle of Pascal’s Law because it can work in a non-contact manner. If a hollow, balloon-shaped material is embedded into the spheroid, pressure applied to the culture medium will contract the material. Repeated applying and releasing pressure, the balloon-like material will be expected to show a pump-like action. After several attempts, we were able to make a spheroid embedding the hollow ball made with silicone tube. When we used 500,000 cells to form a spheroid with pump, the diameter became almost 3 mm. We were concerned that by embedding a hollow structure in the spheroids, the spheroids might float instead of settling. The spheroid we formed this experiment, however, was not drifting in the culture medium when we used 500,000 cells per a single spheroid. We confirmed the balloon material was contracted in the spheroid like a pump by the applied pressure. This method was able to drive the pumps of multiple spheroids simultaneously with maintaining sterility. We are now trying to detect effects of the pump on the cell growth/viability and hepatic functions like albumin secretion.

*Conclusion/Significance: We were able to create a giant spheroid embedded with a pump driven by pressure.

References: [1] Kojima, N., Takeuchi, S. and Sakai, Y. Rapid aggregation of heterogeneous cells and multiple-sized microspheres in methylcellulose medium. Biomaterials, 33, 4508-4514 (2012). [2] Kojima, N., Takeuchi, S. and Sakai, Y. Fabrication of microchannel networks in multicellular spheroids. Sensor. Actuat. B-Chem., 198, 249-254 (2014).

257 - Biofabrication Of A Microvasculature-like Network Through Microfluidic-assisted Wet-spinning

A. Paradiso¹, M. Volpi¹, D. C. Martinez¹, E. Walejewska¹, M. Costantini², W. Swieszkowski¹

¹ Warsaw University of Technology, Warsaw, Poland,

² Institution Polish Academy of Sciences, Warsaw, Poland

*Purpose/Objectives: Nowadays, the insufficient development of detailed vasculature in 3D scaffolds is still a significant obstacle in creating tissue constructs that accurately mimic physiologically relevant niches. Consequently, the design and creation of engineered blood vessels at the micro-scale represent a considerable challenge to fully recapitulate microvascularized tissues. To date, several attempts have been made, although complexity and costly strategies limited the transition from bench to bedside. This study introduces a rapid microfluidic-assisted biofabrication technique to create scalable wet-spun hydrogel-based vessel-like bundles to effectively recapitulate the intricate structure of the native microvascular networks.

*Methodology: Two hydrogel formulations were refined for the core and shell compartments of the microfibers. Fibrinogen was chosen for the cell-laden core, while alginate for the sheath. Rheological evaluation on pre-polymer solutions was conducted. Then, the biomaterials were further characterized to investigate the hydrogel mechanical properties, and the morphology and the structure of the yarns by (scanning electron microscopy) SEM and high-res micro-computed tomography (µCT) techniques. Subsequently, the fibrinogen-based formulation was loaded with an optimized co-culture of human umbilical vein endothelial cells (HUVECs)/mesenchymal stem cells (hMSCs) as a bioink to induce pre-vascular network formation. Both pre-polymer formulations were simultaneously extruded from a microfluidic co-axial nozzle immersed in a CaCl2 coagulation bath to produce vessel-like compartmentalized meter-long fibers. A secondary enzymatic-mediated crosslinking on the fibrous scaffolds was performed to stabilize their structure and form a fibrin network in the core. Constructs were cultured up to 21 days. In this framework, both proliferation and cytotoxicity tests were assessed. Then, cell-laden scaffolds were investigated in terms of cell morphology characterization and actin organization. Finally, immunofluorescence analysis revealed the formation of tiny vessel-like structures.

*Results: The pre-polymer formulations exhibited a Newtonian-like behavior. Tissue-specific wet-spun core-shell fibers supported cell adhesion, migration, and alignment over the culture time, forming packed 3D cell-laden constructs that recapitulate the branched microvascular network. The biocompatibility of the scaffolds was proven and the proliferation assay confirmed consistent metabolic activity during the cell-culture period. Remarkably, the scaffolds shown a CD31 expression increasing over the three-week culture, revealing the potential role of the fibers in the localized pre-vascularization of tissue-specific constructs. The proposed formulations can be used to engineer low viscous fiber-based hydrogels to promote cell-specific proliferation. Encapsulated cells spread within the microfiber core, displayed metabolic activity and did not show cytotoxic response. CD31⁺ cells innervated the core after one week of culture, thus forming highly interconnected microvessel-like networks after 2- and 3-week time. Such network branched along the fiber axis direction, confirming the synergistic interplay between human MSCs and HUVECs in the microvascular function. Taken together, such results validated the microfluidic system for the biofabrication of functionalized soft fiber-based hydrogel constructs for microvascular tissue engineering.

*Conclusion/Significance: This study aims to highlight a new wet-spinning model to promote the formation of microvascular-like networks via a microfluidic-assisted wet-spinning approach. Thus, such strategy can be considered a potential alternative to 3D bioprinted microvascularized constructs. Finally, this hydrogel fiber-based technology can be tuned to recapitulate soft tissue-specific morphologies and functions.

260 - Chemotherapeutic Assessment On A Novel, Dynamic, Multicompartment, Multicellular Pancreatic Cancer Model: Towards Engineering Of Personalised Tools For Cancer Treatment Screening

P. Gupta¹, B. Singh², M. J. Wilkinson², H. Kocher³, P. Perez-Mancera⁴, E. Velliou¹

¹ UCL, London, United Kingdom,

² Kirkstall Limited, York, United Kingdom,

³ Queen Mary University, London, United Kingdom,

⁴ University of Liverpool, Liverpool, United Kingdom

*Purpose/Objectives: With a 5-year survival rate of only 11% in the USA and less than 7% in the UK, pancreatic cancer (PDAC) is considered to be one of the deadliest diseases. This is partly attributed to the tumour’s resistance to currently available treatment, resulting from a complex and highly heterogeneous tumour microenvironment (TME). A key challenge in cancer tissue engineering is to mimic the different key features of the TME. In this work we have developed robust, PDAC biomimetic models for therapeutic assessment in vitro with the goal to optimize patient specific treatment regime.

*Methodology: We have advanced our previously developed 3D polyurethane (PU) based polymeric scaffold assisted PDAC model by incorporating biological complexity (multiple cell types), spatial complexity (scaffold compartmentalization) and fluid flow (perfusion). Specifically, we have developed a multi-cellular system of pancreatic cancer, pancreatic activated stellate and endothelial cells. Two different two architectural configurations were used: (i) a single scaffold and (ii) a zonal scaffold comprised of an inner (cancer) compartment and an external (stroma) compartment. Furthermore, via using a perfusion bioreactor we achieved mimicry of the interstitial flow, which enables more accurate drug delivery. Chemotherapeutic assessment using Gemcitabine (GEM) was carried out to validate our models. Imaging of cellular proliferation/spatial organization, apoptosis of the different cell types and ECM secretion was carried out along with q-PCR assessment of various biomarkers, e.g. EMT or metastatic markers pre- and post- treatment.

*Results: Within our static models, we observed that the dual scaffold showed a higher resistance to GEM in comparison to the single scaffold. More specifically, there was no reduction of cell viability in the zonal model, especially for the cancer mass, and only a slight induction of apoptosis was observed for the stromal compartment. In addition, we also observed selective depletion of cytokeratin expressing cancer cells and stellate cells post chemotherapy in both models. Furthermore, Collagen I distribution in the zonal scaffold was unaffected 24 hours post-treatment, in contrast to the single multicellular scaffold. These results highlight that the spatial arrangement of the cells, within a 3D model, affect the response to chemotherapy. Furthermore, the introduction of dynamic flow affected the cell spatial organization, and biomarker expression involved with EMT, matrix remodeling highlighting the importance of fluid and its role in PDAC’s resistance to chemotherapy.

*Conclusion/Significance: Overall, we have developed a robust, versatile PDAC model that allows us to recapitulate and test multiple critical aspects of the pancreatic TME including cell-cell and cell-ECM interactions, interstitial fluid flow and the development of an in vivo-like niche by the cancer cells. Our work highlights the importance of spatio-temporal cellular arrangement and interstitial fluid flow for accurate in vitro studies of the chemoresistance for PDAC.

Acknowledgements: E.V. (PI) and P.P.M. (co-I) are grateful to the Medical Research Council UK for a New Investigator Research Grant (MR/V028553/1), which also financially supports P.G.

261 - Defining A Gene Signature To Augment Patient Stratification Using An Engineered Model Of Patient-derived Pancreatic Cancer Cells And Primary Macrophages

A. P. McGuigan, I. L. Co, C. P. Yu, K. P. Campbell

University of Toronto, Toronto, ON, Canada

*Purpose/Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. Patients are typically stratified into classical or basal disease sub-types, where basal sub-type associates with poorer prognosis. In PDAC, the tumour microenvironment (TME) plays a critical role in dictating disease behaviour and potentially the balance of classical versus basal tumour sub-populations. In particular, interactions between tumor associated macrophages (TAMs) and cancer cells in the hypoxic TME have been shown to play significant roles in disease progression, chemotherapy resistance, and immunosuppression. However, the precise dynamics of TAM-tumour interactions in the presence of varying hypoxia that drive aggressive tumour phenotypes remains unknown, owing largely to a lack of relevant experimental systems. Here we set out to develop a model of TAM-tumour interactions in the TME and to use this model to explore the influence of TAMs on PDAC tumour cell states under different TME conditions.

*Methodology: The TRACER platform models the TME and involves rolling a cellulose scaffold infiltrated with tumour cells in a matrix around an oxygen-impermeable mandrel to create a layered tissue structure in which progressive cellular consumption and production establishes gradients of oxygen, nutrients, waste, and metabolites; after a defined timepoint, TRACER is then unrolled for layer-specific analysis of cell properties associated with specific TME conditions. Here we assembled TRACER cultures from primary human macrophages and patient-derived PDAC organoids. We then characterized the layer specific properties of both cell types in co-culture TRACERs and used layer specific RNAseq analysis to explore the impact of TME gradients on tumour-TAM interactions.

*Results: We confirmed that co-culture TRACERs establish cell-generated hypoxic gradients and exhibit expected transcriptional and phenotypic responses to these gradients. In line with previous literature, macrophages and organoids in the inner hypoxic layers of co-culture TRACERs were more resistant to chemotherapies and produced secreted factors that attenuated T-cell inflammatory response. When we performed bulk transcriptional analysis of cells isolated from the different co-culture TRACER layers we observed that macrophages exhibited PDAC TAM-like transcriptional profiles defined from patient data. Further, the transcriptional profiles of PDAC organoid cells isolated from co-culture TRACERs were significantly less sensitive to the hypoxia and gradient-driven changes compared to organoids from monoculture TRACERs suggesting that the presence of TAMs dominated organoid phenotypes. Significantly, gene signatures defined using the hypoxia-sensitive genes unique to or disrupted in co-culture TRACERs enabled stratification of patient outcomes in the TCGA patient dataset. Gene signatures from mono-culture TRACERs or co-cultures grown in a hypoxia chamber alone however failed to stratify patient outcomes.

*Conclusion/Significance: Overall, our results suggest that co-culture TRACERs capture important aspects of the PDAC TME that cannot be modelled in monocultures or using hypoxia chambers. Our model will therefore be useful for the development of novel therapeutics that target TAMs, for example. Further, our work provides an early example of the use of an engineered tumour to generate TME defined datasets that can be easily filtered in controlled ways to enable the identification of gene signatures for augmenting analysis of existing and often complex patient datasets.

263 - Delineating The Role Of Bone Marrow Adipocytes And Therapy Response On Metastatic Prostate Cancer Growth Using Humanized 3d Coculture Platforms In Vitro And In Vivo

A. Bessot^1,2,3, S. Bhatia^1,2, J. Gunter^1,4, L. Badin⁵, J. A. Clements¹, D. Waugh^1,6, D. W. Hutmacher^2,3,7, J. McGovern^1,2,3, N. Bock^1,2,3

¹ Queensland University of Technology / Translational Research Institute, Woolloongabba, Australia,

² Max Planck Queensland Centre, Brisbane, Australia,

³ Centre for Biomedical Technologies, Brisbane, Australia,

⁴ Australian Prostate Cancer Research Centre - Queensland (APCRC-Q), Brisbane, Australia,

⁵ School of Bioengineering Polytech Sophia, Nice, France,

⁶ Centre for Cancer Biology, University of South Australia, Adelaide, Australia,

⁷ Engineering Faculty , Queensland University of Technology, Brisbane, Australia

*Purpose/Objectives: Current preclinical bone cancer models show limitations in replicating the structural and functional features of the human bone tumor microenvironment (hBTME), restricting the discovery of novel contributors to bone metastatic cancers. To overcome these limitations, our previous work has leveraged gelatin-based hydrogels (gelatin methacrylyol (GelMA)) and biomimetic cultures to successfully engineer humanized 3D co-culture models in vitro and in vivo that better replicate the native hBTME. Using these well-characterized platforms, we hypothesized that; 1) bone marrow adipocytes (BMA) would enhance prostate cancer (PCa) growth and underpin resistance to the androgen receptor antagonist Enzalutamide (Enz), and 2) the combination of Enz with repurposed drugs (anti-cholesterol (Simvastatin, Sim) and anti-diabetic (Metformin, Met) drugs) would modulate tumor response to Enz due to their pro-apoptotic capacity on cancer cells, and their inhibitory effect on adipogenesis and lipolysis on adipocytes.

*Methodology: To delineate the crosstalk between BMA and PCa cells (C4-2B and LNCaP), we cultured 3D mineralized, adipose and tumoral microtissues together for one week. We tested different treatments; Enz, Enz+Met and Enz+Sim combinations on these co-culture models to 1) determine the effect of BMA on Enz resistance and 2) evaluate the efficiency of combining Enz with repurposed drugs to limit cancer cell growth in vitro. The 3D microtissues were also implanted ectopically in immunodeficient mice to form a hBTME for in vivo drug testing. After tumor engraftment and growth, mice were surgically castrated to mimic androgen-deprivation therapy and orally treated with the different drug combinations (Vehicle, Enz, Enz+Met, Enz+Sim) for up to four weeks.

*Results: Preliminary (2 out of 3 biological replicates) in vitro data have shown delipidation and morphology changes in adipocytes when co-cultured with PCa cells, similar to those observed in cancer-associated adipocytes. The presence of human adipocytes did not significantly impact the size of PCa spheroids but reduced their sensitivity to the anti-tumor effects of Enz. The complementary treatments (Enz+Met, Enz+Sim) attenuated the increase in spheroid size contributed by BMA. Similarly, the addition of human adipocytes to the in vivo humanized PCa models clearly demonstrated the capacity of Enz to exhibit modest anti-tumoral effects in the hBTME. Furthermore, the co-administration of Sim with Enz showed superior effects in diminishing tumor burden and size in presence of BMA, which was not observed with Met. Our observations are consistent with Sim attenuating the impacts of the cholesterol-orientated metabolism of BMA and current clinical trials. Further RNA sequencing is underway to identify specific pathways involved in the BMA-PCa cells crosstalk.

*Conclusion/Significance: The chosen gelatin-based hydrogel platforms have modelled the impacts of human BMA on PCa tumors in a humanized bone microenvironment for the first time. This study shows that the model can be used to study the impact of current and repurposed drugs on tumor growth and may be used prospectively to identify effective therapeutic interventions to test in future clinical trials.

265 - 3D Bio-printed Immune-responsive Microtissues To Develop Patient-specific Non-animal Technologies (NAT) In Leukaemia Drug Development

S. Hockney¹, J. Parker¹, B. Tzivelekis², K. Dalgarno², D. Pal¹

¹ Northumbria University, Newcastle-Upon-Tyne, UNITED KINGDOM,

² Newcastle University, Newcastle-Upon-Tyne, UNITED KINGDOM

*Purpose/Objectives: The lack of biomimetic and tractable preclinical in vitro models for studying Acute Lymphoblastic Leukaemia (ALL) greatly impacts the ability to test new therapies on clinical samples leading to significant drug attrition. There is still a heavy reliance on the current gold standard models for leukaemia drug studies- patient derived xenograft models in immune-compromised mice. These models have sustainability issues, variable sample engraftment success and data delivery to clinics is slow. Additionally, these models typically have impaired macrophage function. Developing a translatable preclinical model must capture the complexities of the leukemic bone marrow niche with cellular interactions including immune responsiveness and extracellular matrix (ECM) considerations. These aspects are established in driving the disease and influencing treatment response. Such models have even more importance given the Food and Drug Administration’s (FDA) recent announcement that it will no longer require all drugs to be tested on animals prior to in-human trials.

*Methodology: Here we describe an animal component-free prototype hydrogel formulation that comprises of ECM components of the human bone marrow in defined formulations. We show its ability to be utilised as a bioink for 3D bioprinting within a Reactive Jet Impingement (ReJI) 3D bioprinting platform and its compatibility with tissue culture protocols and microvalve printing processes. We also show generation of iPSC-derived Mesenchymal Stem Cells (iMSC) and iPSC-derived monocytes and macrophages (iMAC) and demonstrate effective incorporation into the hydrogel generating an immune responsive 3D bio printed MSC-ALL organoid. We show the ability of such models to support long term in vitro culture of patient-derived leukaemia cells and that these MSC-ALL organoid models demonstrate clinically translatable drug response whilst using these models to investigate potential mechanisms of treatment resistance via tunnelling nanotubules (TNTs) and testing potential novel drug combinations.

*Results: Culture compatibility assays show that the prototype hydrogel can be maintained in all required culture media; that degradation of the gel material is minimal and that the formulation can withstand strain associated with the microvalve-based bioprinting process. We show successful generation of iMSC and iMAC through relevant protein and gene marker expression. We show that patient derived leukaemia cell co-culture with iMSC microtissues show significant increase in cell proliferation over long term cultures (21 days) compared to 2D approaches. We observe a response to dexamethasone, a commonly used therapy in clinics, in patient derived samples with known sensitivity and resistance- matching patient data. In addition, we have investigated a potential treatment resistance mechanism in the form of TNTs potentially via a CDH2- mediated mechanism. We show that CDH2 inhibitor ADH-1, an FDA-approved orphan drug used in Phase I trials for solid tumours, reduces TNT formation. Finally we have used the model to identify synergy between a clinically used combination of dexamethasone and BCL-2 inhibitor ABT199 with ADH1.

*Conclusion/Significance: Generation of such tractable models in a high throughput, and defined manner lends itself to wider uptake and furthermore demonstrates the flexibility of the model to generate patient-specific data. This results in the potential for use in personalised medicine strategies.

266 - Platelets Are A Key Driver Of LYST-mediated TEVG Stenosis

M. E. Turner, J. Che, J. T. Leland, S. Rajesh, T. Yi, J. W. Reinhardt, C. K. Breuer

Nationwide Children's Hospital, Columbus, OH

*Purpose/Objectives: A tissue-engineered vascular graft (TEVG) that grows with the patient could transform the surgical management of infants and children requiring congenital heart surgery. However, evaluating TEVG efficacy revealed a high incidence of stenosis. The purpose of this study is to investigate the mechanisms underlying TEVG stenosis. Our lab observed reduced TEVG stenosis in mice carrying a mutation in the lysosomal trafficking regulator gene (LYST), which codes for a protein critical to innate immune cell functions. LYST mutations impair the release of platelet dense granules, which carry small molecules that recruit and activate other platelets and immune cells. Increasing evidence suggests that platelets coordinate TEVG stenosis: i.) platelet-rich thrombi developed on wild-type mouse TEVGs that remodeled into collagen-rich neotissue, persistently narrowing or occluding the lumen and ii.) antiplatelet drugs and platelet depletion partially improved TEVG performance. We predict that TEVG stenosis is a complex, multistage process initiated by platelet signaling. Our goal is to understand how LYST-mediated platelet signals contribute to the immune microenvironment of remodeling TEVGs and in excess, cause stenosis.

*Methodology: To investigate the lineage of the cell type initiating stenosis, we irradiated wild-type (WT) mice and LYST mutant mice and transplanted i) WT mice with LYST mutant bone marrow and ii) LYST mutant mice with WT bone marrow. After immune reconstitution and TEVG implant, we evaluated TEVG patency using ultrasound and Micro-CT at 3- and 14-days post-implantation. To determine whether LYST-mediated platelet signaling is sufficient to cause stenosis, we generated and implanted Pf4-Cre driven platelet LYST mutant mice and assessed TEVG performance at 3- and 14-days. Dissected TEVGs were stained using Carstairs’ (platelets and fibrin) and H&E for morphometric analysis. Finally, we used platelet aggregometry to determine the effects of LYST mutations on platelet function.

*Results: 33% of WT TEVGs (n=25) were patent and 96% of global LYST mutant TEVGs (n=25) were patent. WT mice transplanted with LYST mutant bone marrow were largely resistant to stenosis (96% of mice patent, n=24) whereas transplanting WT bone marrow in LYST mutant mice caused increased stenosis (38% of mice patent, n=22) (p<0.05 | Fisher’s Exact Test). Conditional mutant studies revealed that the platelet specific LYST mutation reduced acute TEVG narrowing at 3 days post-implantation (97% of mice patent, n=37) and the incidence of TEVG stenosis did not change by day 14 (p<0.05 | Fisher’s Exact Test). Aggregometry tests confirmed that LYST mutant platelets do not release granules in response to ADP, collagen, and thrombin and are activated by but do not aggregate in response to collagen.

*Conclusion/Significance: The study highlights the significance of platelet-immune signals in TEVG stenosis. Additionally, aggregometry experiments confirm that LYST mutation impairs platelet function by preventing dense granule exocytosis. This suggests that the LYST-mediated platelet response to the TEVG requires purinergic signaling to allow for platelet recruitment and further activation. Additional mechanistic experiments are required to determine the specific platelet-immune interactions guiding neotissue formation and inward remodeling. In conclusion, LYST modulation, particularly in platelets, could be a promising avenue to improve TEVG outcomes in human patients.

269 - Microfabricated Anisotropic Cardiac Myobundles For The Modular Assembly Of Cardiac Tissue Grafts

M. Jewett, A. Bluem, S. Xi, S. Bhirud, S. DePalma, B. Baker

Biomedical Engineering, University of Michigan, Ann Arbor, MI

*Purpose/Objectives: Following myocardial infarction, tissue ischemia and resulting cardiomyocyte (CM) death cause myocardial tissue remodeling, disruption of mechanical and electrical properties, and impaired function. The field of cardiac tissue engineering aims to produce engineered tissues that could replace injured or diseased myocardium and restore normal cardiac function. Two features critical to healthy myocardial function are the ECM’s high degree of structural anisotropy and the resulting organization of resident cell populations; however, replicating these features at scales relevant to tissue replacement therapies remain a major challenge. As such, there is a critical need for new biomanufacturing technologies that can organize ECM and multiple, distinct cell types with microscale precision in large tissue grafts. Here, we propose a new method for fabricating individual anisotropic myofiber bundle-like tissues that can be modularly assembled into larger tissue grafts.

*Methodology: Microchannel device molds were 3D printed and cast with polydimethylsiloxane (PDMS). PDMS microchannels were placed channel side down on a 2 wt% gelatin treated glass coverslip. Induced pluripotent stem cells (iPSCs) containing a GFP fusion tagged titin reporter (Allen Institute) were differentiated into iPSC-CMs using a previously established protocol. Channels were perfused with fibrin (10 mg mL^-1 fibrinogen with 1 U mL^-1 thrombin) and 2 mg mL^-1 rat tail collagen type I containing 20 M mL^-1 cells (25% normal human cardiac fibroblasts (CFs) and 75% iPSC-CMs) and 2 vol% dextran-vinyl sulfone (DVS) electrospun fibers (Figure 1A). Following gelation at 37°C for 30 min, PDMS molds were removed, and tissues were cultured for up to 14 days.

*Results: Perfusing DVS fibers suspended in fibrin through a range of channel sizes, we observed a decrease in fiber alignment with increasing channel size (Figure 1B, C), likely stemming from diminished mechanical boundary effects provided by the channel walls and/or reduced flow rates resulting in lower shear forces. However, all channels tested led to increased fiber alignment compared to non-perfused controls. Since CMs are minimally migratory, we hypothesized that mechanical and topographical cues provided by DVS fibers would promote CM spreading and organized myofibril assembly. Additionally, we anticipated that tissues supplemented with CFs would improve CM spreading due matrix proteolysis and deposition provided by CFs. Indeed, we observed improved CM spreading and contractile synchronicity when CFs and DVS fibers were incorporated (Figure 1D-F). Additionally, the inclusion of fibers improved contraction synchronicity (Figure 1E, left). Further, the inclusion of fibers enhanced myofibrillogenesis (quantified by the number of z-discs) while myofibrils rarely formed in tissues lacking fibers (Figure 1E, right). These findings suggest that mechanotopographical cues provided by synthetic fibers and CF-mediated biochemical cues may work synergistically to improve CM spreading, myofibril formation, and calcium handling properties.

*Conclusion/Significance: Taken together, we developed a microfabrication approach to produce anisotropic, myofiber-like tissue bundles using a combination of naturally derived ECM and synthetic, polymeric fibers that can be flow-aligned within microfabricated microchannels. This approach allows us to fabricate unit cell tissues of desired size and alignment that can be modularly assembled to produce functional cardiac tissues for future therapeutic applications.

271 - Micro-RNA Mediated Reprogramming Of Human Cardiac Fibroblasts Into Induced Cardiomyocytes Via Bioengineering Strategies In Vitro And In Vivo

C. Paoletti¹, L. Nicoletti¹, M. Coletto¹, E. Marcello¹, G. Ruocco¹, B. Stella², S. Bollini³, E. Di Pasquale⁴, C. Divieto⁵, C. Mattu¹, V. Chiono¹

¹ Politecnico di Torino, Turin, Italy,

² Università di Torino, Turin, Italy,

³ University of Genoa, Genoa, Italy,

⁴ Humanitas Research Hospital, Rozzano, Italy,

⁵ Istituto Nazionale di Ricerca Metrologica, Turin, Italy

*Purpose/Objectives: Myocardial infarction (MI) causes the irreversible loss of cardiomyocytes and the formation of scar tissue, which may lead to heart failure. Recently, the delivery of microRNAs (miRNAs) able to induce direct cell reprogramming has emerged as a new potential strategy for myocardial regeneration [1]. We demonstrated that in vitro transient transfection of human adult cardiac fibroblasts (AHCFs) with four miRNA mimics (miRs-1, 133, 208 and 499, termed “miRcombo”) was able to trigger their direct reprogramming into induced cardiomyocytes (iCMs) using commercial transfection agents and novel lipoplexes [2,3]. Direct reprogramming efficiency was also found to increase by culturing transfected cells in a 3D cardiac tissue-like hydrogel [4]. In this work, we studied the role of extracellular matrix (ECM) proteins on in vitro cell reprogramming to select those suitable for integration into injectable alginate-based hydrogels for the in situ delivery of miRcombo-loaded nanotherapeutics. Indeed we also designed novel miRcombo-loaded polymer-lipid nanoparticles, surface functionalized with ligands for specific delivery to target AHCFs.

*Methodology: MiRcombo-transfected AHCFs using DE-DOPE lipoplexes [3] were cultured on ECM proteins, including laminin, fibronectin, collagen I and biomatrix, produced in vitro by AHCFs. Direct reprogramming was analyzed at different times. Hydrogels, based on alginate, alginate dialdehyde and gelatin, were functionalized with selected ECM proteins. Polymer-lipid hybrid nanoparticles (NPs) were prepared by coating DE-DOPE lipoplexes [3] with synthetic polymers and, then, surface functionalized with specific ligands (F-NPs) for AHCFs. NPs and F-NPs were tested for in vitro cell uptake, biocompatibility and reprogramming ability. Selective F-NP uptake by AHCFs versus cardiomyocytes (CMs) was analyzed. NPs and F-NPs loaded with siRNA-Cy5 (for biodistribution trials) or miRcombo (for efficacy tests) were intracardially injected one week after myocardial infarction (MI) in a mouse model. Biodistribution was analyzed by ex vivo IVIS and total fluorescence of explanted organs. Efficacy was evaluated at 30 days by echocardiography and histological analysis of explanted hearts.

*Results: MiRcombo-transfected AHCFs showed decreased reprogramming into iCMs when cultured on fibronectin and collagen I coatings, while laminin and biomatrix coatings greatly enhanced cell reprogramming. YAP/TAZ activation, dependent on protein coatings, influenced cell reprogramming. F-NPs were cytocompatible and showed higher intracellular uptake by AHCFs compared to NPs, while limited uptake by CMs. In vitro AHCFs reprogramming into iCMs was efficient using F-NPs loaded with miRcombo. F-NPs were retained in the heart after in vivo intracardiac injection and, by delivering miRcombo, improved cardiac functionality at 30 days post-injection. In vivo tests using hydrogels loaded with F-NPs are in progress.

*Conclusion/Significance: Laminin and biomatrix enhanced direct reprogramming efficiency. Novel F-NPs were designed for miRNA delivery to target AHCFs, showing in vitro and in vivo efficacy. In the future, F-NPs will be delivered through alginate-based injectable hydrogels, functionalized with laminin or biomatrix, to improve local retention (minimizing off-target effects) and direct reprogramming efficiency. References: [1] Jayawardena et al, Circ Res 2011; [2] Paoletti et al, Frontiers Bioeng Biotech 2020; [3] Nicoletti et al, Nanomedicine: NBM 2022; [4] Paoletti et al; Cells 2022. Acknowledgments: BIORECAR ERC project (H2020; 772168) and POLIRNA ERC-PoC (Horizon Europe; 101113522) are acknowledged.

273 - Investigating Dural Cell Mechanoresponse Under Ultrasound Stimulation For Bone Repair

H. Anderson¹, K. Grassie¹, D. S. Hersh², Y. Khan³

¹ University of Connecticut, Storrs, CT,

² Connecticut Children's Medical Center, Hartford, CT,

³ University of Connecticut Health Center, Farmington, CT

*Purpose/Objectives: Skull development and growth are associated with the underlying dura mater, a meningeal layer covering the brain. Previous studies indicate the importance of intact dura in promoting calvarial defect healing; however, mechanisms through which the dura participates in cranial repair remain unclear. Dural cells appear to be mechanosensitive, as tensile strain imparted by brain expansion during early development can trigger dural cells to release cytokines that potentially contribute to skull growth. Similar loading may be exerted by ultrasound-generated acoustic radiation force (ARF), which generates a measurable and controllable mechanical force that can stimulate mechanosensitive cells like those of the dura. Our preliminary characterization of dural cells has revealed mesenchymal stem cell (MSC) marker expression (Fig. 1, top left), suggesting a potential for multilineage differentiation that may include osteogenesis, as we and others have seen in MSC populations. Given this, we hypothesize that ARF could be used to mechanically stimulate dural cells to promote cranial bone healing. The objectives of this work were to: 1) determine how dural cell gene expression is influenced by ARF-induced mechanical stimulation via RNA Sequencing analysis, and 2) identify potential mechanisms through which dural cells contribute to osteogenesis during cranial bone healing.

*Methodology: Rat dural fibroblasts (RDuF) were isolated from the dura mater of 4-6 weeks-old Sprague Dawley rats and expanded to passages 1-3. For bulk RNA Sequencing experiments, RDuF seeded in 24-well plates received 20 minutes of ARF (150 mW/cm²) once daily for 28 days. Control RDuF received no ARF stimulation. Total RNA extraction was performed using RNeasy^® Mini Kit and QIAcube. Raw reads were trimmed with Trimmomatic and mapped to Homo Sapiens genome (GRCh38) with HISAT2. Differential gene expression between conditions was evaluated using DESeq2. Genes with false discovery rate (FDR) < 0.05 were considered significant. Gene Set Enrichment Analysis (GSEA) was performed with Ingenuity pathway analysis. Volcano plot thresholds were set to |log₂Fold Change (FC)| > 1 and padj < 0.05.

*Results: We have shown that dural cells respond to ARF by upregulating several cell functions, including cellular movement, cellular assembly and organization, and cell-cell signaling, as revealed by GSEA (Fig. 1, bottom). During development, dural cells contribute to suture patency and skull remodeling through biochemical signaling with osteoblasts and stem cells; this response may be partially stimulated by the mechanical environment of the expanding cranial vault. Our findings suggest ARF may elicit similar paracrine signaling from dural cells that may evoke osteoblast and stem cell activity for bone repair. Importantly, a significant increase in mRNA expression of Wnt5a was also observed in ARF-treated RDuF compared to the control (Fig. 1, top right). Wnt5a, an extracellular protein, is involved in osteogenesis through noncanonical Wnt signaling; therefore, dural cells may enact Wnt5a-induced osteogenesis directly or may act as a Wnt5a source for surrounding osteoblasts.

*Conclusion/Significance: This work posits dural cells as a mechanosensitive cell population that might participate in cranial osteogenesis through cellular crosstalk, and therefore may be an untapped tool that could be exploited for calvarial reconstruction when combined with ARF stimulation.

275 - Development Of A High-frequency Ultrasound System To Assess The Microstructural, Contractile, And Mechanical Properties Of Engineered Cardiac Tissues

J. A. Sebastian^1,2, E. M. Strohm³, E. Chérin⁴, Z. Gouveia^1,2, J. P. Santerre^1,2, C. Démoré^4,1, M. C. Kolios^3,5,6, C. A. Simmons^1,2,7

¹ University of Toronto, Toronto, ON, Canada,

² Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, Toronto, ON, Canada,

³ Toronto Metropolitan University, Toronto, ON, Canada,

⁴ Sunnybrook Research Institute, Toronto, ON, Canada,

⁵ Institute of Biomedical Engineering, Science and Technology (iBEST), a partnership between Toronto Metropolitan University and St. Michael’s Hospital, Toronto, ON, Canada,

⁶ Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, ON, Canada,

⁷ Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, ON, Canada

*Purpose/Objectives: Current drug screening often fails to identify effective therapeutics for heart disease. Heart-on-a-chip models, integrating 3D microtissues and stem cells, better replicate human cardiac (patho)biology and drug responses but are limited by the invasiveness of current assessment methods. Ultrasound has the potential to non-invasively measure cardiac microtissue microstructure, contractility, and stiffness, but current methods lack the spatiotemporal resolution to image dynamic microscale engineered tissues. To overcome these challenges, we developed a high-frequency ultrasound (HFUS) system to assess engineered tissues’ acoustic properties, contractility, and stiffness in vitro, with sub-millimeter spatial and microsecond temporal resolutions.

*Methodology: The HFUS system is composed of imaging transducers of center frequencies from 40-200 MHz to perform acoustic (e.g., speed of sound, acoustic impedance, acoustic attenuation), and contractile (e.g., beat rate and rhythm) measurements and a 15 MHz excitation transducer for elastography (e.g., shear modulus) measurements. Sampling rates from 0.625-10 GHz and pulse repetition frequencies of 10-500 kHz provide unprecedented ultrasound imaging spatial (8 µm) and temporal (1000 frames per second) resolutions. We demonstrated HFUS acoustic property measurements using thin (<1.25 mm) fibroblast-seeded fibrin hydrogels as engineered tissue models and correlated tissue thickness-independent acoustic properties with intrinsic tissue structure and cellularity in vitro. We also demonstrated versatile cardiac contraction measurements by HFUS with single cardiomyocytes, 3D cardiac organoids, and 3D cardiac microtissues compared to standard optical methods. Lastly, we determined the stiffness of 1 mm-thick agar-silica biomaterials using a viscoelastic Lamb wave model that considered multiple wave modes in thin biomaterials, with validation by shear rheometry.

*Results: Standard US measurements of the acoustic properties of engineered tissues can be confounded by cell-mediated contraction, which alters tissue thickness in the axial direction. Using an analytical approach to account for thickness-dependent interfacial effects, we found that most acoustic properties (e.g., speed of sounds, impedance) of the cell-seeded tissues were independent of cell concentration (p>0.11). The exception was acoustic attenuation, which correlated with cell concentration (p<0.001) but not tissue thickness (p>0.1), demonstrating that the HFUS measurement of acoustic properties reflect intrinsic microtissue physical properties of interest. Cardiac single cell, organoid, and microtissue contraction kinetics were all readily measured by HFUS, with higher temporal resolution than standard optical imaging. Notably, HFUS provided insights into the contraction of 3D organoid and microtissue models, reflecting tissue maturity and microstructure that was not observable optically. Moreover, the shear moduli of model biomaterials measured by HFUS elastography and shear rheometry were comparable (p>0.05). Thus, our ultrasound system enables non-invasive, non-destructive, and label-free measurements of material properties and function of microscale cardiac models and biomaterials, with good agreement to gold-standard measurement techniques. Ongoing studies are applying the HFUS system for longitudinal monitoring of the material properties and contractile force of cardiac microtissue models to assess anti-fibrotic drug effects.

*Conclusion/Significance: Our HFUS system enables high spatiotemporal resolution measurement of the intrinsic material properties and function of static and dynamic thin microtissues via Lamb wave analysis. Its versatility will enable broad applications for the non-invasive assessment of organ-on-a-chip models for pre-clinical cardiac disease modelling and drug testing.

276 - A Next Generation Glomerulus-on-a-chip Promotes In Situ Tissue Patterning And Morphogenesis

X. Mou, J. Shah, Y. Roye, C. Du, S. Musah

Biomedical Engineering, Duke University, Durham, NC

*Purpose/Objectives: Organ-on-chips has gained profound interest in disease modeling, drug testing, and therapeutic discovery for various diseases. Existing organ-on-chips rely on synthetic polymers, such as polydimethylsiloxane (PDMS), to engineer porous membranes as cell culture substrates and barriers between cell layers. However, such membranes are limited by their thickness, which presents a significant physical barrier that hinders intercellular crosstalk, leading to suboptimal tissue patterning and morphogenesis. This works presents an ultrathin biomimetic membrane that can be used to replace PDMS membranes for enhanced intercellular crosstalk and tissue morphogenesis in a human kidney glomerulus model.

*Methodology: An ultrathin biomimetic membrane was engineered by electrospinning of a naturally derived material - silk fibroin. The resulting membrane was integrated into PDMS-based microfluidic chips through PDMS glue and oxygen plasma treatment. Human induced pluripotent stem (hiPS) cell-derived kidney glomerular cells was incorporated into the resulting microfluidic devices on both sides of the membrane and further differentiated into their terminal cell types - podocytes and glomerular endothelial cells. The resulting devices were evaluated based on cell morphology, barrier function, drug-induced injury response, and intercellular signaling.

*Results: The final glomerulus-on-a-chip devices demonstrated in vivo-like tissue patterning with cell-specific marker and extracellular matrix marker expression, size-selective filtration function under healthy and diseased conditions, and tissue morphogenesis with endothelial fenestrations that have never been observed previously in organ-on-chips. Further analysis focusing on podocyte-endothelial cell crosstalk revealed VEGF-A signaling between these two cell layers, indicating presence of intercellular crosstalk across the membrane to enhance tissue morphology.

*Conclusion/Significance: This work for the first time demonstrated successful engineering of a functional next-generation organ-on-a-chip with an ultrathin membrane, showing in-vivo like tissue organization, function, and disease phenotypes. Additionally, for the first time, this work showed successful induction of endothelial fenestrations from hiPS cell-derived cells, through in situ intercellular signaling, resembling in-vivo tissue morphogenesis of the kidney glomerulus. The resulting device presents a novel platform for studying kidney development, drug testing, and kidney disease modeling. The device can also support modeling of various organs through incorporation of their corresponding cells, thus providing a blueprint for development of body-on-a-chip in the future.

277 - Effects Of Combined Recombinant Protein Therapy And Mechanical Loading On 3D-Bioprinted Patient-Specific Biomimetic Bone Organoids For Healthy And Osteogenesis Imperfecta Donors

A. M. de Leeuw¹, C. Steffi¹, G. N. Schädli¹, P. J. Lim², M. Rüger^1,2, M. Rohrbach², C. Giunta², R. Müller¹

¹ ETH Zürich, Zürich, Switzerland,

² University Children’s Hospital Zurich, Zürich, Switzerland

*Purpose/Objectives: Osteogenesis imperfecta (OI) is a rare, genetically heterogeneous connective tissue disorder characterized by bone fragility and deformities. Despite extensive research in animal models, the disease pathomechanisms are not yet fully understood, thereby impairing the development of targeted therapies; there is currently no cure for OI. Patient-derived organoids offer faster and more personalized platforms for drug testing in comparison to animal models. The Wnt signalling pathway is being explored as a potential therapeutic target for OI, to increase bone formation, mineral density, and strength. Here, we propose a proof-of-concept in vitro drug testing platform for recombinant protein therapy to model OI pathomechanisms, investigating anti-Dickkopf-related Protein 1 (anti-DKK1) treatment combined with mechanical loading.

*Methodology: Primary osteoblasts were isolated from surgical bone waste of a 3-year-old female with FKBP10-OI and a metabolically healthy 15-year-old male. Cells were encapsulated in alginate-gelatin-graphene oxide hydrogels and extrusion 3D-bioprinted. Healthy and OI bone organoids were cultured under static conditions or in compression bioreactors for 8 weeks and subjected to uniaxial cyclic compressive loading. After 4 weeks, organoid surfaces were reseeded with osteoblasts from the same donor and treated with 1 ug/mL anti-DKK1 (treated group) or isotype IgG control (control group). Treatment response was investigated by weekly time-lapsed micro-computed tomography (micro-CT), collagen secretion (ELISA) and mechanical testing. Endpoint histology, and immunohistochemistry enabled characterization of cellular phenotypes and mineralized extracellular matrix formation.

*Results: Anti-DKK1 treatment significantly increased procollagen I secretion in OI bone organoids (Fig 1A). An average procollagen concentration of 0.41 ± 0.16 ug was found for healthy controls, 0.70 ± 0.16 ug for treated healthy organoids compared to 0.94 ± 0.23 ug for OI controls and 1.73 ± 0.63 ug for treated OI organoids. Similarly, endpoint immunostaining results suggested more collagen deposition in the extracellular matrix of treated OI bone organoids (Fig 1D). A significantly higher average tissue mineral density was found in OI organoids compared to healthy controls (p<0.05), recapitulating the clinical disease phenotype (Fig 1B). However, no significant differences were found in treated versus untreated organoids. Endpoint compression testing revealed no significant difference in average stiffness amongst groups (Fig 1C). Nevertheless, macroscopically, anti-DKK1-treated OI organoids maintained their shape fidelity as organized lattice structures similar to that observed in healthy organoids; this is in contrast to IgG control OI organoids that presented with a fractured phenotype with broken filaments (Fig 1E).

*Conclusion/Significance: We successfully established 3D-bioprinted personalized bone organoids for healthy and OI patients. This in vitro platform recapitulates aspects of the disease phenotype, offering clinically relevant readouts for assessing anabolic treatment response over time. While anti-DKK1 treatment was shown to upregulate collagen secretion and deposition, there was no effect on mineralization. Notably, anti-DKK1 treatment preserved OI organoid shape fidelity, which may be attributed to higher collagen secretion, however further investigation is needed. The established bone organoid may become a powerful tool to study pathomechanisms for numerous OI mutations and facilitate the development and testing of advanced therapies on a patient-specific level.

278 - Increased Hydrostatic Pressure But Not Shear-stress Promotes Ve-cadherin Internalisation: A Blood Vessel-on-chip Microfluidic Model Of Microvascular Dysfunction

P. Vasanthi Bathrinarayanan¹, C. Anderton¹, N. George¹, D. Vigolo², L. M. Grover¹, M. J. Simmons¹

¹ University of Birmingham, Birmingham, United Kingdom,

² University of Sydney, Sydney, Australia

*Purpose/Objectives: Orthopaedic traumas, especially tibial fractures can lead to a limb-threatening condition called Acute compartment syndrome (ACS) wherein the pressure within the affected osteo-muscular compartment exceeds the blood perfusion pressure leading to microvasculature collapse and subsequent muscle necrosis due to rapid depletion of oxygen and nutrients. Early diagnosis and surgical intervention are critical to prevent permanent neuro-muscular damage. Currently, ACS diagnosis is heavily dependent on clinical signs which are subjective and not useful in unconscious patients, thus giving rise to a high percentage of false positives leading to unnecessary invasive surgeries. The pathophysiological mechanisms underlying ACS remains unclear which hinders the development of objective diagnostic methods. The current study aims to investigate microvascular dysfunction mechanisms which are an important contributor to ACS using an in vitro microfluidic model of the blood vessel. Specifically, the current study aims to investigate the internalisation dynamics of VE-cadherin, a membrane-bound endothelial protein under conditions of low shear stress and high hydrostatic pressure, conditions characteristic of ACS.

*Methodology: Blood vessel-on-chip microfluidic devices were prepared using standard polydimethylsiloxane soft lithography procedures. Human umbilical vein endothelial cells were seeded into gelatine-coated devices (100,000 cells/device) and treated to either low shear-stress (0.08 dyne/cm²) or high shear-stress (0.8 dyne/cm²) in the presence or absence of hydrostatic pressure head (40 cm H₂O/3960 Pa) for 48h. The cells were then fixed to spatially locate VE-cadherin in the cells via standard immunofluorescence staining. Cells were also lysed to quantify the membrane and cytoplasmic VE-cadherin protein expression levels via standard Western Blot procedures. In addition, cell culture supernatants were analysed via ELISA to quantify the secreted VE-cadherin levels.

*Results: Endothelial cells exposed to low shear stress were smaller and demonstrated polygonal morphology with VE-cadherin and actin predominantly maintained at the cell membrane. Application of hydrostatic pressure caused a marked increase in cell area, decrease in circularity and a significant increase in VE-cadherin internalisation from the membrane into the cytoplasm in the form of endocytic vesicles. VE-cadherin membrane-to-cytosol internalisation was confirmed by Western Blot analysis. Moreover, actin stress fibres were found to be aligned parallel to the direction of the flow and demonstrated lamella formation at the cell edges. High shear-stress demonstrated a similar result wherein the application of hydrostatic pressure caused an increase in VE-cadherin internalisation, although the effect was less prominent compared to the low shear-stress condition. ELISA analysis revealed decreased levels of secreted VE-cadherin shedding upon hydrostatic pressure application compared to the shear stress alone conditions, possibly due to the low levels of membrane-bound VE-cadherin available for shedding into the supernatants.

*Conclusion/Significance: In conclusion, this study demonstrates that increased hydrostatic pressure alters endothelial morphology and upregulates VE-cadherin internalisation, which has been shown to increase microvascular permeability and inflammation, thus elucidating a possible mechanism of ACS development. Early detection of VE-cadherin internalisation markers could lead to novel ACS diagnostic/therapeutic markers. Future work would involve investigating the cell-signalling events that lead to VE-cadherin internalisation by focusing on α-catenin dynamics which links VE-cadherin and actin via β-catenin in a Src-kinase mediated phosphorylation.

279 - ORGANOID-BASED HUMAN STOMACH MICRO-PHYSIOLOGICAL SYSTEM TO RECAPITULATE THE DYNAMIC MUCOSAL DEFENSE MECHANISM

H.-J. Jeong¹, J.-H. Park², J. Kang¹, J. Sabaté del Río³, S.-H. Kong⁴, T.-E. Park¹

¹ Ulsan National Institute of Science & Technology, Ulsan, Korea, Republic of,

² Gachon University Gil Medical Center, Incheon, Korea, Republic of,

³ Institute for Basic Science, Ulsan, Korea, Republic of,

⁴ Seoul National University College of Medicine, Seoul, Korea, Republic of

*Purpose/Objectives: Several stomach diseases arise from the disruption of the normal physiological functioning of the gastric mucosal barrier caused by pathogens. The potential of gastric organoids in developing treatments for gastric infections is significant. However, their ability to replicate the functional characteristics of the in vivo gastric mucosal barrier and interactions between the host and microbes is limited by the lack of physiological stimuli. Herein, we demonstrate a human stomach micro-physiological system (hsMPS) with a physiologically relevant defense mechanism, achieved through the integration of organoid and MPS technologies.

*Methodology: The hsMPS, comprising epithelial cells derived from human antral organoids (hAOs) and primary gastric mesenchymal stromal cells (gMSCs) extracted from stomach tissue, is cultivated under controlled fluid flow, recapitulates gastric epithelial homeostasis for maintenance of the functional mucosal barrier. Within a microfluidic device, controlled fluid flow facilitates interaction between epithelial and mesenchymal niches, leading to the functional maturation of gastric epithelial cells. Utilizing our hsMPS, cellular responses triggered by Helicobacter pylori (H. pylori) infection are monitored, by also adding co-cultures with immune cells.

*Results: Epithelial-mesenchymal interactions, activated by fluid flow, induced asymmetric division of stem cells, encourages stem cells to divide in a way that increases the number of progenitor cells. This balance is vital for the renewal and differentiation of epithelial cells. Accordingly, the resulting hsMPS demonstrates a physiologically relevant mucosal defense system, featuring a mesh-like mucus layer that continuously covers the gastric mucosa, containing protective TFF peptides, and enhanced gastric epithelial junctional complexes. Additionally, we developed an H. pylori infection model within the hsMPS to observe how the gastric mucosal defense reacts to pathogen invasions. Notably, the hsMPS exhibited cellular responses similar to those seen in patients infected with the H. pylori, such as releasing inflammatory cytokines and activating immune cells. A novel finding was the unique pattern of TFF peptide expression in response to H. pylori, forming an effective barrier against bacterial colonization. This highlights the potential of hsMPS in deepening our understanding of the mucosal defense mechanisms.

*Conclusion/Significance: Consequently, the hsMPS emerges as a novel in vitro tool for research endeavors where understanding the defense mechanisms of the gastric mucosa is of paramount importance in developing therapeutic strategies. Furthermore, it highlights the substantial importance of gMSCs in the field of gastrointestinal epithelial regeneration therapy.

281 - Can Gelma And Biomimetic Cultures Support The Establishment Of Mineralized 3d Osteocyte Networks?

N. Bock^1,2, T. Herath^1,2, A. Bessot^1,2, D. W. Hutmacher^1,2

¹ Queensland University of Technology, Brisbane, Australia,

² Max Planck Queensland Centre, Brisbane, Australia

*Purpose/Objectives: Rooted in the change of paradigm from 2D to 3D culture, osteocytes should not be studied in vitro in the absence of their surrounding extracellular components, including their unique collagen-minerals arrangement. We applied extracellular matrix (ECM)-derived 3D hydrogels composed of tuneable gelatin methacrylyol (GelMA)¹, for the establishment and study of 3D osteocyte networks in vitro. Gelatin is applicable for tissue engineering microenvironments as it chemically resembles native collagenous bone matrix, contains integrin attachment sites that facilitate interaction of the cytoskeleton and ECM, and is conducive to osteoblastogenesis and mineralization^2,3. Yet it is unknown whether GelMA can support osteocytogenesis of human primary cells or recapitulate mineralized osteocyte cell networks of established osteocyte cell lines. Thus we hypothesized that combining GelMA and biomimetic culture would provide new avenues to control the formation and mineralization of osteocyte 3D networks in vitro and identify how ECM and culture cues support network formation and functions.

*Methodology: We used either human primary osteoprogenitor cells (pOB) or murine DMP1-GFP OCY454 cell lines (OCY454), and encapsulated cells in 4-5% w/v GelMA (Figure 1A top). We assessed the use of growth medium (GM) or osteogenic medium (OM) up to 28 days with/without the use of a mineralization medium (MM) boost for 3 days following cell encapsulation (Figure 1A bottom), known to enhance nanoscale biomineralization⁴, and compared normoxic/hypoxic conditions (20%/2% O₂) due to bone tissue being highly hypoxic. The MM-supplemented condition refers to OM+. Mineralization was assessed by micro-computed tomography (µCT); osteocyte phenotype by brightfield and confocal laser microscopy; and protein and gene expression by immunofluorescence, Western blots and RT-qPCR, respectively.

*Results: Ongoing experiments showed that biological cues from biomimetic cultures such as OM and OM+ positively modulated mineralization and osteocyte phenotype over time. GM was insufficient to provide mineralization in either normoxia or hypoxia for both cell types, as assessed by µCT. Compared to OM, the use of OM+ provided superior hydrogel mineralization (Figure 1B). DMP1 expression, an intermediate marker for osteocytes was observed in OM/OM+ only (not GM), a positive sign of osteocytogenesis onset.

*Conclusion/Significance: While mineralization was positively affected by biomimetic cultures for both cell types, OCY454 cells presented more typical osteocyte-like phenotype features, suggesting that GelMA and biomimetic culture may be a suitable platform for osteocyte cell lines but that additional media supplements may be needed to drive full osteocytogenesis of primary human osteoprogenitors. Further optimization of in vitro 3D osteocyte culture models will enable understanding the effects of ECM-cell interactions and addressing fundamental osteocyte biology questions.

References 1. Loessner, D. et al. Nat. Protoc. 11, 727-746 (2016). 2. Mestres, G. et al. Biomed. Mater 17, 65027 (2022). 3. Bessot, A. et al. Adv. Healthc. Mater. 2201701 (2023) doi:10.1002/adhm.202201701. 4. Thrivikraman, G. et al. Nat. Commun. 10, 3520 (2019).

282 - Fabrication & Physicochemical Characterization Of Lactoprene-βTCP(60:40) Composite Scaffolds For Scaffold Guided Bone Regeneration (SGBR)

E. SEIFI^1,2, M. Mohseni^1,2,3, S. Cavelier^1,2, S. Taylor^4,5,6, B. Gaerke⁴, D. Hutmacher^1,2,7

¹ Mechanical, Medical and process Engineering, Queensland University of Technology, Brisbane, AUSTRALIA,

² ARC Training Centre for Cell and Tissue Engineering Technologies, Queensland University of Technology, Australia, Brisbane, AUSTRALIA,

³ Centre for Biomedical Technologies, Queensland University of Technology, Australia., Brisbane, AUSTRALIA,

⁴ Poly-Med, Inc, Anderson, SC,

⁵ School of Mechanical, Medical and Process Engineering, Faculty of Engineering, Queensland University of Technology, Australia, Brisbane, AUSTRALIA,

⁶ Australian Research Council Training Centre for Multiscale 3D Imaging, Modelling, and Manufacturing, Queensland University of Technology, Australia, Brisbane, AUSTRALIA,

⁷ Australian Research Council Training Centre for Multiscale 3D Imaging, Modelling, and Manufacturing, Queensland University of Technology, Australia, Brisbane, AUSTRALIA

*Purpose/Objectives: Polylactide (PLA) has been extensively utilized in the 3D printing of scaffolds. However, in the context of scaffold-guided bone regeneration (SGBR) applications the low hydrophilicity and poor osteoconductivity PLA requires it to be used in combinations with ceramic biomaterials. Nevertheless, achieving a high bioceramic phase (> 20%) in 3D printed medical grade composite scaffolds remains a challenge as a higher quantity of ceramic additive increases viscosity, impacting both the accuracy and speed of the 3D printing process. The use of medical grade biomaterials in the development pipeline is an important guideline for moving a lab based technology to a higher Technology Readiness Levels (TRL), since FDA approval and CE Marking typically require the prospective therapy to be in its final formulation throughout the validation phase. One development over the past decade is the increased supply of Good Manufacturing Practice (GMP)-manufactured biomaterials of different formulations, notably available in filament form specifically for 3D printing. The earlier in the TRL process researchers use medical grade biomaterials instead of industrial-grade equivalents, the more likely the improvement of both reproducibility and the time required to move into a higher TRL. The present study aims to characterize the mechanical properties and 3D printability of a medical grade 60/40 blend of Lactoprene, a PLA derivative, and β-TCP.

*Methodology: In this study, a Voronoi CAD model was employed to fabricate cylindrical scaffolds using Lactoprene/β-TCP filaments. Once fabricated, the scaffolds were Micro Computed Tomography (µCT) scanned and compared with the CAD model to assess the quality and reproducibility of 3D printing. The scanned scaffolds were subjected to uniaxial compression testing in air and aqueous medium to study the mechanical properties under different conditions. Then, accelerated and real-time degradation studies were performed in accordance with EN ISO 10993-13:2010 and ISO 13781 norms to evaluate the mass loss, PH alternations and release of degradation by-products over time.

*Results: The geometrical comparison between the CAD design and scaffolds showed standard deviations for scaffold volume and surface area of 1.3% and 1.5% respectively, demonstrating the high level of reproducibility. However, based on compression test results, a significant reduction in scaffolds strength occurred when tests were performed under simulated physiological conditions. The accelerated degradation study revealed a significant drop in mass and PH level from day 7 to day 14 which might be related to cleavage of polymeric bonds.

*Conclusion/Significance: The evaluation of fabrication accuracy, compression tests, and accelerated degradation outcomes suggest the need for a comprehensive examination of scaffold performance in accordance with standard norms, particularly under physiological conditions.

283 - Novel Biomimetic Surface Treatment For Improved Regeneration Of Endovascular Implants

H. Level¹, D. Mantovani², C. Hoesli¹

¹ McGill University, Montreal, QC, Canada,

² CHU de Quebec Research center, Laval University, Quebec City, QC, Canada

*Purpose/Objectives: With the progressive aging of the population, cardiovascular diseases will continue to impose a significant burden on our societies. Consequently, endovascular implants remain crucial tools for treating patients who lack alternative options. However, these devices are susceptible to both short-term and long-term complications, potentially posing a threat to the patient's life. Currently, most solutions focus on preventing cell proliferation on the implant's surface to mitigate complications, but this approach limits the device’s integration in the vascular environment.We hypothesized that we could take advantage of circulating endothelial colony-forming cells (ECFC) to improve the regenerative potential of cardiovascular implants. Our main goal is to provide a biomimetic microenvironment to promote the rapid ECFC adhesion and proliferation on the implant’s surface. Furthermore, we chose to focus our work on biodegradable metallic scaffolds, which received a growing interest thanks to their great mechanical properties. Our work will pave the way for a new generation of regenerative and biodegradable endovascular implants.

*Methodology: We developed a drug-free surface treatment technology applicable to a range of materials, including biodegradable alloys. We used a biocompatible metal oxide film that served as a platform to covalently graft biomolecules on the implant. We focused our efforts on two different substrates: stainless steel as a durable alloy and Hadfield steel (iron-manganese alloy) as a biodegradable metal. Titanium and zirconium oxide films were dip-coated on the substrates using a simple and scalable sol-gel process. It was then followed by the formation of a phosphonic acid monolayer containing carboxylic groups for subsequent biomolecule grafting. Two molecules were attached on the surface: a biomimetic peptide containing the RGD sequence and a highly hydrophobic fluorinated small molecule.The quality of the surface modification was assessed using X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). To study the cell-surface interactions, ECFC were first isolated from the peripheral blood of several donors. Cells were then seeded onto the modified surfaces for 2h, before fixation and staining of the cytoskeleton.

*Results: Surface characterization with XPS shows successful modification of the two different substrates, with significant amounts of nitrogen detected on the RGD-modified surface that is typical of peptide bonds. We also confirmed that both substrates were properly covered with the oxide films. SEM imaging also supports the homogeneous coating of the surface. Cell adhesion assays yielded two key findings: first, the presence of RGD sequence on the metal oxide surface significantly enhances ECFC spreading, resulting in a remarkable 360% increase in cell surface area compared to the unmodified surface. Second, the hydrophobic molecule prevented cell adhesion on the surfaces, with a 56% lower number of cells present. Interestingly, the type of oxide used didn’t influence the efficiency of cell spreading after RGD modification.

*Conclusion/Significance: These results suggest that our approach enables the efficient modification of metallic implant surfaces using a metal oxide layer interface. Notably, we developed a unique strategy to bring the regenerative potential of ECFC on biodegradable implants. Future work includes the addition of immobilized antibodies on the surface to selectively target circulating ECFC post-implantation.

284 - Porous Microwell Scaffolds For Promoting Aggregation And Insulin Secretion Of Pancreatic Beta Cells

H. Chen¹, T. Zeng², T. Yoshitomi¹, N. Kawazoe¹, G. Chen¹

¹ National Institute for Materials Science, Tsukuba, Ibaraki, Japan,

² University of Tsukuba, Tsukuba, Ibaraki, Japan

*Purpose/Objectives: Type 1 diabetes mellitus (T1DM) is a chronic disease that seriously impedes patients’ life due to dysregulation of blood glucose homeostasis and other serious complications. Cell therapy has emerged as an alternative and prospective strategy for treatment of It has been well recognized that the malfunction of endocrine cells in pancreatic islets causes T1DM, especially insulin-secreting pancreatic beta cells. Therefore, transplantation of pancreatic beta cells has been expected as a novel cell therapeutic strategy that can treat T1DM once and for all. However, it remains a challenge to maintain cell-cell interaction and promote functions of pancreatic beta cells.

*Methodology: To address these issues, a biodegradable gelatin-PLGA mesh porous microwell scaffold was prepared for 3D culturing of pancreatic beta cells. Ice particulate templates were used to fabricate microwell array on the surface of scaffolds. Templates were prepared by depositing water droplets on hydrophobic substrate and frozen in a deep freezer. The size of ice particulates could be adjusted by moisture exposure time of substrate. PLGA mesh immersed in 5% (wt%) gelatin solution was pre-cooled in a low temperature chamber and covered by ice particulate templates of four types of different sizes of ice particulates to prepare flat scaffold, small, middle and large microwell scaffolds. The ice particulate templates were removed by freeze-drying and microwell scaffolds were cross-linked by amidation reaction.

*Results: Microwells of different sizes were formed on the surface of the small, middle and large microwell scaffolds. The microwells had a concave morphology separated by thin ridges. The size of microwells in the small size, middle size and large size microwell scaffolds was 221.1 ± 34.8, 453.1 ± 64.0 and 874.3 ± 89.4 μm, respectively. Observation at a high magnification of SEM showed that the walls of the microwells had dense small pores. RIN-5F cells were seeded on the porous microwell scaffolds and cultured for 7 days. Live/dead staining results showed high viability of RIN-5F cells in the scaffolds after 7 days of culture. DNA amount in the porous microwell scaffolds showed significant increase. The cells cultured in the small, middle and large microwell scaffolds formed small grape-like aggregates and the small aggregates further connected together. Insulin secretion of RIN-5F cells in the scaffolds were investigated by ELISA test. The results showed that insulin secretion of RIN-5F cells in the microwell scaffolds after 7 days culture was significantly higher than that in 2D culture. These results indicated that the gelatin-PLGA mesh porous microwell scaffolds could promote functions of pancreatic beta cells.

*Conclusion/Significance: In summary, porous microwell scaffolds prepared by biodegradable biomaterials had controllable microwell size and could support the proliferation of pancreatic beta cells. More importantly, the porous microwell scaffolds promote aggregation and insulin secretion of pancreatic beta cells. The porous microwell scaffolds showed a high potential serving as a 3D culture model for T1DM treatment.

285 - Moderate Mechanical Ventilation Results In Enhanced Re-epithelialization Of Decellularized Lung Scaffolds

S. Bian^1,2, J. Say³, T. K. Waddell^2,4,5, G. Karoubi^1,2

¹ Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, CANADA,

² University Health Network, Toronto, ON, CANADA,

³ Woodsworth College, University of Toronto, Toronto, ON, CANADA,

⁴ Institute of Medical Science, University of Toronto, Toronto, ON, CANADA,

⁵ Institute of Biomedical Engineering, University of Toronto, Toronto, ON, CANADA

*Purpose/Objectives: Lung transplantation, the current treatment for advanced lung diseases, is hindered by the scarcity of suitable donor organs. To address this challenge, bioengineered lungs, created via re-epithelialization of acellular scaffolds with universal cell lines, have emerged as a promising alternative. However, the existing recellularization protocols are not adequate and do not fully recapitulate functional epithelium. While protocols predominantly rely on chemical stimuli, the mechanical environment, especially the influence of mechanical ventilation, remains inadequately explored. This study aims to investigate the effects of cyclical stretch in bioengineered lung scaffolds on distal lung epithelial cells, with a focus on enhancing attachment, proliferation, and function of the reconstituted epithelium.

*Methodology: We utilized a Negative Pressure Wet Ventilation System (NPWVS) for a decellularized/recellularized mouse lung model. 36 acellular mouse scaffolds were subjected to various ventilation conditions, including native, decellularized but non-perfused/non-ventilated, perfused-only, and combinations of perfusion and ventilation with varied tidal volumes and rates (300μL and 1500μL tidal volume; 16/32 breath per minute (BPM) ventilation rate) for 5 days. Structural changes, including alveolar integrity and scaffold thickness, were assessed through histological analysis. Mechanical properties were evaluated using uniaxial tensile strength testing and atomic force microscopy. Additionally, we examined the cellular response of human lung epithelial cells (A549 cells) to cyclically stretched scaffolds and cyclical stretch, using Precision Cut Lung Slices (PCLSs) and whole-organ bioreactor-based recellularization. 40 PCLSs were created by sectioning nonventilated/ventilated decellularized lungs and were repopulated with A549 cells for 5 days. Similarly, A549 cells were seeded into each of 15 decellularized lungs (5 different ventilation conditions) for a 5-day whole-lung recellularization. The assessment of surface coverage and cell viability were conducted with Live/Dead Imaging; the assessment of cell metabolic activity was achieved by PrestoBlue Assay; and proliferation was assessed by Ki67 immuno-staining.

*Results: The stress-strain relationship revealed that moderate ventilation parameters did not alter the structure of the acellular graft and maintained ECM proteins (p>0.05). In contrast, we observed significantly reduced stiffness (64%±22% loading stress decrement at 0.50 strain, p<0.05) in the positive control of excessively ventilated lungs (with 1500μL/16BPM ventilation), indicating expected structural changes suggesting potential damage. Cellular response on PCLS showed significantly improved outcomes in cell adhesion (increased by 89%±29%), viability (increased by 16%±9%), metabolic activity (increased by 22%±15%), and proliferation (increased by 14%±8%) for A549 cells seeded on PCLS from moderately ventilated conditions (with 0.3mL/16BPM ventilation) at Day 5 (all p<0.05). These results, indicated the potential of moderate ventilation in enhancing cell spreading, survival, and proliferation in decellularized lungs. Thus, ventilation conditions were incorporated to our whole organ recellularization protocols with results showing that 5-day moderate ventilation (300μL/16BPM ventilation) results in enhanced A549 recellularization of whole lungs, increasing the cell coverage rate by 28%±21% (p<0.05).

*Conclusion/Significance: Moderate ventilation enhanced the coverage of human lung epithelial cells within mouse lung scaffolds, without causing significant structure damages or changes in mechanical properties. Future research should focus on exploring effects of ventilation on primary human lung epithelial cell function and associated mechano-transduction signaling pathways during prolonged culture periods within our NPWVSs.

286 - Biomimetic Fibrous Scaffolds As Artificial ECM: A Novel Matrix To Animal-free Tissue Equivalents

T. Weigel¹, C. Fey¹, M. Wussmann¹, N. Pallman², M. Steinke¹, J. Schwebler¹, A. Appelt-Menzel^1,2, B. Christ¹, J. Probst¹, F. Groeber-Becker¹

¹ Translational Center for Regenerative Therapies, Fraunhofer Instisute for Silicate Research, Wuerzburg, GERMANY,

² Chair of Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, Wuerzburg, GERMANY

*Purpose/Objectives: Stromal tissues as well as interfacial basal membranes are key elements of engineered tissues applied as alternative for animal experimentation or advanced biological implants. Due to the lack of synthetic and recombinant alternatives, the focus relies on animal derived materials such as collagen¹, decellularized tissues² or Matrigel as a pre-coating strategy. However, besides ethical concerns, biological scaffolds are prone for batch-to-batch variances, increased costs as well as undesired dependencies in supply shortages. The challenge within the development of suitable synthetic 3D scaffold materials is to fulfil and combine requirements such as biomimetic structure, high porosity and bioactivity³. Here, we present the generation of highly porous fibrous scaffolds and demonstrate the applicability for a wide variety of stromal, epithelial and endothelial tissues without use of animal/human derived extracellular matrix.

REFERENCES: 1. Montalbano G. et al., Mater. Sci. Eng. C Mater Biol Appl. 91, 2018. 2. Kort-Mascort et al., Biomater. Sci., 11, 2023. 3. Weigel T. et al., Adv. Mater. 2106780, 2022

*Methodology: Scaffolds were generated by a modified electrospinning process. From the variety of biocompatible polymers that are applicable, we focused on polyamide. The porosity was controlled by the addition of NaCl porogen particles in defined proportions and size distributions. The resulting highly porous scaffolds were characterized concerning structure and micro-scaled mechanical properties by nano indentation. For biologization, the 3D scaffolds were seeded with human stromal tissue cells from the target tissue (fibroblasts, mesenchymal stromal cells - MSC). After 1-4 weeks of migration, proliferation, differentiation and matrix synthesis, the resulting human ECM tissues were combined with epithelial cells (e.g. skin, intestine, airway, cornea), tumor cells or endothelial cells (e.g. peripheral or blood-brain-barrier) to form monolayered, multi-layered or hierarchically structured tissues.

*Results: The proportion and size distribution of the porogen particles were defined and the resulting structural properties, like pore sizes, mesh sizes and porosities determined. The mechanical properties on the cellular level with a young’s modulus of 3 kPa were comparable to conventional hydrogels e.g. collagen gel. Since the scaffolds allow cellular migration as well as the dynamic rearrangement of the fibers, primary human fibroblasts were able to homogenously populate the scaffold and remodel the structure and secreted tissue specific ECM components. Furthermore, MSC were able to be differentiated inside the 3D structure (e.g. adipogenic or osteogenic) to transform the synthetic scaffold in further applicable types of biologized tissues. The biologized scaffolds were suitable for combination with human epithelial cells to generate physiological and functional models of skin, airways, intestine, cornea and endothelia. The resulting tissue equivalents were characterized concerning physiological cellular composition, polarization as well as characteristic barrier functionality.

*Conclusion/Significance: The described modular principle, starting from a synthetic open meshed scaffold (with a variety of applicable polymers), various biologization possibilities and following combinations with epithelial/endothelial layers, generated a platform technology, which can address nearly all human types of tissues. This enables finally a simultaneous replacement of animal derived materials, an increase in tissue complexity and improved physiological readout.

287 - Bio-fabrication Of A Human IPSC-based Vascularized Endocrine Pancreas For The Treatment Of Type 1 Diabetes

F. Campo^1,2, A. Neroni^1,2, C. Pignatelli¹, S. Pellegrini¹, I. Marzinotto¹, F. Manenti¹, L. Valla^1,3,4, M. Policardi¹, V. Lampasona¹, L. Piemonti^1,2, A. Citro¹

¹ San Raffaele Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milan, ITALY,

² Università Vita-Salute San Raffaele, Milan, ITALY,

³ Chair for Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, Munich, GERMANY,

⁴ Center for Innovative Medical Models (CiMM), Oberschleißheim, GERMANY

*Purpose/Objectives: Intrahepatic islet transplantation in patients with type 1 diabetes is limited by donor availability and lack of engraftment. To overcome these limitations, new sources of β cells and alternative sites are needed. Organ decellularization is an emerging strategy in organ regeneration. Based on our experience with decellularized rat lung as scaffold for the generation of Vascularized Endocrine Pancreas (lung scaffold repopulated by neonatal pig islet and BOEC cells), we used the same platform to engineer an iPSC-based version named iVEP (iPSC-derived Vascularized Endocrine Pancreas).

*Methodology: iPSC-derived β (iβ) and endothelial (iEC) cells were characterized in flow cytometry and then aggregated into functional vascularized iβ spheroids (SPH). Rat lung was decellularized by vascular perfusion with 1% SDS and 0.1% Triton and seeded with iECs and SPH from vascular and air accesses. The recellularized scaffold matured in vitro for 7 days in a customized perfusion bioreactor specifically designed to allow cell/compartment integration. The iβ death was estimated during ex vivo organ maturation compared to 7 days of iβ in vitro culture evaluating miR-375 expression by droplet digital PCR. At day 7, fluorangiography assay was performed to evaluate the vascular compartment structure and function while for the endocrine compartment, insulin production was measured by dynamic glucose perifusion and insulin quantification (ELISA/IF). Matured iVEPs were then transplanted subcutaneously into NSG diabetic mice followed for 30 days and compared to the deviceless (DL) implantation site. Explanted iVEPs were used for IF evaluation.

*Results: iEC/iβ maintained for 7 days in vitro their phenotype expressing endothelial (>95% CD31+/CD105+/CD73+/CD90- cells) and β-cell (>60% PDX1+/insulin+ cells) markers respectively. Matured iVEPs showed regenerated vascular network (CD31+) able to sustain the direct distribution of a perfusate with SPH (Chromogranin A/Insulin+) fully integrated in the engineered vasculature. Also matured iVEPs significantly reduced β cell death: the amount of lost iβ was ≤15% during organ maturation, while >60% during in vitro culture (p<0.05). In iVEPs, vascularized ECM was able to significantly sustain iβ engraftment, survival and promote phenotypical maturation of iβ with physiologic insulin secretion (AUC first phase iVEPs: 3.233±1.391 vs. batch matched SPH: 0.9483±0.4069, p<0.05). Preliminary preclinical results demonstrated the ability of iVEP to engraft and restore normoglycemia in diabetic recipient mice compared to SPH implanted in the DL site (iVEP 100% vs DL 25%) with a median time of 8 days.

*Conclusion/Significance: Ex vivo iVEPs enables iβ engraftment, survival in a pre-vascularized ECM. promoting iβ phenotypic maturation and function. In vivo, iVEPs showed immediate function restoring normoglycemia in diabetic NSG mice. To our knowledge, we assembled the first human entirely iPSC-derived Vascularized Endocrine Pancreas able to provide both controlled insulin secreting phenotype ex vivo and in vivo function.

288 - Establishment Of A Human 3d Kidney Tissue Model Using Decellularized Rat Kidney Slices

H. Salti¹, L. Kramer¹, S. Nelz¹, S. Lichtwark¹, M. Lorenzz², C. Pohl³, J. Hofrichter¹, A. Breitrück¹, S. Mitzner^1,4, R. Wasserkort^1,4

¹ Extracorporeal Therapy Systems (EXTHER), Fraunhofer Institute for Cell Therapy and Immunology (IZI), Rostock, GERMANY,

² Wismar University of Applied Sciences, Wismar, GERMANY,

³ Department of General Surgery, Visceral, Thoracic and Vascular Surgery, Greifswald University Medical Center, Greifswald, GERMANY,

⁴ Division of Nephrology, Department of Internal Medicine, Rostock University Medical Center, Rostock, GERMANY

*Purpose/Objectives: The global rise in chronic kidney disease underscores the pressing need for innovative treatments for end-stage renal disease (ESRD) beyond transplantation and dialysis. The use of tissue engineering to develop bioartificial kidneys may offer an alternative path. The extracellular matrix (ECM) obtained by decellularization provides scaffolds with the complex architecture of the organ. However, whole kidney de- and recellularization by perfusion are highly complex processes that require intensive optimization of various parameters. Precision-cut kidney slices (PCKS) were utilized as a source of natural scaffolds. Since chemical reagents have a damaging effect on the ECM, this study aimed to investigate the impact of physical treatments on the efficacy of PCKS decellularization by immersion in chemical reagents.

*Methodology: Physical methods involved pretreatment of PCKS with high hydrostatic pressure (HHP) or freezing-thawing cycles (FTC) followed by immersion decellularization with a protocol involving only chemical reagents (CHEM-Imm). PCKS were also decellularized using the CHEM-Imm protocol in an ultrasonic bath system (UBS). The evaluation of decellularization included assessing the preservation of ECM components and removal of residual DNA, alongside histological assessment. Furthermore, to assess variations in the ability of decellularized rat PCKS to support recellularization, the scaffolds were seeded with human immortalized renal proximal tubular epithelial cells (RPTEC/TERT1). The assessment of recellularized scaffolds involved evaluating cell viability using the resazurin assay and examining the expression of the tight junction protein zonula occludens-1 (ZO-1). Furthermore, artificial neural networks (ANN) were utilized for a location-specific assessment of the seeded RPTECT/TERT1 cells. This involved employing ANN to automatically identify Bowman capsules (BC) in scaffolds stained against collagen type IV (Col-IV). Subsequently, the number of seeded cells within the BCs relative to the total number of seeded cells were counted, serving as an indicator of specific recellularization.

*Results: In comparison to decellularization by CHEM-Imm alone, FTC-Imm (FTC + CHEM-Imm) was the most effective in reducing DNA while preserving glycosaminoglycan content (GAG) in the ECM. While UBS-Imm resulted in a comparable reduction of DNA, it was the least effective in retaining GAGs. Conversely, despite pretreatment with pressures up to 200 MPa, HHP200-Imm (HHP200 + CHEM-Imm) was the least effective in DNA removal. All recellularized scaffolds, regardless of the decellularization method, demonstrated cell growth over time (7 days). Notably, FTC-Imm scaffolds exhibited the highest expression of ZO-1, suggesting a better cell interaction with the scaffold. Moreover, assessment with ANN demonstrated that, at least 96% of RPTEC/TERT1 cells on all decellularized scaffolds avoided unspecific attachment to the BCs. All results were integrated into a scoring system, which revealed that FTC-Imm received the highest score.

*Conclusion/Significance: This study underscores the potential of using FTC in whole kidney tissue engineering. It also provides valuable insights into the instructive memory within the decellularized ECM, influencing the migration of immortalized RPTEC/TERT1 cells and preventing location-unspecific attachment. In summary, this research highlights the significance of using PCKS as a model for the initial investigation of de- and recellularization methods, thereby reducing the number of sacrificed animals. Moreover, this model can potentially be used in nephrotoxicity assessments.

289 - Compassionate Use Of Allogeneic Self-assembled Skin Substitutes For Recessive Dystrophic Epidermolysis Bullosa Wound Closure

M. A. Barbier^1,2, A. Dakiw Piaceski^1,2, D. Larouche^1,2, J. Fish³, E. Pope³, L. Germain^1,2

¹ The Tissue Engineering Laboratory / LOEX and Department of Surgery, Faculty of Medicine, Université Laval, Québec, QC, CANADA,

² CHU de Québec-Université Laval Research Centre, Québec, QC, CANADA,

³ Hospital for Sick Children and University of Toronto, Toronto, ON, CANADA

*Purpose/Objectives: Recessive dystrophic epidermolysis bullosa is a rare genetic disease in which loss-of-function mutations leads to the absence or truncation of type VII collagen, an essential component of the epithelia to stroma junctions. This results in a decrease in adhesion strength. In the skin, patients could suffer from large open wounds that are extremely challenging to heal. A patient with a severe form of RDEB presenting open wounds covering 30% of his total body surface area was treated with tissue-engineered skin substitutes (TES) in a n-of-1 compassionate clinical trial. The hypothesis was that the addition of normal cells within TES could produce enough C7 to improve the dermo-epidermal adhesion. In the present study, we assessed both in vitro and in vivo the potential of allogeneic skin cells as a therapy for RDEB wounds.

*Methodology: Combinations of autologous RDEB keratinocytes (RDEB-K) and fibroblasts (RDEB-F) and healthy allogeneic keratinocytes (H-K) and fibroblasts (H-F) were used to produce autologous (RDEB-F/RDEB-K), mixed (H-F/RDEB-K) or chimeric (H-F/RDEB-K/H-K) TES using the self-assembly approach. C7 production was analyzed by immunofluorescence staining and the dermo-epidermal strength was quantified through mechanical peeling tests.

*Results: Mixed and chimeric TES were applied on chronic wounds situated on the back of the patient. In vitro, the deposition of C7 at the dermo-epidermal junction and the adhesion strength of the dermo-epidermal junction of mixed TES were higher compared with autologous TES. The chimeric TES, containing both allogeneic and autologous keratinocytes, allowed for an intermediate increase in C7 in vitro. Two weeks after grafting, a loss of the epidermis was observed in chimeric TES. Keratinocytes are known to be highly immunogenic. Interestingly, the epidermis rapidly reepithelialized thereafter. At the site grafted with mixed TES, a high graft take (98%) was observed, and the epidermis persisted for the first 6 weeks, with only 2% of wounded surface per grafted area. However, wounds started to reappear after 8 weeks (13%) and 6 months (47%) but decreased after 1 year (12%). CD45-positive cells were observed in the reconstructed dermis 3 months after grafting, indicating a potential rejection of allogeneic fibroblasts. However, this did not lead to rejection of the entire skin substitute.

*Conclusion/Significance: The use of mixed TES allowed for the closure of chronic wounds and help the general health of the patient. TES did not persist long term in a n-of-1 compassionate clinical trial, indicating that it is not a permanent treatment for RDEB. However, it led to significant improvements in quality of life and total wound surface area in the short term.

290 - De Novo Formation Of Sebaceous Glands In Self-assembled Skin Substitute: Impact Of Human Epithelial Stem Cells

B. Cattier^1,2, B. Magne^1,2, M. Lemire-Rondeau^1,2, A. Morissette^1,2, É. Philippe^1,2, D. Larouche^1,2, L. Germain^1,2

¹ Département de Chirurgie, Faculté de médecine, Université Laval's Research Centre: The Tissue Engineering Laboratory (LOEX), Québec, QC, CANADA,

² Regenerative Medicine Axis, Centre de recherche du CHU de Québec, Québec, QC, CANADA

*Purpose/Objectives: Sebaceous glands are skin appendages distributed throughout the body, either as part of pilosebaceous unit or as free structures. They maintain skin hydration by secreting sebum, a lipid mixture produced by sebocytes. However, upon disease or injury, these skin structures can be altered or lost, compromising cutaneous homeostasis. This could lead to inflammatory diseases or result in irreversible loss of function, as observed in severe burn victims. To date, de novo regeneration of sebaceous glands does not occur in human tissue-engineered skin substitutes. Several studies suggest that the cell source used to produce the epidermis can influence the type of adnexa that can form when they are associated with neonatal fibroblasts. The objective is to explore the conditions (environmental, cell source) that promote the differentiation of epithelial stem cells into sebocytes in human tissue-engineered skin substitutes (TESs) produced by the self-assembly approach.

*Methodology: Human bilayered TESs comprising a dermis and an epidermis were produced by the self-assembly approach using keratinocytes from three anatomical sites (scalp, foreskin, breast) and neonatal foreskin fibroblasts. Keratinocytes were cultured on fibroblasts sheets and matured for 10 days at the air-liquid interface. TESs were then transplanted onto athymic mice for 21 days. TESs biopsies taken before and after grafting were analyzed through histology. The resulting glandular structures were characterized using Oil Red O and immunofluorescence staining, as well as transmission electron microscopy (TEM). Differentiation into sebocytes and epidermis was assessed using antibodies against keratin7, EMA/mucin-1, keratin15 and loricrin. The human origin of the structures was confirmed using antibody raised against HLA-ABC antigen.

*Results: Prior to grafting, clusters of enlarged epidermal cells were noticed in some sections from TES produced using scalp epithelial cells but not in TES produced with another epithelial cell source. Their morphology suggested that these cells have not undergone normal commitment to epidermal lineage. Following grafting, glandular structures were only observed in TESs produced with scalp epithelial cells suggesting that enlarged cells observed in vitro developed into sebaceous glands after grafting. TEM confirmed ultrastructural characteristics specific to sebaceous glands, including a stratified morphology and lipid droplet content. Glandular structures exhibited red staining with Oil Red O and an excretory canal filled with lipids connected them to the skin's surface suggesting that the newly developed glands were functional. Immunofluorescence staining revealed the presence of basal precursors (keratin15), early and mature sebocytes (keratin7, EMA/mucin-1) within these structures. Adequate differentiation of the epidermis was evidenced by loricrin expression. The expression of HLA-ABC, specific to human cells, confirmed that these sebaceous gland-like structures originated from human cells.

*Conclusion/Significance: We have developed a human model enabling the de novo formation of sebaceous-like glands after grafting on mice. Our findings indicate that the origin of epithelial stem cells plays a crucial role in the appendage’s differentiation. Identifying key molecular and cellular signals involved in this interaction is a strategy to pursue in the future. These findings open exciting new avenues for understanding stem cell differentiation toward the sebaceous gland lineage and the development of drugs targeting sebaceous glands.

292 - Development Of A Functional Vascularized Skin Graft Using Gelatin Methacryloyl As Bioink

H.-Y. WAN^1,2, A. M. Blocki^1,2,3

¹ The Chinese University of Hong Kong, Hong Kong, Hong Kong,

² Center for Neuromusculoskeletal Restorative Medicine, Hong kong, Hong Kong,

³ Institute for Tissue Engineering and Regenerative Medicine, Hong kong, Hong Kong

*Purpose/Objectives: Chronic wounds fail to heal due to dysregulated wound healing processes. While autograft and allograft skin transplants are the gold standard treatments, they are limited by donor availability and immune rejection issues. Skin tissue engineering (STE) provides a novel approach for skin wound treatments, aiming to fabricate skin grafts that facilitate healing and restore skin function. Especially 3D bioprinting enables the delivery of skin grafts with high precision, potentiating the development of STE. Nevertheless, current bioprinted grafts lack vasculature, limiting successful engraftment and leading to necrosis of these skin grafts. Hence, 3D bioprinting of a vascularized skin graft was proposed. In order to produce successful vascularized skin grafts, the bioink of choice should (I) exhibit good printability, biocompatibility and biodegrdability, while (II) supporting microvasculature formation and maintenance and (III) dermal and epidermal maturation. Herein, we propose to engineer vascularized skin grafts by using Gelatin methacryloyl (GelMA) as bioink. GelMA is an established bioink with good printability, mechanical strength and biocompatibility. We hypothesized that GelMA will support the formation of stable microvascular networks (MVNs) within, while promoting differentiation of keratinocytes seeded on top, thereby paving the way for bioprinting of vascularized skin grafts.

*Methodology: Human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDF) were embedded in GelMA hydrogels to form the dermal layer. Formation of hollow interconnected MVNs was observed by fluorescent microscopy and number of vessel nodes and branch length was quantified over time. Next, keratinocytes were seeded on top of optimized dermal layers and cultured in an air-liquid interface. Keratinocyte differentiation and formation of epidermis was evaluated by histological evaluation by H&E staining and immunohistochemistry for epidermal markers, including cytokeratin 10 and 14 after 2 weeks.

*Results: GelMA was synthesized using a facile one-pot method, achieving at least 95% degree of substitution. The synthesized GelMA exhibited no cytotoxicity, as confirmed by encapsulation of HUVECs followed by live-dead cell staining. HUVECs seeded in GelMA hydrogels self-assembled into tubule-like structures spontaneously by vasculogensis and displayed an interconnected vascular network when encapsulated in 2 to 4%, but not in 5% (w/V) GelMA. While microvessels in 2% (w/V) GelMA exhibited a dilated lumen, microvessels cultured in 3 to 4% GelMA exhibited smaller diameters of less than 50 µm, consistent with capillaries in vivo. Co-culture of HUVECs and HDF at ratios of 10:1 to 60:1 in GelMA hydrogels promoted MVNs stability over time comparing to HUVECs cultured alone. In addition, co-cultured HDFs often took up perivascular location, suggesting pericyte-like behavior. Keratinocytes cultured on top of these dermal layers proliferated, forming a basal layer and differentiated thereby forming an epidermal layer. The successful differentiation was further confirmed by the expression of cytokeratin 14 and 10 in basal and suprabasal keratinocyte, respectively, after 2 weeks of culture.

*Conclusion/Significance: We demonstrated GelMA is a suitable bioink, as it supports microvasculture formation in the dermal layer and formation of an epidermal layer. Moving forward, this bioink will be employed for 3D bioprinting of vasularized skin grafts, advancing the development of STE for skin wound healing.

293 - Pre-organized Cellular Components In Bioprinted Skin Improving Long Term Functionality Of Full Thickness Wounds

K. Willson¹, A. Gorkun², N. Mahajan², E. Potts², N. Edenhoffer¹, A. Jorgensen¹, C. Clouse¹, S. Soker¹, A. Atala²

¹ Wake Forest University, WINSTON SALEM, NC,

² Wake Forest Institute for Regenerative Medicine, WINSTON SALEM, NC

*Purpose/Objectives: Bioengineered skin constructs currently used for treatment of full thickness skin wounds contain 1-2 cell types resulting in minimal development of sub-epidermal structures (hair follicles, glands, pigmentation, and vasculature). We printed multi-layered skin constructs with 6 cell types containing dermal, hypodermal, and epidermal layers which integrate and form functional skin with pigmentation in full thickness wounds.

*Methodology: We compared printed hydrogel (non-cellularized), tri-layered bioprinted skin, and tri-layered skin patterned with pre-cultured skin organoids (reorganized skin) constructs for their capabilities to regenerate native skin with a wound only control (Figure 1A). For re-organized skin, 25% of the cells were pre-cultured as organoids, then added to the dermal layer during printing. Printed constructs were matured prior to implantation in a 2*2 cm skin wounds on mice and analyzed after 14, 21, 56, and 91 days.

*Results: Wound sites were monitored and imaged throughout the study to analyze for contraction and epithelialization. Contraction was measured using Image J, normalized to the original wound size for each animal. Cellularized constructs showed significantly less contraction at the final timepoint, maintaining 56.65% and 44.28% of the original wound size (reorganized and bioprinted) compared to 13.9% and 7.27% (hydrogel, wound only). Representations of wound only and reorganized skin are shown in Figure 1B and 1C. Epithelial thickness, measured at each explant, was increased in both cellularized groups, comparable to normal human skin. Histochemical analysis of the epithelial layer showed the development of rete ridges (characteristic of non-scarred human skin) in reorganized constructs only (Figure 1D/E). Staining of these regions has indicated different patterns of collagen deposition for collagens I and III compared to areas in the same animal that did not exhibit rete ridges. Furthermore, cellularized constructs showed lower coefficients of both collagen fiber alignment and shorter fiber length, typical of normal skin compared to the non-cellularized control groups. Visible pigmentation was observed in mice treated with reorganized skin beginning at 28 days. Images of the animals were analyzed using mono-spectral analysis, with higher pigmentation being maintained in the reorganized group compared to all other through 91 days. Lastly, differences have been observed in inflammation-related gene expression not only between cellularized and non-cellularized implants but between the bioprinted and reorganized samples continuing throughout the 91-day study.

*Conclusion/Significance: This study marks not only the development of a multi-layered skin construct which can aid in minimally contractile wound closure but also an important step towards fully functionalizing bio-printed skin in vivo. While both cellularized constructs showcased epithelial thickness and collagen alignment comparable to healthy, non-scarred human skin, the addition of organoids allowed for the development of human characteristic rete ridges and visible pigmentation. Further analysis into the alterations that pre-organized cells provided in specific collagen deposition and genetic regulation may provide important insight towards developing fully functional skin replacements for the clinic.

294 - 3d-printed Bilayer Scaffold/membranes For Tailored Differentiated Full-thickness Skin Models

M. Rielland

Skin Tech Platform, L’Oréal Advanced Research, Aulnay Sous Bois, FRANCE

*Purpose/Objectives: Over the past 50 years, in vitro skin and hair models have been developed and used for various clinical and research applications including skin grafting for wound or burn healing as well as studies on skin physiology and pathophysiology. Hydrogel models exhibit low extracellular matrix (ECM) neosynthesis, along with weak mechanical strength and stability and fall short in replicating the complexity and organization of natural ECM, limiting their ability to predict in vivo responses. An alternative approach is to use porous materials, or scaffolds. However, current porous scaffold techniques exhibit limited ability to faithfully replicate the ultrastructural architecture of the skin, we believe due to manufacturing limitations. The 3D-printed bilayer scaffold/membrane model described here overcomes some of these deficits by incorporating an electrospun (SES) membrane on a melt electrowritten (MEW) scaffold which provides an optimal open-pore environment, resulting in a high-quality in vitro skin model.

*Methodology: MEW and solution electrospinning (SES) of polycaprolactone (PCL) were combined to produce a bilayer straight or wavy SES-MEW scaffold/membrane consisting of microfibers and nanofibers. Normal human fibroblasts or keratinocytes from skin plastic surgery wastes are then seeded in the bilayer scaffolds. Our results are based on extensive histological, immunofluorescence, microscopy (second harmonic) and mechanical property measurements to characterize and statistically analyze the formed skin models.

*Results: The scaffold/membrane fabricated using MEW and SES were designed to mimic the skin while addressing the limitations of current scaffolds and is the first instance of developing a bilayer scaffold/membrane using SES and MEW for skin reconstruction models. The outcome was striking, with a properly stratified and differentiated epidermis. Furthermore, this scaffold/membrane model is, to the best of our knowledge, the only one capable of creating a properly differentiated, full thickness skin model with neosynthesized extracellular matrix in just 18 days. Both the wavy and straight MEW fiber scaffold designs exhibited a well-organized dermis with presence of collagen IV, VII and perlecan at the dermal-epidermal junction and collagen I, fibrillin and elastin in the dermal part. The morphology of the straight and wavy MEW scaffolds encouraged different ECM dermal collagen organization. Overall, the addition of cells and vitamin C to the scaffolds improved the viscoelastic mechanical properties, moving towards mimicking native human skin.

*Conclusion/Significance: To the best of our knowledge, the bilayer scaffold/membrane model described in this study is the only one capable of creating a properly differentiated, full thickness skin model with neosynthesized ECM in only 18 days. Both the wavy and straight fiber scaffold designs create a well-organized dermis, but dermal collagen organization differs between designs. Adding cells and vitamin C to the scaffolds improves the mechanical properties to more closely mimic native human skin. The versatility and adaptability of this model give it many uses including studying how the biological and physical microenvironment impacts the skin, creating pathological skin models, and testing dermo-cosmetics and pharmaceutical treatments on different ages of skin. Furthermore, it could be an excellent new tool for studying wound healing in vitro, as graft or wound dressing.

295 - Fgf8 Induces Bone And Joint Regeneration At Digit Amputation Wounds In Mice

L. Yu, M. Yan, H. M. Smith, J. D. Knue, C. P. Dolan, R. Brunauer, L. A. Dawson

Texas A&M University, College Station, TX

*Purpose/Objectives: Humans and mice have limited limb regeneration capacity after amputation, with complete regeneration restricted to amputations transecting the distal digit tip, the terminal phalanx (P3). Conversely, proximal amputations of the digit and limb, such as amputation through the adjacent skeletal segment, the middle phalanx (P2), fail to regenerate, resulting in bone truncation at the amputation level and soft tissue scar formation. In mice, P2 serves as a non-regenerative wound site to investigate regenerative failure and as a model system to screen regenerative approaches aimed at stimulating mammalian limb regeneration. Here, we used this growth factor screening approach and identified FGF8 as a potent inducer of synovial joint regeneration at the P2 amputation wound. Specifically, FGF8 transitions P2 fibrotic scarring into a spatially appropriate multi-tissue regenerative response characterized by bone, articular cartilage, synovial cavity, tendon, and ligament regeneration.

*Methodology: Using male and female neonate ICR (n = 60 digits) and Prg4^{GFPCreERt2+/+};Ai9 (n = 28 digits) mice, P2 amputations were performed on hind limb digits at post-natal day 3 (PN3), followed by implantation of FGF8 (500ng/µl) or BSA (0.1% in PBS) coated beads at PN7. Digits at various time points were analyzed for gene expression, histology, in-situ hybridization, and immunohistochemistry. A t-test was used to determine significance. All animal use and techniques followed the standard operating procedures of Texas A&M University's IACUC.

*Results: FGF8-induced joint regeneration is a multi-step process. First, quantitative RT-PCR analysis demonstrates FGF8 stimulates the upregulation of several joint development genes, including Gdf5, Prg4, and Chrdl2, joint cavity development genes CD44 and Has2, as well as hyaline cartilage genes Sox9, Acan, and Col11a2. Next, at a frequency of 62% of treated digits (n = 32), FGF8 induces the formation of a synovial cavity expressing Prg4, the gene encoding the proteoglycan Lubricin, followed by the formation of a cartilaginous nodule distal to the amputation plane and the subsequent induction of cartilage that caps the amputated P2 stump. The FGF8-induced cartilage that caps the P2 stump serves as a template for distal bone elongation to restore 14% of the amputated skeletal length (t-test; P = .001439; n = 12 digits). Lastly, the nodule ossifies centrally, while the cartilage adjacent to the synovial cavity matures into articular chondrocytes. Lineage tracing analysis using Prg4^{GFPCreERt2+/+};Ai9 mice revealed that the multi-tissue regenerative response is predominantly derived from the FGF8-induced Prg4-lineage.

*Conclusion/Significance: An important conclusion from these studies is that regenerative failure at proximal amputation levels is not a consequence of absent regeneration-competent cells within the wound environment. Instead, we propose that regenerative failure is a consequence of absent or attenuated pro-regenerative signaling at the amputation site. The significance of this conclusion is vast in that it identifies non-regenerative amputation wounds as a rich source of regeneration-competent cells that are otherwise dormant but can be targeted to develop novel strategies for enhancing mammalian limb regeneration.

296 - Induction Mechanism Of Axolotl Limb Regeneration

A. Satoh

Okayama University, Okayama, Japan

*Purpose/Objectives: BACKGROUND: The axolotl, an amphibian, is well-known for its remarkable regenerative abilities. This capability extends to the organ level, enabling axolotls to regenerate parts such as limbs, tails, and even brains. Such high regenerative capacity is basically not observed in mammals, including humans. Hence, deciphering the mechanism behind axolotl regeneration could potentially lead to significant advancements in regenerative medicine for complex organs of mammals. The study of limb regeneration in axolotls has a history spanning over 200 years. However, the initiation of this regenerative process remained largely unknown until recent times. The essential role of nerves in starting regeneration was demonstrated by Todd in 1723, but the specifics of how nerves molecularly and dominantly control limb regeneration remained unclear. Clarification of the initiation mechanism of limb regeneration regulated by nerves had been a long standing issues in this field.

*Methodology: METHODOLOGY/RESULTS I:To clarify the nerve-derived molecules, we have developed a unique experimental model, the 'accessory limb model.' Usage of this model allowed us to identify the nerve-derived molecules. We identified BMP and FGF as the nerve-derived molecules, and discovered that their synergistic action initiates regeneration. While FGF signaling plays a crucial role in axolotl regeneration, complete limb regeneration cannot occur without the complementary BMP signal. The individual roles of FGF and BMP signals in this process are still not fully understood. The neural factors we identified (FGF+BMP) can induce organ regeneration in axolotls and have shown to be effective in inducing organ regeneration in other species such as newts, chickens, and frogs. This suggests the potential for a universally applicable mechanism for organ regeneration across different organisms.

*Results: METHODOLOGY/RESULTS II: We have expanded our investigation to comprehend endogenous reprogramming in axolotl limb regeneration. Nerve-derived molecules induce undifferentiated cells in this process, previously believed to originate from fully differentiated fibroblasts. However, the determination of fully differentiated fibroblasts had not been established in the axolotl research field. Consequently, we defined differentiated fibroblasts before delving into endogenous reprogramming. We employed a single-cell labeling method and successfully identified differentiated fibroblasts. The definition of fully differentiated fibroblasts enables the investigation of endogenous reprogramming by the defined factors (FGF+BMP).

*Conclusion/Significance: CONCLUSION: The discovery of regeneration inducers (FGF+BMP) in axolotl limb regeneration provides an opportunity to test their effects in other vertebrates and/or organs and to explore endogenous reprogramming.

297 - Cellular And Molecular Profiling Of Critical Bone Fractures In Axolotls

A. Polikarpova¹, I. Rivero Garcia², T. Gerber³, J. Wang¹, M. Torres Sánchez², F. Sánchez Cabo², E. M. Tanaka¹

¹ Institute of Molecular Pathology, Vienna, Austria,

² CNIC (Spanish National Center for Cardiovascular Research), Madrid, Spain,

³ EMBL (European Molecular Biology Laboratory), Heidelberg, Germany

*Purpose/Objectives: Fractures are prevalent traumatic injuries, with small aligned fractures typically healing through the formation of fibrocartilaginous callus. However, larger and complex fractures may lead to non-union (pseudoarthrosis) in 5-10% of long-bone fractures. This adversely affects patients' mobility, quality of life and imposes a substantial clinical, economic, and social burden. To establish effective cell-based or molecular therapy methods, the critical size defect (CSD) model is employed to study fracture non-union. Axolotls, known for limb regeneration via a transient progenitor pool, the blastema (BL), present a unique regenerative capacity. Paradoxically, axolotls cannot heal critical size bone defects. Our study aims to elucidate whether soft connective tissue (SCT) cells migrate to CSD and identify the factors prompting SCT cells to contribute to bone regeneration.

*Methodology: To examine SCT cell migration and their potential contribution to cartilage in CSD, we transplanted tissues containing fluorescently labeled CT cells into wildtype hosts before fracture surgery. To identify the factors involved in dedifferentiation of SCT cells to blastema versus CSD, we employed bulk and single-cell transcriptomics. We compared single-cell atlas across all cell types for the two conditions, including SCT cells, epidermis, macrophages, neutrophils and other cell types. To examine if blastema-specific signaling has an impact on CT cell transcriptome profiles, we analyzed CT GRNs , based on blastema and CSD scRNAseq.

*Results: The transplantation of tissues with fluorescently labelled CT cells demonstrated SCT cells migrating to the CSD gap, with some expressing the cartilage progenitor marker, SOX9. Subsequent assessment of cell proliferation revealed a diminished rate in CSD compared to the blastema.

The bulk and single-cell transcriptome analysis revealed changes in proliferation- and dedifferentiation-specific gene expression, along with differences in interaction with wound epidermis and macrophages. As interplay between SCT cell, epidermis and immune cells was shown to be important for regenerative blastema establishment, we examined ligand-receptor interactions in early regenerating limb and CSD using the NicheNet algorithm. It showed strong interaction of early (3-5 dpa) blastema CT cells with epidermis and macrophages. The interaction between wound epidermis (WE) and stump fibroblasts is essential for activation of the latter to migrate and form blastema. The epidermal subpopulation in BL samples, but not in CSD samples, manifested WE signature. Contrarily, in CSD, fibroblasts and epidermis did not exhibit robust interaction and lacked ligand-receptor pairs present in the blastema.

Whole transcriptome GRN inference uncovered a shift in transcriptional factor hubs, such as the WNT and TGFb pathway transcription factors. We discovered differential usage of TCF7L2 and LEF1 TFs in blastema and CSD and revealed members of WNT pathway possibly modulating SCT cell behavior between homeostatic and regenerative mode.

*Conclusion/Significance: Our findings, derived from tissue transplantation and GRN analysis, demonstrate that SCT cells can migrate to axolotl CSD but fail to proliferate and establish blastema tissue. This may stem from differences in fibroblast and wound epidermis interaction and an insufficient TGFb and WNT signaling milieu in CSD. We propose the rewiring of GRN in CSD to induce a blastema-like phenotype and to ultimately facilitate bone bridging.

298 - Fetal Tendon In Vivo And Ex Vivo Healing Models Based On The Chick Embryo

P. Nguyen, C. D. Hart, C. K. Kuo

University of Maryland, College Park, MD

*Purpose/Objectives: Injured adult tendons heal as fibrotic scar tissue and possess high re-injury rates, whereas fetal tendons appear to heal scarlessly. Our long-term goal is to elucidate mechanisms of fetal scarless tendon healing, and use this knowledge to inform therapeutic approaches to regenerate injured adult tendons. However, knowledge of fetal tendon wound healing, previously studied in a sheep model, is limited due in part to the need for an accessible animal model. The chick embryo is an attractive option to study mechanisms of fetal tendon healing. A unique advantage of the chick embryo is that it develops independently within an eggshell in only 21 days and can be accessed directly during development without the complications of in utero surgery that are associated with mammalian models. The chick embryo has been used to elucidate key aspects of tendon development that share significant overlap with mammal (including human), including collagen fibrillogenesis, collagen crosslinking, extracellular matrix (ECM) deposition and remodeling, and mechanical property elaboration. Our objective was to develop and use in vivo and ex vivo chick embryo models to study fetal tendon healing. We hypothesized that embryonic tendons heal rapidly after injury in vivo and ex vivo, and that healing responses are dependent on developmental stage at time of injury. We pursued an in vivo model because it provides a physiological environment, and pursued an ex vivo model because it enables greater control over the culture environment.

*Methodology: Calcaneal (Achilles) tendons of embryonic day (D) 14 and D17 chick embryos were injured by transecting 25% of the tendon width at the midpoint of the midsubstance. Healing responses were assessed using histological staining and histomorphometry, gene expression analyses, and mechanical testing. N=3 to 5 chick embryos were required for each assay. Two-tailed Student t-test was used to analyze statistical differences between injured and non-injured tendons at the same timepoint and injured tendons at different timepoints, with p<0.05 considered significant.

*Results: In both the in vivo and ex vivo models, injury sites filled rapidly with cells and extracellular matrix during healing, with wound closure occurring faster in vivo. Wounds were filled with a mix of cells and ECM by 48h in vivo and by 120h ex vivo. Expression levels of tendon phenotype markers, collagens, collagen crosslinking regulators, matrix metalloproteinases, and pro-inflammatory mediators exhibited embryonic stage-dependent changes during healing. Apoptosis occurred during healing in vivo and ex vivo, but ex vivo injured tendons exhibited approximately 6-times higher levels of apoptosis than in vivo injured tendons.

*Conclusion/Significance: The establishment of these in vivo and ex vivo chick embryo tendon injury models enables future studies to examine scarless and regenerative healing by fetal tendons. Each model offers unique advantages, but differences in their healing rates should be considered carefully. Elucidation of key mechanisms and regulators of fetal tendon healing could significantly advance therapeutic approaches to regeneratively heal adult tendons.

299 - Xenograft Of Bio-3D Printed Scaffold-free Cartilage Constructs For Regeneration Of Articular Cartilage In Immunodeficient Pigs

T. Nonaka¹, D. Murata¹, A. Nakamura¹, H. Yoshizato¹, S. Kashimoto¹, M. Itoh¹, M. Ikeya², J. Toguchida², M. Mawatari¹, K. Nakayama¹

¹ Saga University, Saga, Japan,

² Kyoto University, Kyoto, Japan

*Purpose/Objectives: Arthroplasty is currently the only option for the reconstruction of large articular cartilage defects, mainly due to osteoarthritis. However, artificial materials have problems such as degradation, foreign body reaction, and bacterial infection. The objective of this study is to establish a new method for articular cartilage reconstruction that fundamentally solves the problems associated with artificial materials by creating scaffold-free bio-three-dimensional (3D) printed human induced pluripotent stem cell (iPSC)-derived cartilage constructs and implanting them into extensive osteochondral defects in immunodeficient mini-pigs without artificial materials.

*Methodology: Tubular cartilage constructs were produced by a bio-3D printer with a Kenzan using human iPSC-derived mesenchymal stem cells (iPSC-MSCs) which have chondrogenic differentiation potential. The constructs were partially cut to form a patch and implanted into osteochondral defects created in the femoral trochlear groove of immunodeficient mini-pigs. Histological assessments were scored using the modified O’Driscoll histologic scoring system. The compressive rupture strength of cartilage tissue after implantation was measured.

*Results: Implanted human iPSC-derived cartilage constructs matured without rejection in immunodeficient mini-pigs, and cartilage tissue was confirmed to be donor-derived by human-specific anti-Vimentin antibody staining. Histological examination of the constructs showed many chondrocytes and a glycosaminoglycan-rich extracellular matrix, indicating that the cartilage properties were maintained and matured after implantation. The scaffold-free cartilage constructs were strong enough to endure handling during the surgery. Furthermore, the mechanical property of the implanted sites were significantly higher than that of the constructs before implantation, and their compressive rupture strength increased to a value approximating that of normal cartilage within three months, indicating that maturation had also proceeded in vivo.

*Conclusion/Significance: As this study suggests that human iPSC-MSC-derived cartilage constructs may contribute to the reconstruction of extensive cartilage defects, we will also evaluate the safety of the constructs for future clinical application.

300 - An Ex Vivo Culture Model To Study Zonal Articular Cartilage Calcification

J. Xu¹, E. Farrell², N. Kops¹, P. A. Brama³, G. J. van Osch¹

¹ Orthopedics and Sports Medicine, Erasmus Medical Center, Rotterdam, NETHERLANDS,

² Oral and Maxillofacial Surgery, Erasmus Medical Center, Rotterdam, NETHERLANDS,

³ 3.School of Veterinary Medicine, University College Dublin, Dublin, IRELAND

*Purpose/Objectives: In healthy joints, non-calcified cartilage serves as a low-friction and wear-resistant surface, while the calcified cartilage layer that is located between the articular cartilage and the subchondral bone plays a vital role in transmission of mechanical loading and diffusion of nutrients, growth factors and metabolic waste products. Regeneration of this zone might be crucial for consistent and stable cartilage repair but appears challenging. Research specifically focusing on the effect of candidate factors on the formation of a zone of calcified cartilage for regenerative medicine is scarce. We developed an ex vivo culture model to investigate candidate factors that would regulate the formation of a calcified cartilage layer.

*Methodology: Full-thickness cartilage explants (4 mm diameter) were harvested from metacarpal-phalangeal joints of 6- to 8-month-old calves that had not developed the zone of calcified cartilage yet. Prior to initiation of the culture, all cartilage explants were scanned under micro-CT to confirm the absence of any pre-existing calcification. To study the effect of different zones, full-thickness cartilage explants were carefully separated into two distinct layers - the top layer and the bottom layer and cultured separately or together. The explants were cultured in the presence of β-glycerophosphate (10mM, β-GP) for 3 weeks to allow calcification. Calcium concentration in the medium was measured every two days throughout the experimental duration. Calcification volume was quantified on Micro-CT and tissue morphology was investigated by histology at the end of the experiment.

*Results: After 3 weeks, calcification was observed in the bottom zone of full thickness cartilage explants, confirmed by Micro-CT scanning and Von Kossa staining. When top layers were removed from full thickness cartilage, more calcium uptake (4.8 ± 2.6 vs. 1.1 ± 1.6 µmol, P<0.001) and higher calcification volume (1.7 ± 1.3 vs. 0.2 ± 0.5 mm³, P=0.002) was observed in the bottom layer (n=12) compared to full thickness cartilage (n=32). No calcification was observed in the top layers. Co-culturing a single bottom layer with four top layers (n=12) significantly reduced calcium uptake (5.9 ± 4.1 vs. 11.0 ± 1.4 µmol, P=0.002) and calcification in bottom layers (1.6 ± 1.2 vs. 2.5 ± 0.5 mm³, P=0.043) compared to bottom layer only (n=6). The addition of Parathyroid hormone-related protein (PTHrP, 0, 10, 50, 100 nM) to bottom layers (n=20), also significantly and dose-dependently reduced the calcium uptake (7.2 ± 5.3, 3.7 ± 3.5, 2.5 ± 2.7, 0.4 ± 1.5 µmol for the respective dosages, P<0.001) and calcification (1.6 ± 1.1, 0.6 ± 0.8, 0.4 ± 0.5, 0.1 ± 0.2 mm³, for the respective dosages P<0.001).

*Conclusion/Significance: Using immature bovine full-thickness cartilage as an ex vivo model enabled us to study formation of a zone of calcified cartilage. We found that the top layer of cartilage and PTHrP can inhibit calcification of the bottom layer. This model can be used as an ex vivo screening tool for candidate factors that affect formation of a zone of calcified cartilage in articular cartilage repair procedures.

301 - An Injectable Composite Hydrogel System For The Controlled Release Of Platelet Lysate For The Repair Of Cartilage Defects

A. S. Atwal¹, T. P. Dale¹, M. Snow^1,2, N. R. Forsyth^1,3, P. Davoodi¹

¹ Keele University, Stoke-on-Trent, United Kingdom,

² The Robert Jones and Agnes Hunt Hospital, Shropshire, United Kingdom,

³ University of Aberdeen, Aberdeen, United Kingdom

*Purpose/Objectives: Cartilage damage presents a substantial clinical challenge, resulting in persistent pain and impaired joint function. Existing treatments have limitations in fully restoring cartilage function, sparking growing interest in regenerative medicine solutions. Platelet rich plasma (PRP) has demonstrated potent regenerative effects, but its practical application is hindered by the need for frequent injections and clearance in the synovium. Herein, we present the development of a platelet PL-encapsulated alginate microparticle-hydrogel composite for the sustained release and targeted delivery of PL to cartilage lesions.

*Methodology: To obtain control over platelet lysate (PL) release, we utilized sodium periodate to oxidize alginate, resulting in the formation of hydrolytically degradable microparticles. These microparticles had a degree of oxidation ranging from 1-10% and were generated through drop-on-demand printing into a calcium chloride bath. Mechanical and rheological testing was carried out as well as the release of PL over time. Chondrocytes were embedded in MPs with Live/Dead, GAG/DNA, Histology and immunohistochemical techniques carried out to assess chondrogenesis.

*Results: The addition of microparticles to some extent increased the initial compressive modulus whilst the sustained release of PL correlated with the degradation of these microparticles, further facilitated by the Schiff-base linkage between the aldehydes on the alginate microparticles and residual amines found in PL. Scanning electron microscopy analysis confirmed the formation of spherical microparticles within the size range of 100-200 µm. These microparticles were embedded in gelatin methacryloyl (GelMA) and hyaluronic acid methacryloyl (HAMA) hydrogels. This integration established a protective and supportive matrix, creating an environment conducive to chondrocyte growth and facilitating controlled PL delivery. Protein release assays conducted over various time intervals confirmed sustained release over 28 days. The rate of PL release was directly correlated with the degree of alginate oxidation, with higher oxidation resulting in faster release. Simultaneously, the secreted extracellular matrix (ECM) from chondrocyte-laden hydrogels was assessed via histological analysis, a DMMB assay, and immunofluorescence. Notably, the released PL significantly increased chondrocyte proliferation compared to the control group, as evidenced by elevated GAG/DNA and collagen type II content in the hydrogel. This highlights that the microparticles served synergistically as a sacrificial porogen and as a drug delivery vehicle.

*Conclusion/Significance: Our study underscores the potential of a PL-encapsulated alginate microparticle-hydrogel composite for the repair of cartilage lesions. The tunable release of PL via alginate oxidation offers a versatile approach to tissue engineering. The hydrogel system provides a supportive matrix, promoting chondrocyte proliferation and the formation of hyaline neotissue. Importantly, the composite system can be delivered arthroscopically to cartilage lesions, potentially enhancing the clinical management of cartilage damage.

302 - Cartilage Organoid Production And Fusion Using Human Articular Chondroprogenitor Cells

D. M. Menssen¹, J. C. Feenstra¹, R. P. Janssen^1,2,3, F. Abinzano¹, K. Ito¹

¹ Orthopaedic Biomechanics, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, NETHERLANDS,

² Paramedical Sciences, Fontys University of Applied Sciences, Eindhoven, NETHERLANDS,

³ Orthopaedic Surgery and Trauma, Máxima Medical Center, Eindhoven-Veldhoven, NETHERLANDS

*Purpose/Objectives: The 2D expansion of autologous chondrocytes (ACs) for cartilage repair strategies is time-consuming and induces dedifferentiation, resulting in fibrocartilaginous repair tissue. Recently, spinner flasks (SFs) were shown to be an efficient method for generating large amounts of cartilage organoids, allowing ACs to proliferate without dedifferentiating. However, porcine notochordal cell-derived matrix (NCM) was necessary for the ACs to aggregate, limiting its application. Additionally, the number of organoids is constrained by the number of ACs obtained during a biopsy. Therefore, the objective of this study was to create cartilage organoids using articular chondroprogenitor cells (ACPCs) without NCM. These cells can differentiate towards the chondrogenic lineage after more than 30 passages. Furthermore, the ability of these ACPC organoids to fuse and form cartilage tissue was investigated.

*Methodology: Human ACPCs and ACs were harvested from redundant articular cartilage tissue of TKA patients (n=4) at the Máxima Medical Center (METC #N16.148). Cartilage organoids using P1 ACs were created with NCM following a standard protocol. P4 ACPC organoids were created in SFs by allowing the cells to self-aggregate for 3 days, followed by stimulation with 100 ng/ml BMP-9 for 11 days. After 14 days, the size was measured and organoids were mechanically tested. Fusion was evaluated by fusing ∼20 organoids in low adhesive wells for 21 days. Finally, organoids and fusions were assessed using biochemical, histological, and immunohistochemical analysis.

*Results: The projected area of the ACPC organoids was similar to the AC organoids, however, there were four times more AC organoids per SF culture than ACPC organoids. Although the ACPCs aggregated without NCM (high in GAG content), there were no significant differences in either GAGs per DNA (Fig. 1A) or in Young’s modulus (Fig. 1B) between the ACPC and AC organoids. Immunohistochemistry revealed type II collagen in all organoids. However, type I collagen was present in some ACPC organoids, unlike the AC organoids (Fig. 1C). Both types of organoids fused successfully. The ACPC fused constructs were 1.5 times larger than the AC constructs after 21 days. Histology showed that the fusion region between organoids consisted of GAGs and collagen, and biochemistry showed similar GAG per DNA content with both cell types. There appeared to be more GAGs and type II collagen in the fused regions between ACPC than AC organoids. However, some type I collagen was found at the outer periphery of the fusion masses from both cell types (data not shown).

*Conclusion/Significance: ACPCs can self-assemble into cartilage organoids without the need for NCM and grow with BMP-9 supplementation, expanding application possibilities. Despite disparities compared to the AC organoids, such as fewer ACPC organoids per culture and the presence of type I collagen, advantages using ACPCs were observed. Specifically, the ACPC organoids fused to larger cartilage-like tissues than the AC organoids. The method could be improved by optimizing SF culture conditions, e.g. to reduce type I collagen production by ACPCs. Optimization of the ACPC organoids without the need for NCM could lead to a qualitative improvement and increased scalability of regenerative treatments for cartilage defects.

303 - Towards Scalable And GMP-compliant Cartilage Microtissue Production In Bioreactors

F. Staubli^1,2, D. Gawlitta^1,2, A. Garcia Gonzalez^1,2, A. Rosenberg¹

¹ University Medical Center Utrecht, Utrecht, Netherlands,

² Regenerative Medicine Center Utrecht, Utrecht, Netherlands

*Purpose/Objectives: In endochondral bone regeneration (EBR), bone defects are treated by implanting cartilaginous intermediates, which are remodelled by the recipient body into bone tissue. To address the clinical challenge of regenerating large bone defects, cartilage microtissues can act as modular building blocks for the bottom-up fabrication of large implants. Current protocols for generating cartilage microtissues are predominantly based on static culture systems like microwells. These systems are laborious in terms of cell handling, require time-consuming medium exchanges, have low throughput, and are challenging to automate. Dynamic bioreactor systems overcome these limitations and offer a GMP-compliant solution for clinical scale-up. Here we aim to generate aggregates from single-cell suspensions through collision-based self-assembly, followed by chondrogenic differentiation within the same system. This was not previously explored for mesenchymal stromal cells (MSCs), which are most often used for generating cartilage intermediates in EBR.

*Methodology: MSCs (p4) in single-cell suspension (10⁶ MSCs/ml; 50ml) in chondrogenic medium were seeded in spinner flasks (Corning) and cultured at 37°C and 5% CO₂. 50% of the medium was changed every day for the first 3 days and then every 3 days thereafter. Optimal stirring conditions were determined by comparing different regimens during the first 7 days in terms of aggregation and viability, with 80 rpm for the first 2 days and 120 rpm thereafter selected for the 21-day culture (n = 1). A positive control involved static culture in a 96-well plate (10⁵ cells/well). Aggregate size and shape (brightfield microscopy) and viability (Live/Dead assay) were regularly examined, and chondrogenic progression was assessed on days 7, 14, and 21 using glycosaminoglycan quantification and histological staining.

*Results: Over 21 days, MSCs formed stable aggregates in both static and dynamic conditions. Comparable viability was observed between static and dynamic groups. Although the average projected aggregate area was similar, dynamic culture aggregates displayed greater variability (2.055 ± 0.176 and 1.985 ± 2.733 mm² for static and dynamic, respectively). Toluidine Blue staining (see figure) on days 7, 14, and 21 showcased successful chondrogenesis and cartilage matrix deposition in both groups. However, dynamic culture exhibited high variation in chondrogenic differentiation among samples in one culture run, accompanied by generally lower glycosaminoglycan levels, as evidenced by quantification normalized to DNA (49.02 ± 11.00 and 12.69 ± 8.28 for static and dynamic, respectively).

*Conclusion/Significance: This study demonstrates the feasibility of aggregation and chondrogenic differentiation of MSCs in the same closed dynamic system. Improving control over aggregate size and reproducibility among MSCs of different donors is ongoing. To do so, a stirred tank mini-bioreactor system will be employed for the screening of optimal conditions. Optimization of this approach will bring us closer towards upscaled, GMP-compliant production of building blocks for endochondral bone implants—an essential advancement for their clinical translation to treat large bone defects.

304 - A HUMAN-SCALED KNEE BIOMIMETIC BIOREACTOR FOR PATIENT-SPECIFIC OSTEOCHONDRAL TISSUE ENGINEERING

N. Campillo¹, S. Micó¹, J. A. Marchal^2,3, J. M. Baena^1,4

¹ REGEMAT 3D S.L., Granada, Spain,

² Instituto de Biopatología y Medicina Regenerativa, Granada, Spain,

³ Instituto de Investigación Sanitaria, Granada, Spain,

⁴ BRECA Health Care S.L., Granada, Spain

*Purpose/Objectives: Musculoskeletal medical conditions are one of the leading causes of human disability worldwide, accounting the lesions of the osteochondral interface for a great proportion of these disorders (Hermes et al., 2020). The lack of effective regenerative approaches for a growing elderly population accentuates the need to generate new strategies based on cutting-edge technologies for regenerative medicine. In recent years, the emergence of 3D bioprinting has allowed great advances in tissue engineering, making feasible the automatized and controlled ex vivo generation of bioartificial tissue substitutes with a certain degree of complexity (Oberdiek et al., 2021). However, the application of biochemical, physical and mechanical stimuli biomimetizing those found in a given tissue, by custom-designed bioreactors, is critical to promote the maturation of the 3D constructs towards clinically relevant products (Selden et al., 2018). Despite the need for such devices, the availability of dedicated bioreactors for tissue engineering, with the ability to recreate the complex microenvironment to which cells are subjected in vivo, is currently scarce. Therefore, the aim of this work was to develop a novel bioreactor able to mimic the anatomy and physiology of the human knee joint for the maturation of patient-shaped 3D bioprinted cartilage substitutes under a physiologically relevant microenvironment.

*Methodology: Functionality testing was performed by monitoring the temperature, CO₂, pH, and relative humidity during the actuation of the bioreactor. In addition, load was monitored during flex-extension cycles. On the other hand, sterility and cytotoxicity tests were performed following ISO standards for medical devices throughout the operation of the device. 3D bioprinted polycaprolactone scaffolds loaded with a cell-laden hydrogel containing chondroprogenitor cells were subjected to mechanical loading under physiologically relevant regimes to determine cell viability through the stimulation period.

*Results: Functionality tests revealed the ability of the device for controlling all the parameters within a range suitable for cell culture. Load monitoring demonstrated the feasibility of the bioreactor to apply loads within the tibiofemoral surfaces of the reconstructed knee joint, suitable for the mechanical stimulation of chondroprogenitor cells. No microbial contamination nor cytotoxic effects throughout the operation of the device were detected. The viability of the cells through the stimulation period was demonstrated via Live/Dead stain and confocal microscopy.

*Conclusion/Significance: In this study, a human-scaled knee joint biomimetic bioreactor was developed for the engineering of patient-shaped 3D printed osteochondral tissue substitutes. The functionality of the device was demonstrated, as well as its ability to support the culture of chondroprogenitor cells under physiologically relevant loading regimes. Future work will address the chondrogenic effects of physiological-loading within the bioreactor.

305 - In Vivo Validation Of A Continuous Gradient Porous Scaffold For Osteochondral Defect Repair In A Rabbit Model

K. W. Smith^1,2, S. Ali Akbari Ghavimi³, S. Logterman⁴, P. M. Gehret⁵, E. Capuana⁶, G. Conoscenti⁶, V. Brucato⁶, V. La Carrubba⁶, J. T. Lawrence¹, R. Gottardi¹

¹ Children's Hospital of Philadelphia, Philadelphia, PA,

² University of Pennsylvania, Philadelphia, PA,

³ Harvard Medical School, Boston, MA,

⁴ Arnold Palmer Hospital for Children, Orlando, FL,

⁵ L.E.K. Consulting, New York, NY,

⁶ University of Palermo, Palermo, Italy

*Purpose/Objectives: Osteochondral defects, characterized by damage to the articular cartilage and subchondral bone, are a major health concern affecting 60% of knee surgery patients. Standards of care include microfracture, which creates fibrocartilaginous scar tissue, or osteochondral transplant, which suffers from limited donor supply. Tissue engineering represents an exciting alternative, but a key hurdle is ossification of engineered cartilage. To overcome this, we developed a porous osteochondral scaffold with pore sizes tailored to promote chondrogenesis or osteogenesis on opposite sides. We used an established biphasic bioreactor that allows parallel chondrogenic and osteogenic differentiation of the osteochondral constructs, which were tested in vitro for strong osteochondral differentiation capacity. Then we validated the osteochondral construct in vivo using a rabbit osteochondral defect model.

*Methodology: Porous gradient PLLA scaffolds were fabricated by Thermally Induced Phase Separation and uniformly seeded with rabbit mesenchymal stem cells (MSCs). To determine the effect of pore size on differentiation, the gradient scaffold was perfused with either chondrogenic, osteogenic, or growth medium. To develop the complete osteochondral construct, the scaffold was placed in the biphasic bioreactor and the small pores (cartilage region, ∼70μm) was perfused with chondrogenic medium, while the large pores (bone region, ∼200μm) was perfused with osteogenic medium. Differentiated constructs were assessed for morphology via histology, RT-qPCR, and immunofluorescence. For in vivo validation, bilateral osteochondral repair was performed on 21 rabbits with empty defects, acellular scaffolds, MSC-seeded scaffolds, or pre-differentiated engineered osteochondral constructs. After three months, rabbits were euthanized, knees were examined for gross appearance, integration, and repair, then excised for microCT, histology, and immunofluorescence.

*Results: In vitro, glycosaminoglycan and chondrogenic gene expression were observed in the chondral region, while calcium and osteogenic gene expression were observed in the osseous region. After three months, healed osteochondral defects exhibited complete defect closure and integration in the cell-seeded and differentiated groups, and high variability of closure in empty and acellular groups. Histology shows glycosaminoglycan deposition in the small pore region of the scaffold and surrounding integrated tissue, and mineral deposition in the large pore region. Immunofluorescence shows Collagen II deposition in the cartilage region, and Collagen I deposition in the bone region. Examinations by microCT show both cartilaginous and trabecular repair in healed defects, proving successful biphasic morphologies, and initial analyses of the in vivo model suggest specific chondrogenic and osteogenic differentiation analogous to in vitro results.

*Conclusion/Significance: With this work we explored the use of local pore geometry to replicate developmental processes—namely mesenchymal condensation—that drive chondrogenesis, establishing a powerful approach for robust cartilage engineering. Our pore gradient scaffold avoids the risk of delamination common with biphasic models, promoting dual chondro- and osteogenic differentiation to align with the osteochondral bilayer in the knee. Construct maturation within the dual-flow bioreactor allows the simultaneous differentiation of each tissue type within a monolithic structure ready to be implanted. The effectiveness of our engineered osteochondral construct to repair a rabbit osteochondral defect shows tremendous promise for this technology to heal osteochondral defects and provide a solution to a major health problem.

306 - Bioengineering Multi-tissue Musculoskeletal Interfaces For Enhanced Tissue Regeneration

V. K. Lee, T. Lee, S. Sengupta, H. Jang

Brigham and Women's Hospital, Harvard Medical School, Boston, MA

*Purpose/Objectives: In vitro cultured musculoskeletal tissues have numerous important applications, including tissue engineered implant for musculoskeletal regeneration, disease modeling for pathological mechanistic studies, and preclinical drug testing platform. The intricate architecture of the musculoskeletal system, consisting of bone, muscle, and connective tissues, plays a pivotal role in maintaining structural integrity and facilitating tissue regeneration. However, existing in vitro models often lack a comprehensive representation of these interconnected components, limiting their ability to recapitulate the dynamic cellular tissue regeneration processes observed in vivo. Our study aims to develop a 3D in vitro musculoskeletal tissue interfaces that simulates the synergistic relationship between bone, periosteum, and muscle tissues, focusing on their role in tissue regeneration.

*Methodology: Human mesenchymal stem cells (MSCs) were embedded in a 3D Gelatin Methacryloyl (GelMA) hydrogel matrix with bone minerals (calcium phosphate nanoparticles) to form the bone tissue component. A GelMA hydrogel layer with endothelial cells (ECs) and MSCs was added to simulate the periosteum. Additionally, GelMA hydrogel with human myoblasts was layered on top of periosteum to create a multi-tissue structure of bone, periosteum, and muscle (Figure 1A). This multi-tissue interface was fabricated using 3D printing and layer-by-layer deposition techniques. Culture media conditions were optimized to support osteogenic differentiation of MSCs, myogenic differentiation of myoblasts, and vascular formation of ECs simultaneously. The engineered multi-tissue interfaces, cultured in this optimized media for 2-3 weeks, were mechanically stimulated in custom bioreactors to mature into functional musculoskeletal system.

*Results: We engineered in vitro musculoskeletal interfaces with varying tissue compositions to analyze the impact of multi-tissue interactions on the regeneration of musculoskeletal system. Adding periosteum tissue significantly enhanced osteogenic marker expressions of cells in bone tissue, and this effect was further amplified by the incorporation of muscle tissues (Figure 1B). Components of each tissue, such as bone minerals in the bone tissue or ECs/MSCs distribution in periosteum, played a distinct role in facilitating osteogenesis (Figure 1C). This work demonstrated the capability to develop each tissue component to not only function independently but also coexist synergistically with other tissues, effectively mimicking the dynamic interplay involved in regeneration of musculoskeletal system. The control over individual elements within each tissue type enabled us to fine-tune the optimal conditions for co-culturing multiple tissues and effectively stimulate their maturation through interfacial interactions in vitro, similar to musculoskeletal system.

*Conclusion/Significance: Our research presents an innovative 3D in vitro model that simulates the complex interactions among bone, periosteum, and muscle tissues. The advanced model enhances tissue regeneration capacity, outperforming that of single-type tissue models. This notable enhancement in tissue regeneration highlights the important role of interfacial interactions between different tissue types in the musculoskeletal system. The model serves as a valuable tool for advancing research in musculoskeletal tissue regeneration, disease modeling, and therapeutic drug testing.

Figure 1. Engineering In Vitro Musculoskeletal Interfaces. (A) Fabrication of stratified multi-tissue construct. (B) Enhanced osteogenic gene expression in multi-tissue conditions over single-tissue conditions. (C) Cross-sectional and top views of individual tissue type matured within the musculoskeletal interfaces.

307 - Evaluating The Pre-vascularization Of Biomimetic Collagen Matrices For Enhanced Humanized Bone Tissue Engineering

S. Bhatia^1,2,3, L. Hipwood^1,2,3, B. Claxton^1,3,4, A. Bessot^1,2,3, A. Weekes³, K. Sokolowski⁴, N. Bock^1,2,3, J. Mcgovern^1,2,3

¹ School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Australia,

² Max Planck Queensland Centre on the Materials Science for Extracellular Matrices, QUT, Brisbane, Australia,

³ Centre for Biomedical Technologies, QUT, Brisbane, Australia,

⁴ Translational Research Institute, Brisbane, Australia

*Purpose/Objectives: Osteogenesis (bone formation) and vascularization (blood vessel formation) are two crucial and interconnected physiological-relevant processes in bone formation. Previous work from our team has demonstrated the successful implementation of an orthotopic humanized tissue-engineered bone with human vascular niche. In this study, we hypothesized that pre-vascularization within appropriate biomimetic hydrogel of a mineralized humanized bone niche would enhance the formation of a tissue engineered bone in vivo, compared to a bone niche without pre-vascularization.

*Methodology: A vascular niche was established by co-culturing human umbilical vein endothelial cells (HUVECs) with bone marrow mesenchymal stem/stromal cells (MSCs) within 3D extracellular matrices composed of gelatin methacryloyl hydrogels derived from bovine, porcine or piscine sources. Mechanical analyses were conducted on the hydrogels to determine the relationship between gelatin source and hydrogel stiffness. Further, mPCL (medical grade poly(ɛ-caprolactone)) scaffolds coated with calcium phosphate (CaP) and seeded with human osteoblast cells (hOBs) were cultured under osteogenic conditions (OM), with or without a 3-day mineralization boost (OM+) period to compare osteogenic capacity for 4 weeks. The OM and OM+ cultured hOBs scaffolds were subcutaneously implanted into immunocompromised rats with or without 3-day pre-cultured piscine vascular hydrogels. This procedure resulted in four distinct subgroups: OM, OM+, OM/Vas, and OM+/Vas. The subgroups were analyzed every 2 weeks using µCT to assess bone volume and mineralization density. Histological analysis was conducted on the excised scaffolds to characterize bone morphology, ECM properties and blood vessel formation.

*Results: Interestingly, piscine-derived hydrogels exhibited the lowest stiffness compared to bovine and porcine equivalents, providing the optimal ECM parameters for angiogenic-like alignment, facilitating the superior formation of elongated vascular networks as early as day 3. Preliminary data indicate an increased bone volume and accelerated biomineralization in vivo in rats for both OM+ and OM+/Vas conditions, in comparison to OM and OM/Vas. Notably, the OM+ group exhibited more rapid osteoinduction and enhanced osteogenic differentiation within rat ectopic tissue engineered bone constructs as compared to the other subgroups. Histological analyses via H&E further supported the effective formation of bone construct in the OM+ group. We also observed that the in vivo bone volume and density in vascular containing bone construct groups (the subgroups OM/Vas and OM+/Vas) were lower as compared to the bone construct only groups (OM and OM+ subgroups), with the lowest overall bone volume and density in the OM/Vas subgroup. This raises speculation that the presence of a pre-formed vasculature niche may have outcompeted osteogenic outgrowth, which will be confirmed via histology.

*Conclusion/Significance: The present study demonstrates the feasibility of fabricating humanized vascularized bone in animal models. Our study is imperative to guide and refine the methods needed to establish a successful murine model that more accurately replicates human bone physiology. Ongoing aims to create an optimal humanized vascularized bone tissue model will thus serve as a relevant bioengineered niche with various implications in regenerative engineering, disease modeling and to study bone-resident malignancies such as osteosarcoma.

308 - Mechano-immunomodulation Of Macrophages To Establish A Regenerative Environment For Bone Repair

S. Petrousek, G. Soares Kronemberger, G. O’Brien, S. O’Rourke, L. Shanley, A. Dunne, D. Kelly, D. Hoey

Trinity College Dublin, Dublin, Ireland

*Purpose/Objectives: Successful completion of the initial inflammatory phase is critical for the establishment of a regenerative environment conducive to bone fracture healing. A potent regulator of skeletal repair is mechanical loading, which may offer the potential to influence local inflammation, by acting upon the early fracture hematoma, as suggested by heightened inflammatory levels observed in mechanically unstable fractures. However, surprisingly little is known about the role that mechanical cues play in directing the early immune response and subsequent orchestration of bone repair. Therefore, this study aims to decipher the contribution of local mechanics on macrophages, key mediators of the immune response, and later healing processes, and harness this information to develop novel mechano-biologics to target the early regenerative niche to modulate inflammation for repair.

*Methodology: A purpose-built in vitro model of the bone fracture hematoma was developed utilised a hematoma mimetic fibrin hydrogel and a custom-made bioreactor (Fig.1A-C), that can replicate the different loading conditions experienced within a fixated large bone defect. This model was harnessed to (1) apply dynamic compressive strains of 5% (rigid fixator), 20% (compliant fixator) and 35% (unfixed defect) for 4 hours on unactivated M0 macrophages, and evaluate macrophage phenotype via cell surface marker and cytokine levels (Fig.1D-E), and impact of loaded macrophage secretome on vascular tube formation and mineralisation (Fig.1F-H); (2) explore whether a lower loading (∼5% strain) could modulate a highly inflammatory M1-like macrophage by performing similar phenotype and secretome analyses (Fig.1I-K); (3) investigate the potential of mechano-therapeutics (Forskolin) to mimic the beneficial effect of mechanics on M0 macrophages and assessing regenerative properties as above (Fig.1L-M).

*Results: Initially, the impact of loading magnitude on M0 macrophages was investigated. Lower loading magnitudes (5-20%) generated a hybrid phenotype with increase in both pro and anti-inflammatory cell surface markers and a 3-fold increase in pro-regenerative IL-10 secretion compared to static controls. Interestingly, the secretion of angiogenic factor VEGF-A decreased in an increasing magnitude dependent manner, which corresponded to an inhibitory angiogenic and osteogenic secretome at higher strains (20-35% strain). Next, we examined whether lower loading, which resulted in a regenerative phenotype in M0 macrophages, could dampen an inflammatory M1 primed macrophage. 5% compressive strains induced a partial inhibition of pro-inflammatory IL-6 and TNF-α secretion which was found to reshape blood vessel composition by favouring capillary-size tubes. Finally, utilising the mechano-therapeutic Forskolin on M0 macrophages, we observed higher regenerative properties in a dose-dependent manner with up to a 10-fold and 2-fold increase in pro-regenerative IL-10 and VEGF-A secretion, respectively, which translated to an 8-fold increase in tube formation and greater calcium deposition.

*Conclusion/Significance: This study offers substantial insights into the role that local mechanics play in regulating the early immune response and how it directly correlated with fracture healing processes. We also demonstrated how macrophage mechanosignalling can be modulated therapeutically to promote repair. Together, these findings highlight the tremendous potential of targeting local mechanics and mechanosignalling for immunomodulation and identify a novel therapeutic to regulate the immune response to establish a regenerative environment conducive to bone regeneration.

311 - FAK Inhibition In Ligament Fibroblasts Decreases Collagen Fibril Formation, But Has No Effect On Fiber Or Fascicle Formation

T. Strayer, L. Troop, J. Puetzer

Virginia Commonwealth University, Richmond, VA

*Purpose/Objectives: Collagen fibers are the primary source of strength in tendons and ligaments; however, they do not regenerate after injury or in engineered replacements. Cells organize these fibers hierarchically, from nanometer-wide fibrils to larger fibers and fascicles with increasing mechanical demands. An understanding of the mechanotransduction pathways that regulate cell-driven collagen organization is critical to developing functional repairs. One way that cells sense mechanical cues is through ECM attachments, regulated via focal adhesion kinase (FAK). FAK is reported to play a major role in fibril formation, but its role at larger length-scales is unknown. Previously, we developed a culture device that guides ligament fibroblasts to develop aligned fibrils by 2 weeks, and larger fibers and fascicles by 4 and 6 weeks. Using this system, our objective is to evaluate how mechanical cues transduced via FAK regulate cellular development of hierarchical fibers. We hypothesize that FAK plays a role during fibril, fiber, and fascicle formation, and FAK inhibition will reduce collagen organization at all stages.

*Methodology: To form constructs, bovine ACL fibroblasts were mixed into 20mg/mL collagen at 5x10⁶ cells/mL and cultured in our device for up to 6 weeks to guide hierarchical fiber development (Fig.1A). To investigate the role of FAK during fibril, fiber, and fascicle formation, constructs were dosed with 10µM FAK inhibitor (FAK-I) PF-228 from 0-2 weeks, 2-4 weeks, or 4-6 weeks while fibrils, fibers, and fascicles are forming, respectively (Fig.1B). Controls were cultured with standard DMEM or DMSO vehicle treatment. Post-culture, collagen organization was evaluated using confocal reflectance and images were analyzed with a custom FFT-based code to determine alignment and mean fiber diameter. Construct tensile properties and composition were also assessed. Significance determined via 2-way ANOVA with Tukey’s post-hoc.

*Results: Confocal analysis revealed FAK-I during fibril formation (0-2 weeks) reduced cell-driven organization with significantly lower collagen alignment at 2 weeks than controls (Fig.1C&D). However, FAK-I during fiber (2-4 weeks) and fascicle (4-6 weeks) formation had no significant effect on organization (Fig.1C&D). Mirroring organization, tensile properties (including elastic modulus, UTS, toe modulus, and transition stress) were significantly reduced in 2wk FAK-I constructs, while FAK-I during fiber and fascicle formation had no effect on mechanics (Fig.1E). Similarly, FAK-I had little effect on composition when applied during fiber and fascicle formation, however when applied during fibril formation, construct collagen significantly decreased compared to controls (Fig.1F), and more collagen accumulated in the media (Not shown). Interestingly, LOX activity also increased with FAK-I during fibril formation (Fig.1F).

*Conclusion/Significance: Collectively, this suggests cell-ECM connections via FAK-cytoskeletal signaling are key to fibril formation but have a smaller role later in hierarchical formation. Interestingly, FAK-I also increased collagen degradation and LOX activity during fibril formation, suggesting a loss of tensional homeostasis, shifting cells to a catabolic state. However, once cells were anchored on fibrils, FAK-I fiber formation continued similar to controls, suggesting other mechanosensors play larger roles at the fiber and fascicle length-scale. Understanding mechanotransduction pathways that regulate collagen organization is critical to regenerating or engineering tissues. Ongoing work is investigating other mechanotransduction pathways.

312 - Antihypertrophic Activity Of Heparin During Mesenchymal Stromal Cell Chondrogenesis In Vitro

S. Schmidt, S. Chasan, C. Binder, S. Diederichs

Orthopaedic University Hospital Heidelberg, Heidelberg, Germany

*Purpose/Objectives: Recently, we have developed a breakthrough system allowing us, for the first time, to direct the fate decision of mesenchymal stromal cells (MSCs) in vivo towards either stable chondral or mineralizing endochondral neocartilage tissue formation. The key for directing chondral development and suppressing chondrocyte hypertrophy was heparin, a highly sulfated glycosaminoglycan (GAG) immobilized in a PEG-based hydrogel. However, in vitro MSC chondrogenesis in the hydrogel remained insufficient. Yet, understanding how to engineer cartilage replacement tissue under defined and reproducible conditions is essential for clinical translation. We asked whether free soluble heparin added to standard 3D cultures would allow MSC chondrogenesis in vitro, directing a chondral fate decision and suppressing hypertrophic development. Understanding heparin’s ability to stabilize MSC chondrogenesis could pave the way for novel approaches in articular cartilage engineering and clinical regeneration of cartilage defects and degenerative diseases.

*Methodology: Human bone marrow-derived MSCs were cultured as standard 3D pellets with TGF-β1 (10ng/mL) and insulin (6.25μg/mL) stimulation and treated with heparin or chondroitin sulfate (CS; 10, 100, 700μg/mL) as control (n=3-5). Pro-chondrogenic and pro-hypertrophic signaling activity was analyzed by Western blotting or using target gene expression (qPCR; n=2-4). Chondrogenic differentiation and hypertrophy were assessed via histology, proteoglycan quantification with DMMB, qPCR, and protein detection of specific markers.

*Results: Heparin decreased activation of SMAD3 and SMAD1/5/9 by TGF-β but not of SMAD2; and this appeared to be dose-dependent. Also, insulin-induced phospho-AKT levels were increased. In line with these slight effects on pro-chondrogenic signaling, GAG/DNA levels were similar across all treatment groups at 4 weeks according to DMMB assay. Consistently, ELISA revealed unchanged deposition of type II collagen by heparin compared to the CS controls. However, while gene expression of type II collagen (COL2A1) and SOX9 were dose-dependently reduced, aggrecan (ACAN) was unaffected. Histologically, unlike CS, 100 and 700μg/mL heparin led to compromised pellet integrity. Overall, while 10μg/mL heparin allowed strong MSC chondrogenesis, higher concentrations were unfavorable. Intriguingly, heparin (10μg/mL) decreased pro-hypertrophic HH signaling activity according to target gene expression (GLI1) but increased pro-hypertrophic WNT3A-induced β-catenin levels. Day 28 expression of the hypertrophy markers MEF2C, IBSP, and ALPL was significantly reduced compared to the CS control and COL10A1, IHH, and RUNX3 were reduced by trend, indicating an overall reduced pro-hypertrophic signaling. In line, type X collagen protein levels were not reproducibly affected, while ALP enzyme levels were strongly inhibited. Higher heparin concentrations had stronger effects, yet only at the expense of chondrogenic differentiation. Overall, soluble heparin had slight anti-hypertrophic effects on MSC-derived chondrocytes, however, failed to stably reprogram MSC differentiation into the chondral lineage.

*Conclusion/Significance: We show here that low-dose heparin has promising anti-hypertrophic effects that could in future be combined with known anti-hypertrophic stimuli (PTHrP pulses and WNT inhibition). Reasons why free soluble heparin fails to fully reprogram chondrogenesis in vitro as observed with PEG-immobilized heparin in vivo, need further investigation. Frequent removal of free heparin and bound cytokines produced by the cells, or undefined factors only occurring in vivo could potentially contribute.

313 - Deciphering The WNT Contribution To Hypertrophic Chondrocyte Degeneration

S. Diederichs, C. Binder, V. Trivigno, M. Krueger, S. Chasan, H. Dietmar

Orthopaedic University Hospital Heidelberg, Heidelberg, Germany

*Purpose/Objectives: The application of mesenchymal stromal cells (MSCs) for cartilage tissue engineering is critically limited by their inevitable differentiation into hypertrophic chondrocytes. This predestines the engineered neocartilage to mineralization and endochondral bone formation in vivo, and recapitulates the developmental processes of chondrocyte maturation in growth plates and the pathological changes of articular chondrocytes during osteoarthritis. Importantly, WNT/β-catenin signalling has been implicated in all three systems, however identifying the responsible WNT protein(s) has been surprisingly challenging. Of the few WNT proteins present in growth plate cartilage and MSC chondrogenesis, only WNT11 - that predominantly signals independently from β-catenin - significantly correlated with hypertrophy marker expression during MSC chondrogenesis. Nevertheless, our previous data also implicated the R-Spondins 2 and 3 that can increase cell surface levels of WNT receptors, thus amplifying WNT/β-catenin signalling. To assess whether WNT11 or RSPO2/3 could be the long-sought WNT drivers of chondrocyte hypertrophy, we investigated their ability to stimulate β-catenin accumulation and hypertrophic degeneration of chondrocytes. Deciphering the WNT signals that drive chondrocyte hypertrophy will allow specific intervention to improve MSC-based cartilage tissue engineering approaches and cartilage regeneration.

*Methodology: Standard in vitro chondrogenesis of human bone marrow-derived MSCs stimulated with TGF-β in pellet culture was used as model system (n=3-11). Cultures were treated with recombinant WNT11 protein (300 ng/mL) alone or in combination with the β-catenin-stimulating WNT3A (75 ng/mL), with RSPO2 or RSPO3 (100 ng/mL), or the WNT/β-catenin inhibitor DKK1 (200 ng/mL). Cytosolic β-catenin levels were assessed via Western blotting. Chondrogenesis and hypertrophy were investigated via histology, qPCR and protein detection of specific markers.

*Results: β-catenin accumulation was stimulated by the chondro-inducer TGF-β; and RSPO2/3, like WNT3A, but not WNT11 could further enhance this. Since neither histologically detected proteoglycan and type II collagen deposition nor upregulation of COL2A1 and SOX9 mRNA were changed, all treatments allowed strong chondrogenic differentiation of MSCs, albeit WNT11 reduced SOX9 protein levels according to Western blotting. The expected broad prohypertrophic effects of WNT3A, evident from an increased expression of IHH, MEF2C, ALPL, and IBSP, as well as increased ALP enzyme and IBSP protein levels, were inhibited in short-term by cotreatment with WNT11 (day 14-18), but not over a longer term (day 14-28). WNT11 treatment alone did hardly reduce any assessed hypertrophy markers. Together, this identified WNT11 as a weak anti-hypertrophic signal. In contrast, RSPO3, and less efficiently also RSPO2, showed a limited prohypertrophic activity and enhanced ALP mRNA and protein, while all other hypertrophy markers remained unaffected. DKK1 abolished the ability of RSPO2/3 to stimulate ALP, indicating a WNT/β-catenin-dependent mechanism.

*Conclusion/Significance: Overall, WNT11 seems to be an unlikely driver of chondrocyte hypertrophy because of its weak anti-hypertrophic activity during MSC chondrogenesis, while RSPO2 and RSPO3 appear to have the capacity to enhance chondrocyte hypertrophy via stimulating WNT/β-catenin signalling. Still, their ALP-specific activity indicates that the RSPO-WNT interaction is complex and may involve further signals. Unfortunately, the WNT ligand that is responsible for β-catenin accumulation downstream of TGF-β and whose activity may be amplified by the RSPO-WNT-receptor interaction, still remains elusive.

314 - Biomanufacturing In Low Earth Orbit: Accelerating Breakthroughs In Regenerative Medicine

P. Mesci

Axiom Space, Houston, TX

*Purpose/Objectives: Progress continues toward the development of disease models, stem cell manufacturing and tissue engineered products in microgravity that will change the paradigm of regenerative medicine. This session will provide insights into the ongoing initiatives by Axiom Space, the leading provider of human spaceflight services and developer of human-rated infrastructure, to build dedicated research and manufacturing facilities, establish state-of-the-art capabilities and biomanufacturing applications for Axiom Station that will shape the future of cGMP manufacturing in low-Earth Orbit. This session will discuss how Private/Commercial Astronaut Missions to the International Space Station that Axiom operates today are providing increased opportunities for access to microgravity that are accelerating the pace of development for biomedical applications and share a view of what the future of in-space biomanufacturing will look like on the world’s first commercial space station - Axiom Station, the successor to the International Space Station.

*Methodology: Microgravity has the potential to create a new definition of global for research and biomanufacturing, that includes low-Earth orbit (LEO) 250 miles above the Earth. This session will provide participants with an overview of ongoing biomedical initiatives shaping the next frontier for regenerative medicine and biomanufacturing that stand to provide significant global and societal benefits.

*Results: Speaker: Pinar Mesci, PhD Program Manager, In-Space Manufacturing, Axiom Space, Houston, TX

*Conclusion/Significance: This is a follow-on session to a virtual session conducted at TERMIS 2021.

316 - Production Of Extracellular Vesicles During Spaceflight In Skeletal Muscle Microphysiological Systems For Human Muscle Aging Research

M. Parafati¹, S. Ali¹, P. Coen², M. He¹, S. Malany^1,3

¹ University of Florida, Gainesville, FL,

² Advent Health, Orlando, FL,

³ Micro-gRx, Orlando, FL

*Purpose/Objectives: With extended stays aboard the International Space Station (ISS) and our nations plans for deep space exploration, astronauts may develop aging-like symptoms such as muscle wasting at a faster rate than on Earth. Interestingly, spaceflight-induced muscle atrophy experienced by astronauts, as result of muscle unloading, shares similar physiological changes to muscle wasting in older adults, known as sarcopenia. Sarcopenia is a progressive, involuntary skeletal muscle mass loss and strength linked to physical frailty and elevated risk of morbidity in the aged population and effective therapeutics remain unavailable. The molecular mechanisms behind accelerated aging in space remain to be fully characterized; thus, there is a need to develop tools to better understand the effects of microgravity on muscle function both to maintain human performance in space and also to understand disease pathology and progression to benefit civilians on Earth.

*Methodology: To identify in vitro aging phenotypes, our approach has been to study a human bioengineered skeletal muscle microphysiological system (MPS) cultured in an autonomous remote-controlled CubeLabTM on the ISS and on ground. Evidences show that regular exercise can normalize some aspects of age-related impairment of protein homeostasis by promoting recovery of the atrophic muscles and releasing muscle-specific extracellular vesicles (EVs), important mediators in physical activity adaptation. In our work, we recapitulated many steps of myogenesis by using human myoblasts derived from young active and old sedentary donors, classified as > 40 yrs and engaging in strenuous exercise three times per week and < 65 yrs and exercising once per week or not at all, respectively. The MPS can recapitulate a range of functional and biochemical components of muscle tissue. Furthermore, given the potential role of EVs in adaptation to exercise and the challenges in identifying the contribution of muscle-derived EVs from systemic EVs, we underscored the importance of using myobundle-derived EVs to study the effects of microgravity on muscle-specific EVs in-space production.

*Results: To mimic in vivo exercise, we induced contractions by electrically stimulating the donor-derived myobundles daily. The first goal of the two week on-orbit experiment was to record and quantify real-time muscle contractile function of myobundles in response to electrical stimuli using digital image correlation and microstructural image analysis. Also, we preserved space-flown tissues and serum-free secreted conditioned media for total RNA and EVs isolation, respectively. We investigated differences in the transcriptomic networks of donor myobundles as a function of age and adaptation to microgravity conditions. Pathway analysis revealed unique key pathways related to chemokine-mediated signaling in young- and degradation of extracellular matrix in old-derived myobundles, highlighting distinctive responses to spaceflight. We observed changes in EVs profiles and characterized them biophysically, by size, concentration, and morphology as well as by molecular cargos.

*Conclusion/Significance: We envision that both transcriptomics and EVs profiling would provide age- and microgravity-related signatures to elucidate mechanisms of microgravity-induced muscle aging to predict both risk factors and age-specific outcomes of interventions to mitigate sarcopenia on Earth and muscle age acceleration to protect future space explorers.

317

320 - A Biomimetic Helical Robot Actuated By Rotating Magnetic Field For Targeted Navigation And Localized Nir-based Prodrug Activation To Treat Intestinal Diseases

Z. Wang¹, L. Wang¹, W. Chen², Q. Li¹, P. Cao¹

¹ Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China,

² Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

*Purpose/Objectives: Intestinal diseases (e.g., inflammatory bowel diseases and colorectal cancer) are relatively common but severely affect people’s health. Orally dosed medication is the most acceptable and preferred route for intestinal disease treatment owing to its convenience for self-administration, painless operation, and low cost. However, it is limited by premature drug degradation in a harsh gastral environment, inevitable off-target effects, and insufficient retention at lesion sites, leading to poor therapeutic efficacy and toxic side effects, aggravating patients’ pain and discomfort. To address the aforementioned limitations, we develop a biomimetic helical robot (BHR) inspired by helical bacteria, enabling targeted cargo transport and localized prodrug activation within gastrointestinal tract by systemic integration of magnetism- and photo-responsiveness.

*Methodology: The main components of BHR were constructed by 3D printing with resin as the raw printing material. BHR’s head and torso were coated with the enteric Eudragit L-100 layer integrated with photothermal AuC nanocomposites and hot-melt polycaprolactone to load inactive prodrug and its activator, respectively. A stirring magneton was placed into the torso’s cavity that responded to a rotating magnetic field to actuate BHR propelled forward. BHR’s locomotion performance was investigated in diversely shaped lumens (including the simulated peristaltic small intestine) containing lowly to highly viscous media. The optically controllable drug release, prodrug activation, and the subsequent remission of inflammation were determined in vitro. Moreover, the magnetically guided navigation, drug release and reaction, and the efficacy in relieving intestinal inflammation were demonstrated in vivo using a dextran sulfate sodium salt-induced enteritis rabbit model. Finally, the feasibility of external magnetic field to control BHR movement after oral ingestion and BHR’s biosafety were assessed in Bama mini swine.

*Results: Under the guidance of a rotating magnetic field generated by a rotating NdFeB magnet and the stirring magneton at a distance of approximately 4 cm, bioinspired helical structure allows the BHR to rotationally and smoothly travel through the mimic and real gastrointestinal tract. The external magnetic field readily altered BHR’s locomotion trajectory, enabling it to sustainably anchor at the specific location, turn corners, reverse direction, and move back. Further, the BHR with its two different compartments individually loaded with an anti-inflammatory prodrug and the corresponding drug-activator achieves the NIR-triggered drug release and in situ prodrug activation, which then generates effective anti-inflammatory effects, as evidenced by the suppression of inflammatory indicators (ROS, IL-1β, and TNF-α) in the immortalized human colon mucosal epithelial cells (NCM460), murine macrophage-like cells (RAW264.7), and intestinal tissues from enteritis-bearing rabbits. Oral ingestion of BHR did not elicit detectable anemia, inflammation and organ dysfunction in swine, as the main indicators reflecting blood cells, heart, liver and kidney functions exhibited negligible alterations between BHR-treated and untreated swine.

*Conclusion/Significance: By coupling the advantages of biomimetic architectural design with magnetic and optical equipment, BHR proposed here allows for spatiotemporally controlled navigation and targeted prodrug activation in gastrointestinal system, providing a promising medication mode for intestinal disease treatment. Meanwhile, the BHR allows various physicochemical modifications and photoelectric integration, potentially offering ample opportunities for versatile functional designs demanded by different clinical treatments.

322 - Regional Delivery Of Sdf-1α Loaded Hydrogel Fragments To Supraspinatus Muscle After Rotator Cuff Injury

L. Anderson, J. Pearson, R. Miller, J. Mao, J. Temenoff

Georgia Institute of Technology and Emory University, Atlanta, GA

*Purpose/Objectives: Following rotator cuff tendon injury, the corresponding muscle undergoes degeneration, which has been found to occur asymmetrically. In our rat model of rotator cuff tear, the myotendinous junction (MTJ) (region closest to tendon) exhibits increased de/re-generative processes and increased immune cell presence. The muscle belly (MB) (further into the muscle) exhibits fewer muscle changes and different cellular milieu. Previously, treatment with stromal cell-derived factor-1α (SDF-1α) loaded microparticles increased pro-regenerative cell (M2-like macrophages and mesenchymal stem cells/MSCs) presence in supraspinatus muscle 7 days after injury. Building upon this work, we designed an injection strategy that targeted either the MTJ or MB regions to spatially recruit cells to specific muscle areas. We hypothesized that: 1) spatially localized SDF-1α loaded fragment delivery to the MTJ or MB region would increase pro-regenerative cells in treated regions compared to untreated controls; 2) treatment in the “more active” MTJ region would promote early muscle regeneration and prevent initial degeneration more than treatment in the “less active” MB region.

*Methodology: Based on the muscle changes observed after injury in our model, 0-1000 µm and 1500-2500 µm into the muscle were defined as MTJ and MB regions, respectively. Hydrolytically degradable heparin-based hydrogel fragments were optimized for sustained release of SDF-1α over 7+ days in vivo (IVIS, n=4-6). Spatial injection was confirmed after injection ex vivo via tissue explants (Fig 1A). Immediately after rotator cuff injury, SDF-1α loaded fragments were injected into either region of injured supraspinatus muscle. Presence of inflammatory cells (neutrophils, macrophage and T cell subsets) and stem cells (fibroadipogenic progenitor cells, satellite cells, MSCs) in each region was assessed via flow cytometry 3 and 7 days following treatment (n=7-9). Muscle tissue changes were evaluated via immunohistochemical staining for regenerating muscle fibers (eMHC), fibrosis (collagen) and fatty infiltration (perilipin) 7 days post injury (n=6). Statistical analysis consisted of two-way ANOVA followed by Tukey’s or Sidak’s multiple comparison test where appropriate (p ≤ 0.05 for all tests).

*Results: 20 wt.% Hep^-N hydrogel fragments released SDF-1α over at least 10 days in vivo (Fig 1B). SDF-1α exhibited general effects on cellular recruitment in both muscle regions, including decreased neutrophils and T cells over time, regardless of treatment region. Within the MTJ, there were increased stem cell populations after SDF-1α treatment, regardless of treatment region (Fig 1C). In the MB, spatial differences in early cellular milieu were observed after treatment, characterized by increased T cell and macrophage subsets compared to the untreated MB region (MTJ treatment) at day 3 (Fig 1D). Significantly more early muscle regeneration was observed in the MTJ compared to the MB, regardless of treatment (Fig 1E). No significant differences in fibrous or fatty infiltration were observed between regions after treatment.

*Conclusion/Significance: The regional differences in cell populations observed after treatment demonstrated technical feasibility of spatial material delivery and subsequent cellular recruitment. Thus, localized delivery using this biomaterial platform may be a useful tool to probe the effects of spatial cell recruitment in orthopaedic degenerative diseases and beyond.

324 - Engineering Biomimetic 3D Skeletal Muscle Architectures Using FRESH 3D Printed Collagen Scaffolds

M. A. Stang, A. Lee, J. Bliley, B. Coffin, S. Yerneni, P. Campbell, A. W. Feinberg

Carnegie Mellon University, Pittsburgh, PA

*Purpose/Objectives: Skeletal muscle has an intrinsic regenerative capacity; however, significant impairment or loss of muscle function can result from myopathy or neuromuscular diseases, traumatic injuries, or surgical complications. Therapies such as functional free muscle transfer and transplantation of exogenous myoblasts are only moderately successful. Engineered skeletal muscle tissue is a potential solution to restore muscle function, but current tissue engineering approaches are not able to recapitulate the various native muscle architectures. Here, we report the engineering of skeletal muscle tissues with complex native-like architectures using Freeform Reversible Embedding of Suspended Hydrogels (FRESH) 3D bioprinting. FRESH-printed collagen type I bioink was used as the scaffold material around which C2C12 myoblasts were cast and compacted in a collagen gel to form highly aligned muscle constructs with parallel, unipennate, and bipennate myofiber architectures.

*Methodology: Scaffolds mimicking various muscle architectures were created using Slic3r slicer software and then FRESH 3D printed using an acidified collagen type I bioink at 24 mg/mL (Advanced BioMatrix) in a gelatin microparticulate support bath. These collagen scaffolds were then placed in polydimethylsiloxane (PDMS) wells containing two posts on either end to hold the constructs during casting and cell culture. C2C12 myoblasts were cast around the collagen scaffolds inside the PDMS wells at 30 million cells/mL in a collagen solution (0.5 mg/mL). Constructs were cultured for up to 20 days and then assessed. C2C12 density and alignment were assessed with immunofluorescent staining and confocal microscopy and contractile forces were measured using a custom-mounted optical force transducer. Tissues were subcutaneously implanted into C3H mice for ten days, and histological sections were created from the explanted tissue.

*Results: Three scaffold designs were created to mimic parallel, unipennate, and bipennate muscle architectures (Figs. 1A, 1B) and were printed from collagen with high fidelity (Fig. 1C). Immunofluorescent images indicate the scaffolds’ ability to provide directional cues to the cells as they are organized, aligned along the collagen filaments, and fused into multinucleated myotubes (Fig. 1D). Engineered muscle tissue featuring a parallel microstructure under field stimulation displayed muscle contraction and at higher frequencies (20 Hz) approached tetanus (Fig. 1E). A histological section taken from an implanted tissue with parallel microstructure demonstrated that scaffold architecture is maintained after 10 days in vivo, and in select areas, vascular ingrowth occurred along the printed collagen fibers, as demonstrated by the presence of blood vessels containing red blood cells (Fig. 1F).

*Conclusion/Significance: Together, these results demonstrate that collagen can be FRESH 3D bioprinted in various skeletal muscle architectures with high fidelity to guide tissue organization, generate active contractions, and direct vascularization in vivo. These engineered muscle tissues are the first to demonstrate control of microstructure and myofiber architecture in 3D space and show the potential to recapitulate different muscle types within the body.

325 - Evaluation Of MuscleMatrix™ For Repair Of Volumetric Muscle Loss (VML) Injuries In The Rat Gastrocnemius

S. B. Shriver¹, M. Pfaff², K. Parekh², K. E. Healy², G. J. Christ¹

¹ University of Virginia, Charlottesville, VA,

² University of California, Berkeley, Berkeley, CA

*Purpose/Objectives: Comprising nearly 40% of a human’s body weight, skeletal muscle plays an essential role in force generation for tasks such as walking, breathing, and eating. While skeletal muscle possesses the unique ability to heal itself after injury, volumetric muscle loss (VML) is a condition that exceeds this endogenous capability - shifting the response towards fibrotic deposition and scarring. These injuries affect both warfighters and civilians, resulting in permanent cosmetic and functional deficits that lead to long-term disability and diminished quality of life. The enormity of this unmet medical need has spurred investigation into a variety of preclinical approaches. The overriding purpose of this project is to develop and further optimize implantable therapeutics to improve muscular regeneration and functional recovery following VML injury. Our lab has worked with the Healy Lab at UC-Berkeley to support development of a novel hyaluronic acid-based material system, MuscleMatrix™. Modified to encourage cell adhesion, sequester endogenous growth factors, and dissipate through cell-mediated mechanisms, this material has been synthesized in two form factors reported herein - hydrogel and cryogel.

*Methodology: Animals were divided into the following groups: no repair(n=6), hydrogel-treated(n=8), and cryogel-treated(n=8). At 12-weeks old, animals received surgery to create a 20% VML defect in the middle third of the lateral gastrocnemius (GN) muscle. The contralateral leg of each animal was left uninjured to serve as a control. Each group was taken off study for in situ functional testing at 24-weeks post-VML. GN muscles were removed and frozen to undergo rigorous histologic analysis. Each sample was stained for H&E, Picrosirius Red, and Masson’s trichrome to gain information on muscle morphology and fibrosis. Furthermore, laminin and DAPI co-staining permitted use of an image analysis pipeline to quantify various metrics such as fiber cross-sectional area and the number of fibers per section.

*Results: Functional testing results showed the average force produced by an uninjured, contralateral control GN was 23.2±3.8 N/kg (n=21). At 24-weeks post-injury, the unrepaired and cryogel-treated animals had maximum force readings significantly lower than that of the controls (15.4±2.5 and 16.2±1.9 N/kg, respectively). When treatment conditions were compared, the hydrogel-treated animals had an average force of 18.4±2.5 N/kg, which was significantly higher (p=0.03) than that of the unrepaired animals (one-way ANOVA, Tukey posthoc). Initial staining demonstrated incomplete cryogel degradation. Preliminary fiber analysis revealed that the GN contained an average of 10,172 (n=6) and 8,364 (n=2) fibers in control and unrepaired VML injured animals, respectively. Data analysis is ongoing.

*Conclusion/Significance: These initial findings confirm and extend observations published by Dienes et al. in the rat TA, to demonstrate that the MuscleMatrix™ hydrogel form factor can enhance functional recovery relative to nonrepaired animals—but importantly the GN VML injury is greater than 2X that of the TA (150 mg vs 70mg). While the MuscleMatrix™ cryogel form factor would have greater utility in austere environments, further investigation of design features to optimize cellular invasion and degradation is required. Tissue histology and muscle fiber analysis studies are ongoing to further identify the corresponding mechanisms that may be responsible for these observations.

327 - Repairing Volumetric Muscle Loss With Commercially Available Hydrogels In The Ovine Peroneus Tertius Muscle

E. Y. Su, C. S. Kennedy, E. E. Vega-Soto, B. D. Pallas, S. N. Lukpat, D. H. Hwang, D. W. Bosek, C. E. Forester, C. Loebel, L. M. Larkin

University of Michigan, Ann Arbor, MI

*Purpose/Objectives: Volumetric Muscle Loss (VML) is a 30% or more loss of skeletal muscle tissue that exceeds the muscle’s self-repair mechanism and results in acute inflammation and excessive fibrosis. Current surgical treatments for VML consisting of autologous muscle grafts are limited by tissue availability and donor site morbidity. Biomaterials such as hydrogels (HG) have significant potential for decreasing acute inflammation and fibrosis, and enhance skeletal muscle regeneration. This study assessed the biocompatibility of commercially available poly(ethylene glycol) (PEG), methacrylated gelatin (GelMA), and hyaluronic acid (HA) hydrogels and the efficacy of combining HA with our skeletal muscle units (SMUs) to treat VML following a 6-week recovery from a 30% injury in the peroneus tertius (PT) muscle of an ovine model.

*Methodology: Male wether Polypay sheep were randomly divided into six groups: VML-Only (n=4), PEG (n=5), GelMA (n=5), HA (n=4), SMU-Only (n=5), and SMU+HA (n=5). In all groups, an incision was made along the midline of the lower left leg to expose the PT muscle, and a v-cut longitudinal portion of the PT constituting 30% total muscle volume was dissected. The fascia was closed and the HG was injected into the VML site. The skin was closed with suture and staples. Following a 6-week recovery, both the contralateral (right) and surgical (left) PTs were dissected and prepared for histological analyses.

*Results: At the 6-week recovery period, there was a significant muscle mass deficit between the contralateral and the VML-Only, PEG, GelMA, and HA groups (P=0.0074, P=0.0135, P=0.0433, P=0.0421). In contrast, the SMU-Only and SMU+HA groups were statistically indistinguishable from the contralateral muscle (P=0.3021, P=0.0603). In just 6 weeks, the SMU-Only group recovered approximately 15% of the VML injury, reaching a muscle mass of 85% in comparison to its contralateral. In accordance, the MF20 and Laminin analysis revealed an increased number of regenerating muscle fibers in the SMU-Only and SMU+HA repair sites when compared to the hydrogel groups. Furthermore, more regenerated fibers in the VML injury site were observed in the PEG and HA group (Fig 1). The M1 and M2 macrophage staining revealed a smaller number of pro-inflammatory markers in the PEG and HA hydrogels and an increase in anti-inflammatory markers in the HA, SMU-Only, and SMU+HA groups.

*Conclusion/Significance: The results from the 6-week recovery period showed fibrotic infiltration into all VML sites. However, the PEG, HA, SMU-Only, and SMU+HA groups exhibited decreased inflammation and more regenerated muscle fibers in the repair site. We summarize that the mass recovery in the SMU-Only group cannot solely be attributed to connective tissue deposition, and the contribution of the SMU led to a more effective muscle mass regeneration when compared to the hydrogels alone. In conclusion, these results demonstrate the efficacy of the HA hydrogel in controlling acute inflammation and fibrosis in the repair site during muscle regeneration following a 30% VML injury. Moreover, these finding collectively underscore the synergistic potential of our SMUs in conjunction with HA hydrogel in mitigating inflammatory responses and increasing muscle regeneration in the repair site following VML injury.

329 - Combinatorial Hipsc-Neuron And Engineered Biomaterial Transplantation Therapy ToTreat Chronic Spinal Cord Injury

V. M. Doulames, M. E. Hefferon, N. J. Baugh, R. A. Suhar, C. M. Long, T. D. Palmer, S. C. Heilshorn

Stanford University, Stanford, CA

*Purpose/Objectives: Cervical spinal cord injuries (SCI) sever the circuitry responsible for sensorimotorfunction and there is no cure. Over time, scarring at the injury site and aberrant rewiring of theremaining circuitry create barriers that hinder recovery in chronic injuries. Transplanting humaninduced pluripotent stem cells that have a deep cortical neuron phenotype (hiPSC-DCNs) canimprove anatomical and functional outcomes in rat models of cervical SCI. As maturing neuronsare mechanosensitive, poor transplanted cell survival and retention remain critical obstacles tosuccessful cell therapies. To address these challenges, we designed and optimized a Shear-thinning Hydrogel for Injectable Encapsulation and Long-term Delivery (SHIELD) to reducehiPSC-DCN damage during transplantation and promote engraftment in the spine.

*Methodology: Patient-derived hiPSCs were differentiated towards a deep cortical neuronphenotype. To pattern hiPSCs towards a neuroepithelium fate, cells were cultured usingmedium containing DMEM/F12, Neurobasal-A, B-27, N-2, GlutaMAX, and MEM non-essentialamino acids and TGFb and BMP signaling were blocked using small molecules. After 14 days,the cells were dissociated, plated on poly-D-lysine and laminin-coated plates, and rostrallyshifted towards a dorsal telencephalic fate via Wnt inhibition. By 35 days, this population ispapain-dissociated and prepared for transplantation. Temporal characterization of thedifferentiating cell population was accomplished via immunocytochemistry and RT-qPCR.SHIELD is a physically-crosslinked hydrogel delivery system, comprised of a recombinantengineered protein with fibronectin-derived, cell-adhesive peptides and a synthetic 8-armpolyethylene glycol polymer modified with peptides to induce self-assembly and gelation. Invivo, mixed sex athymic rats (n=58) received a contusion SCI (100 kdyne) at vertebral level C5and, 8 weeks later, were randomized for transplantation: (i) injury, (ii) saline, (iii) fibrinogen, (iv)SHIELD, (v) hiPSC-DCNs in saline, (vi) hiPSC-DCNs in fibrinogen, and (vii) hiPSC-DCNs inSHIELD. Spinal tissue was excised 12 weeks post transplantation and analyzed for hiPSC-DCNsurvival, morphology, distribution, integration, and endogenous inflammatory markers and SCIpathology. Rats were tested for functional sensorimotor improvement across 5 behavioralassays.

*Results: We demonstrate that by delivering hiPSC-DCNs in SHIELD, we can improve transplantsurvival and therefore improve functional outcomes. This hiPSC differentiation protocol resultsin a robust selection for fate-restricted neural cells with a deep cortical neuron phenotype alonga defined time-course that parallels human corticogenesis, as confirmed by morphology andgene and protein expression experiments. In vitro, we found SHIELD biomimetic properties(fibronectin-derived RGD binding domain, physically soft gel) improved mechanoprotectionfollowing injection when compared to saline. In vivo, we found that delivery of hiPSC-DCNs inSHIELD resulted in more consistent transplant volumes compared to cell delivery with saline.Analysis of excised spinal tissue confirms that hiPSC-DCNs can successfully be grafted into anadult chronic cervical injury and overcome the endogenous inhibitory milieu. Grafted hiPSC-DCNs maintain their subtype identity and extend axons to innervate lower spinal segments.

Additionally, hiPSC-DCN graft integration correlated with improvements in sensorimotorfunction.

*Conclusion/Significance: Our results demonstrate that the combination of a biologically functional, injectabledelivery vehicle with a stem-cell derived, neurocircuit-relevant cell type can significantly improveanatomical and functional outcomes within the chronic injured cervical spine.

330 - 3d Bio-printed Photo-clickable Scaffolds Combined With Transplantation Of Human Spinal Cord Progenitor Cells Promote Nerve Regeneration After Spinal Cord Injury

V. S. Ramanujam¹, P. Nunes de Oliveira¹, C. Kwokdinata², K. Lixuan Chai², N. Chueakula², L. David³, S. chew²

¹ CNRS@create/Nanyang Technological university Singapore, Singapore, Singapore,

² Nanyang Technological university Singapore, Singapore, Singapore,

³ CNRS@CREATE Ltd, Singapore, Singapore

*Purpose/Objectives: Spinal cord injury (SCI) leads to irreversible motor, sensory, and functional disorders. Without appropriate interventions, neurons of the central nervous system do not regenerate spontaneously after injury. While soft biomaterials mimicking the spinal cord tissue have been studied to provide guidance for neurons to cross the injury site, transplanting neural stem cells/progenitor cells (NSC/NPCs) into the injury site has been studied to promote axonal regrowth and remyelination. Notably, human-induced pluripotent stem cells (iPSC)-derived cells, specifically those with spinal cord identity known as spinal cord progenitor cells (SCPCs), have demonstrated promising outcomes by reconnecting with host neurons and thereby enhancing functional recovery post-SCI. This study introduces a novel approach that combines these two strategies, utilizing 3D-printed scaffolds for guiding native nerves and transplanting SCPCs around the injury site to replenish lost neuronal cells, thereby promoting nerve regeneration.

*Methodology: Methacrylated gelatin (GeLMA) scaffolds were 3D-bioprinted using stereolithography-aided digital-light printing (DLP), with unidirectional microchannel structure to guide axonal regrowth. The stability of the scaffold was studied in vitro using Fourier Transform Infrared Spectroscopy (FTIR) and its cytocompatibility was tested using live/dead assays.In vitro differentiation profile of SCPCs cultured on GeLMA scaffolds were analysed for two weeks. The effectiveness of these scaffolds was evaluated in vivo in a complete spinal cord transection rat model. A week after implantation of the scaffolds, SCPCs were transplanted rostral and caudal to the injury site. The combined impact of scaffolds and SCPCs on spinal cord regeneration was assessed four weeks following cell transplantation.

*Results: The GelMA scaffolds were stable after two weeks of in vitro degradation testing, with the FTIR spectra showing typical gelatin peaks. The in vitro cytocompatibility of the scaffolds tested with SCPCs, showed high cell viability, as well as neuronal differentiation after two weeks. The scaffolds also demonstrated good host-implant integration after four weeks of in vivo implantation within the spinal cord. Specifically, neurofilament infiltration from the host tissue was supported by the scaffolds, and viable SCPCs were seen on the rostral and caudal sides. Immunohistochemistry staining revealed that the SCPCs not only differentiated into mature neurons, but also migrated towards the microchannels of the implants. These scaffolds, when used with SCPCs, reduced injury size without triggering adverse scarring or inflammation around the injury site. After two weeks of SCPC transplantation, the rats began to show signs of functional recovery.

*Conclusion/Significance: The 3D-printed GelMA scaffolds promote nerve regeneration after spinal cord injury by simultaneously inhibiting the formation of glial scars and guiding axonal regrowth. In addition, the transplantation of SCPCs provides essential cells for regeneration, offering a comprehensive therapeutic strategy for repair.

332 - Elastic And Hemostatic Human Protein-based Surgical Sealant For The Rapid Closure Of Ruptured Soft Tissues

S. Jain¹, M. Ghovvati², G. Cheng³, J. Boys³, N. Kaneko², A. Weiss⁴, N. Annabi²

¹ UCLA, Calabasas, CA,

² UCLA, Los Angeles, CA,

³ UCSD, San Deigo, CA,

⁴ The University of Sydney, Camperdown, Australia

*Purpose/Objectives: Traditional solutions for hemorrhaging injuries are gauzes, sutures, or staples, all of which can complicate the progression of wound repair. Gauzes can cause thromboembolism or secondary bleeding upon removal. Furthermore, conventional means for wound closure are often inappropriate for elastic organs such as lungs, heart, or muscle. Commercially available products exist for both hemostasis and wound closure (e.g., Tisseel^®, Floseal^®, Tachosil^®) utilizing natural polymers as sealing matrices ad biological components to expedite blood clotting. However, the majority possess inadequate mechanical and adhesive properties that limit their efficacy for sealing elastic organs. Other investigated hemostatic sealants with carbon-based materials, nanoparticles, or catechol-containing compounds may expedite hemostasis or tissue adhesion, but most still have limitations concerning their biocompatibility, elasticity, or mechanical tunability. To fill this scientific gap, we have engineered methacrylated tropoelastin (MeTro)-based hydrogels that mimic native tissue elasticity and provide durable adhesion. Silicate nanoparticles (SN, Laponite^®) were introduced to further enhance tissue adhesion and provide highly charged regions which could promote platelet interactions and blood clotting.

*Methodology: The MeTro prepolymer was combined with varying amounts of SN and photocrosslinked with visible light for 80 sec to form hydrogels. SN concentration tuned mechanical, adhesive, and hemostatic properties required for wound treatment. The MeTro/SN hydrogels then underwent extensive in vitro, ex vivo, and in vivo adhesion, hemostatic, and biocompatibility testing on mammalian cells, various organs, and on small and large animals.

*Results: The engineered hydrogels exhibited strong ex vivo adhesion on soft, elastic, and pressurized (ventilated) pig lungs. The prepolymers were applied on a pleural defect and photo-crosslinked to form sealants (Fig. 1A). MeTro/SN exhibited stronger adhesion (3.6 kPa) than MeTro (2.9 kPa) due to the increased potential for ionic, hydrogen-bonding, and electrostatic interactions between Laponite^® and tissue (Fig. 1B). Both engineered hydrogels exhibited stronger adhesion than commercial sealants. In vitro biocompatibility was evaluated through live/dead testing after 7 days on untreated (control) and 2D-seeded lung fibroblast cells onto MeTro and MeTro/SN (Fig. 1C). All conditions experienced high cell viability (> 95%), indicating no biomaterial-induced cytotoxicity (Fig. 1D). To expand on its clinical efficacy, we applied MeTro/SN on a 15mm laceration on pig lungs and examined its hemostatic, adhesion, and biocompatible properties. After 14 days, the hydrogel-tissue was explanted for immunohistology. Hematoxylin and eosin (H&E) staining of the interface showed durable adhesion and retention of MeTro/SN as well as no signs of fibrosis (Fig. 1E). Immunostaining for pan-macrophages (CD68) and activated macrophages (CD80) showed little to no inflammation related to the presence of the biomaterial (Fig. 1F). Furthermore, a 1 min hemostatic test confirmed that MeTro and MeTro/SN were superior to commercial hemsotats when applied to a heavily bleeding lung injury (Fig. 1G). The hydrogels likely caused charged-mediated blood cell aggregation, which expedited hemostasis.

*Conclusion/Significance: Our novel elastic and hemostatic sealant showcased great potential for a wide array of surgical applications. A well-rounded and easy-to-use material such as MeTro/SN that achieves fast hemostasis and durable tissue adhesion may be able to minimize post-operational complications and lighten medical burden for surgeons.

333 - Injectable And Adhesive Hydrogel Formulation For Exosome Delivery In Cardiac Repair And Regeneration: A Minimally Invasive Therapeutic Strategy For Myocardial Infarction

U. Tariq, A. Pandit, A. Kumar

Indian Institute of Technology Kanpur, Kanpur, India

*Purpose/Objectives: Cardiovascular diseases are the leading cause of death worldwide, accounting for 17.9 million deaths annually, the majority of which are attributed to myocardial infarction (MI). MI is a condition in which the coronary artery gets blocked due to a thrombus or atherosclerotic plaque, resulting in myocardial ischemia and damage to the heart muscle. The damage is more extensive as cardiomyocytes are fully differentiated and have a limited ability to regenerate damaged tissue and regain functionality after MI. Current treatment options, which include “pernicious coronary intervention” (PCI), only focus on reperfusion and not regeneration and repair of the damaged heart. Currently, tissue engineering has emerged as a viable strategy to improve heart regeneration after MI. Stem cell-based therapies have been extensively researched for cardiac regeneration, but clinical translation has yet to be achieved because of invasive transplantation procedures, limited engraftment of transplanted cells, and their high tumorigenic and immunogenic potential. Injectable hydrogels loaded with acellular bioactives, particularly stem cell-derived exosomes, which are extracellular cargos secreted by cells, have emerged as potential therapeutic strategies for cardiac repair.

*Methodology: This study is aimed at developing an injectable, adhesive hydrogel system loaded with adipose-derived stem cell (ADSC)-derived exosomes as a minimally invasive treatment strategy for MI. A chitosan-guar gum-borax hydrogel (CS/OGB) was synthesized and characterized for injectable and tissue adhesive properties. Exosomes were isolated from ADSCs, characterized, and loaded into the hydrogel to evaluate the therapeutic efficacy in different in vitro and in vivo models of MI.

*Results: The synthesized CS/OGB hydrogel demonstrated self-healing, injectable, and adhesive properties, which are essential for minimally invasive administration. Rheological studies showed that the CS/OGB hydrogel resists deformation up to 100% strain values, which suggests its viscoelastic behaviour and is a prerequisite for any hydrogels to withstand strain during injection. The synthesized hydrogel displayed excellent biocompatibility with H9c2 rat cardiomyoblast cells. Moreover, the hydrogel facilitated controlled exosome release (80% release in 14 days). This sustained release from the CS/OGB hydrogel demonstrated an increased proliferation rate of H9c2 cells as compared to direct treatment of exosomes. Oxidative stress and fibrosis are the major hallmarks of MI. The exosome treatment rescued cardiomyocyte death after H₂O₂-induced oxidative stress, which was evaluated by the DCFDA assay and live-dead staining. The cardiac fibrosis was induced in primary cardiac fibroblasts by TGFβ1 treatment, and we found that ADSC exosome treatment reduced fibrosis, which was confirmed by the cell morphology analysis and mRNA expression of α-SMA, collagen I, collagen III, and TIMP2. Further, CS/OGB hydrogel was injected into the pericardial cavity of rats to assess its immune compatibility and retention in the beating heart. The observation revealed the formation of an adhesive elastic patch on the heart, and notably, no immune response was observed until 14 days following injection.

*Conclusion/Significance: Overall, it can be concluded that the CS/OGB hydrogel system loaded with ADSC-derived exosomes presents a promising approach for MI treatment and will pave the way towards the development of acellular, injectable, regenerative biomaterials for different tissue engineering applications.

334 - Mechanically Stimulated Piezoelectric Scaffolds Enhance In Vitro Osteogenic Differentiation And In Vivo ECM Formation

N. N. Tavernaraki¹, V. Platania¹, N. Triantopoulou¹, K. Alpantaki², M. Vidaki¹, M. Chatzinikolaidou^1,3

¹ University of Crete, Heraklio, Greece,

² Venizeleion General Hospital of Heraklion, Heraklio, Greece,

³ FORTH, Heraklion, Greece

*Purpose/Objectives: Bone is a highly dynamic tissue that undergoes continuous mechanical forces through lifetime to sustain the balance of bone formation and bone resorption. Mechanical stimuli applied on cell-loaded scaffolds resembling a part of the human bone can have major effect on osteogenesis. Poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) is a piezoelectric material that responds to mechanical stimulation producing an electrical signal that promotes the osteogenic differentiation of pre-osteoblastic cells by opening voltage-gated calcium channels. The aim of this study was to examine the biological behavior of pre-osteoblastic cells when seeded onto piezoelectric PEDOT:PSS-containing scaffolds applying uniaxial compression as mechanical stimulation relevant to physiological body movements.

*Methodology: Two concentrations of PEDOT (0.15 % w/v and 0.10 % w/v) were combined with a 5 % w/v poly(vinyl alcohol) (PVA) and 5 % w/v gelatin, casted into wells, freeze dried and crosslinked with 2 % v/v (3-glycidyloxypropyl)trimethoxysilane (GOPS) and 0.025 % w/v glutaraldehyde. For the control scaffold, a solution without the addition of PEDOT:PSS was prepared. The scaffolds were physicochemically, mechanically and biologically characterized. Scaffolds seeded with pre-osteoblastic cells were subjected to uniaxial compression (by means of MechanoCulture bioreactor) with a frequency of 1 Hz and a strain of 10% for 1 h every day for 21 days, and their osteogenic response was compared to that of a non-mechanically stimulated culture. The loading parameters were selected to resemble the in vivo loading situation. Cell viability and morphology of the pre-osteoblastic cells seeded onto the scaffolds was determined. The alkaline phosphatase (ALP) activity, the collagen and calcium production were quantified. Determination of the calcium and phosphorous mineralization through atomic-resolution mapping and Energy Dispersive Spectroscopy (EDS) was carried out, as well as X-ray diffraction (XRD) analysis, aiming the detection of biomineralized matrix. In vivo experimentation following subcutaneous implantation in mice was also conducted.

*Results: The physicochemical characterization indicated that PEDOT:PSS/PVA/gelatin scaffolds presented favorable properties for bone tissue engineering. The most significant results were their elastic modulus which ranged between 1 and 5 MPa. The degradation rates indicate a mass loss of 15% after 21 days. The cell viability assay indicates an increase of cell number over time, while scanning electron microscopy images display strong adhesion of well-spread cells. The ALP activity at days 3 and 7 is significantly higher in the dynamic culture compared to the static one. Increased collagen and calcium secretion further validate osteogenic response and extracellular matrix formation. Moreover, EDS and XRD analysis revealed the presence of bone-like apatite formation on scaffold surfaces after 21 days. No adverse reactions of the implanted constructs in mice were observed.

*Conclusion/Significance: The results indicate that the PEDOT:PSS/PVA/Gelatin scaffolds support the adhesion, proliferation, and osteogenic differentiation of the pre-osteoblastic cells under mechanical stimulation, favoring bone and other load-bearing tissue engineering with amplified matrix production. Furthermore, the development of a comprehensive 3D mechano-active model establishes an innovative platform for studying osteogenesis under conditions closely resembling the body natural state. This approach contributes to advancing in vitro evaluation methods but also significantly reduces the need for in vivo experimentation.

336 - Designing Injectable Peptide Hydrogels For Cell Delivery

A. F. Miller¹, A. Saiani²

¹ Department of Chemical Engineering, University of Manchester, Manchester, UNITED KINGDOM,

² Division of Pharmacy and Optometry, University of Manchester, Manchester, UNITED KINGDOM.

*Purpose/Objectives: Hydrogels are a fascinating class of materials that find applications across a variety of fields. Their biphasic nature and low level of structural order makes them challenging materials to design and characterise. Supramolecular hydrogels in particular have come to the fore over the last few decades as they allow the building of typically soft hydrogels through the guided self-assembly of small molecules avoiding chemical crosslinking. This is an attractive preparation route for biomedical applications when biocompatibility is a key requirement. In this context self-assembling peptides are a particularly relevant class of molecular building blocks. Built from nature’s 20 amino acid toolbox they can be designed to be biocompatible and with low immunogenicity. In addition their unique shear-thinning properties makes them ideal for the design of injectable hydrogel scaffolds for the in-vivo delivery of cells.

*Methodology: For this purpose we designed a family of novel short (4 to 14 amino acids) β-sheet forming peptides based on alternating hydrophilic (glutamic acid, lysine, aspartic acid, arginine), and hydrophobic (phenylalanine, valine, leucine, isoleucine, tyrosine, tryptophan) residues. The key property of this family of peptides is their ability to self-assemble in water-based media into cross β-sheet fibres that above a critical gelation concentration (CGC) entangle and associate to form 3D swollen networks, in other words hydrogels. First we investigated the shear-thinning properties of these materials using a range of techniques including shear induced polarised microscopy (SIPLI), transmission electron microscopy (TEM) and rheo-SAXS. Subsequently the hydrogels were used to deliver cardiac progenitor cells (CPC) in a rat myocardial infarction model.

*Results: Through this work we elucidated the shear-thinning and recovery mechanism of these hydrogels showing that two dynamic processes occur when the sample recover following injection: - a fast reassembly process which lead to a fast recovery of the hydrogels' mechanical strength and - a slow long term structural rearrangement that leads to a change in properties over times. In-vitro tests of cell injection using these hydrogel gel showed their suitability as they allow cell to be injected and retain high viability. In addition they were used as bioinks for 3D bioprinting applications. Finally they were used to inject CPC in a rat myocardial infarction model showing that injection of the hydrogel leads to a decrease in heart hypertrophy.

*Conclusion/Significance: Our work shows the potential and suitability of these materials as injectable vehicle for the delivery of cells in-vivo. These unique properties makes then also suitable a bioinks for 3D bioprinting applications.

337 - Cell-delivering Porous Injectable Hydrogels Engineered By Liquid-liquid Phase Separation For The Treatment Of Hindlimb Ischemia

A. Nishiguchi¹, S. Ito^1,2, K. Nagasaka^1,2, H. Komatsu^1,2, T. Taguchi^1,2

¹ National Institute for Materials Science, Tsukuba, Japan,

² Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan

*Purpose/Objectives: Injectable hydrogels hold promise as a cell delivery carrier to enable local administration of transplanted cells and improve therapeutic efficacy. However, hydrogels usually do not have micrometer-scale pores due to the densely cross-linked polymer networks, limiting the communication between transplanted cells and host tissues and resulting in low therapeutic efficacy. In this study, we aim to design porous injectable hydrogels utilizing liquid-liquid phase separation (LLPS) for stem cell transplantation (Figure 1a). We found that ureido-4[1H]-pyrimidinone (UPy) group-modified gelatin (GUPy) formed microcapillary network (μCN)-like LLPS structures in hydrogels (Biomaterials, 305, 122451 (2024)). By mixing GUPy with thiolated gelatin (GTH), vinyl sulfonated G (GVS), and cells, cells were encapsulated in hydrogels with LLPS-induced pores. In this presentation, we will report the observation of LLPS hydrogels, encapsulation of mesenchymal stem cells (MSCs), evaluation of cellular functions (adhesion, spreading, migration, proliferation), and therapeutic effects in hindlimb ischemia models of mice.

*Methodology: GTH, GVS, and GUPy were each dissolved in phosphate buffered saline at 10 wt%. After adjusting pH, each solution was mixed and incubated at 37°C to prepare hydrogels. MSCs were encapsulated in the hydrogels and cultured to evaluate cellular functions. In addition, hindlimb ischemia models were created by ligating and cutting the femoral artery of nude mice (Balb/c, female, 6-8 weeks old), and blood flow rate was measured after implantation of MSC-encapsulated hydrogels.

*Results: Confocal laser scanning microscopic observation revealed that LLPS structures were formed in hydrogels and tGUPy dissolved in PBS to form microporous structures. Cells were encapsulated in GTH-GVS gel (non-porous) and GTH-GVS-GUPy gel (μCN), showing enhanced cell adhesion, extension, migration and proliferation in μCN (Figure 1b). CLSM observation showed that the porous structures not only improved mass transport but also functioned as a void for cell infiltration. Furthermore, when MSC-encapsulated μCN hydrogels were administered to ischemic models, blood flow was recovered due to enhanced graft survival via material-host communication through the μCN structures (Figure 1c).

*Conclusion/Significance: We demonstrate that porous injectable hydrogels can enhance material-tissue communication with biological signals and cells through μCN structures. This approach is useful as a cell-delivering method to improve the therapeutic efficacy of cell transplantation therapy.

338 - Reshaping Hydrogel Scaffolds: Microporous Annealed Crescent (MAC) Particles For Enhanced Tissue Integration And Cell Delivery

R. Tang, L. Shang, P. Scumpia, D. Di Carlo

University of California, Los Angeles, Los Angeles, CA

*Purpose/Objectives: Traditional bulk hydrogels that are injected to scaffold tissue regeneration in vivo often hinder cell infiltration and expansion due to their densely woven structure. As an alternative, granular hydrogels comprising packed microparticles have emerged as a new class of biomaterial, providing necessary mechanical strength and maintaining a microporous environment favorable for cell migration and nutrient transport. Yet, the predominant use of spherical particles fundamentally limits scaffold porosity, potentially restraining the level of cell infiltration possible which could enable regenerative medicine and vaccine technologies. To overcome this limitation, we developed a novel microporous annealed crescent-shaped (MAC) particle scaffold design to systematically increase the overall void volume, thereby enhancing cellular infiltration without relying on external biochemical factors.

*Methodology: Computational simulations were initially conducted to assess the impact of particle shape on void volume fraction and pore interconnectivity in random assemblies of packed particles. Crescent-shaped particles were then fabricated using a flow focusing microfluidic device based on aqueous two-phase separation. These hydrogel particles were then annealed into scaffolds through a thiol-ene reaction, followed by pore analysis through negative staining and confocal Z-stack scanning. Scaffold rheology and injectability were examined using a rheometer and an extrusion force measurement system. Cell expansion and proliferation within the scaffold was first observed when loading mesenchymal stem cells (MSCs) by confocal microscopy in vitro. Finally, following subcutaneous injection of hydrogel scaffolds overall cell infiltration along with characterization of cell types was performed in vivo.

*Results: Computational models showed that the crescent-shaped particle assemblies offered increased porosity and pore interconnectivity. Furthermore, in our manufacturing system we demonstrated controllable cavity size via fine-tuning of precursor solution concentrations. Scaffold pore characterization revealed tunable structural features, where we optimized for the highest porosity and pore anisotropy to provide maximum directional cues. Rheology and extrusion force measurements displayed higher viscosity for crescent particles and clinically relevant injectability of MAC scaffolds. In vitro, MSCs encapsulated in MAC scaffolds displayed extensive cellular organization and distinct morphological differences, with an increase of 66% of total cell volume. Metrics of colocalization and cell-cell interaction surpassed spherical formulations by 48% over two weeks. Lastly, 2.8 fold greater infiltration of cells into the MAC scaffold were observed by flow cytometry following one week of subcutaneous implantation. Furthermore, histological analysis displayed more uniform and uninhibited invasion of cells; notably a 2.6 fold increase in myofibroblasts and 2.7 fold increase in leukocytes.

*Conclusion/Significance: Our findings highlighted the prominent impact of building block particle shape and negative space on optimizing interstitial pore features of granular scaffolds, which led to enhanced cell expansion and invasion without relying on external stimulants. These findings shed light on possibilities for improving microporous annealed particle scaffolds with the potential for superior functionalities in regenerative medicine, immunomodulation, and cell delivery systems.

341 - Not All Extracellular Vesicles Are Made Equal: Comparison Of Matrix Binding And Media Vesicles For Hard Tissue Regeneration

G. Anghileri¹, A. Jalal¹, W. DeVoogt², T. Hyden-Shepherd¹, C. S. Seinen², B. Peacock³, S. Mebarek⁴, P. Vader², I. Martin-Fabiani¹, O. G. Davies¹

¹ Loughborough University, Loughborough, United Kingdom,

² UMC Utrecht, Utrecht, Netherlands,

³ NanoFCM, Nottingham, United Kingdom,

⁴ Claude Bernard University Lyon 1, Lyon, France

*Purpose/Objectives: Extracellular vesicles (EVs) encompass a range of cell-derived nanoparticles, including exosomes and microvesicles. We and others previously demonstrated that EVs can drive regenerative events in hard tissues. However, the EV preparations applied have been highly heterogeneous, with no consensus on selecting the most potent pro-mineralising fractions. Matrix-bound vesicles (MBVs) associate with early osteoid and function as an environmental niche for early mineral nucleation. However, MBVs remain insufficiently characterised when compared with vesicles obtained from cell culture medium (media vesicles, MVs) and their comparative regenerative potential remains largely undescribed. This study provides a detailed comparison of the bio-composition and relative pro-mineralisation capacity of MBVs and MVs to identify a potent EV source for hard tissue regeneration.

*Methodology: Bone marrow MSCs were cultured in osteogenic medium. MVs and MBVs were isolated from the conditioned medium and ECM, respectively, at days 7, 9 and 14. MVs and MBVs were characterised by nanoparticle tracking analysis (NTA), nano-flow cytometry (NanoFCM), western blotting, alkaline phosphatase (ALP) assay, dSTORM super resolution microscopy, transmission electron microscopy (TEM) and fourier transform infrared spectroscopy (FTIR). The relative capacity for MBVs and MVs to drive mineralisation was assessed in MSC cultures over a period of 28 days. Finally, ALP⁺ MBVs were selected using immunoprecipitation and their pro-mineralisation potential was compared against unsorted MBVs.

*Results: NTA and NanoFCM revealed higher concentrations of MBVs than MVs throughout cellular mineralisation. MBV concentration increased with the status of cell culture mineralisation. No significant differences in particle size were observed between MBVs and MVs. Increased ALP activity was observed for MBVs when compared to MVs, increasing with the mineralisation status of MSC cultures (p<0.5). Western blotting revealed expression of both pro-mineralisation markers (ALP, Annexins A2 and A5, Pit1) and exosome markers (CD9, CD81, ALIX, TSG101) in MBVs and MVs. Super-resolution microscopy revealed for the first-time co-expression of ALP with calcium channel Annexin A2 and exosome associated tetraspanin proteins (CD9, CD63 and CD81) at the single MBV level, increasing as mineralisation advanced (p<0.05), with significantly higher co-expression in MBVs (p<0.05). This increase in proteins associated with mineralisation mirrored the pattern of electron-dense material observed in MBVs and MVs via TEM which was validated via NanoFCM. FTIR confirmed the competency of MBVs to generate de novo hydroxyapatite. The addition of MBVs to MSC cultures significantly enhanced mineralisation over 28 days when compared to MVs. Immuno-isolation of ALP⁺ MBVs was validated via western blotting and super-resolution microscopy. ALP⁺ MBVs co-expressed proteins associated with calcium internalisation (ANXA5, ANXA2) and exosome markers (CD9, CD63, CD81) and further enhanced mineralisation in a dose dependent manner when delivered to MSC cultures.

*Conclusion/Significance: Through the application of advanced imaging we demonstrated that MBVs were enriched in ALP and calcium channelling proteins required for mineralisation. We show for the first time that these markers of mineralisation co-localise at the single vesicle level with tetraspanins associated with exosomal biogenesis. Lastly, MBVs were comparatively enriched in hydroxyapatite and able to significantly enhance mineralisation in MSC cultures. MBV mineralisation potential was observed to correlate with ALP expression.

342 - Intravenous Delivery Of Mesenchymal Stem Cells-derived Extracellular Vesicles (msc-evs) As A Potential Treatment For Tendon And Cartilage Degeneration In Type Ii Diabetes (t2d)

Z. Shamim¹, O. Maged¹, S. Tan¹, N. Aizah¹, Q. Daniel Looi², J. Foo³, T. Kamarul¹

¹ Universiti Malaya, Kuala Lumpur, Malaysia,

² My CytoHealth Sdn. Bhd., Kuala Lumpur, Malaysia,

³ Taylor’s University, Selangor, Malaysia

*Purpose/Objectives: Many preclinical and clinical studies have shown a therapeutic benefit of MSC in diabetes complications due to its secreted biological factors, i.e. extracellular vesicles(MSC-EVs). However, to date, none have reported the potential effects of intravenous delivery of MSC-EVs in musculoskeletal disorders. This study aimed to determine in an HFD and STZ-induced T2D rat model, the effect of T2D on tendon, articular cartilage and subchondral bone; and subsequently to determine the effects of intravenous MSC-EVs administration in tendon and cartilage degeneration in T2D rats.

*Methodology: Sprague Dawley(SD, n=15, IACC number:UPM/IACUC/AUP-R047/2020) rats were used; three rats were fed with a normal diet(G1, as control rats), whereas the other 12 rats were induced to be T2D using a high-fat diet(HFD) followed by 60 mg/kg of STZ injection and 120mg/kg of nicotinamide. At week 8, three different doses of MSC-EVs(G3:100ug or 0.25 mg/kg bw, G4:200ug or 0.50 mg/kg bw, and G5:400ug or 1.00 mg/kg bw), were administered intravenously in a 3-day interval until week 12. The oral glucose tolerance test was performed to confirm the T2D status of all the experimental rats, before MSC-EVs intravenous administration to the T2D rats(n=3 for each dose). Three T2D rats were used as a negative control(G2), where no MSC-EVs were administered. All rats were euthanized by week 12, and all rat knees were harvested and fixed in formalin for histopathology analysis of subchondral bone and cartilage. Achilles tendon(AT) and supraspinatus(Ssp) tendons were collected for histopathology analysis, total collagen quantification and biomechanical testing.

*Results: In this study, T2D significantly increased the total cell number in AT and Ssp tendons. Besides, collagen fibre crimping in was also apparent in G2, with a significant increase in total collagen content(in Ssp tendons) compared to G1. However, no significant changes were observed in the biomechanical properties of the AT in G2 compared to G1. MSC-EVs treatment significantly reduced the number of rounded cells and total cell number in both AT and Ssp tendons, in addition to restoring the histological microscopic appearance of both tendons closer to that of G1. No significant differences were observed in the total collagen content in MSC-EVs-treated groups. In X-ray microscope(XRM) imaging analysis, a significant difference in the bone volume(BV, mm³) was observed only in the non-load-bearing region in the distal femur; no significant differences were observed in the other bone morphometric measurements between load-bearing and non-load-bearing regions in both proximal tibia and distal femur. In histology analysis, the modified Mankin scores were significantly higher in G2 compared to G1. A significant increase in the cartilage thickness was observed in G5 compared to G1, G3 and G4.

*Conclusion/Significance: In conclusion, T2D disrupts the histological and cellular characteristics in tendon and cartilage. Intravenous delivery of MSC-EVs in T2D rats restored the histological features to that closer to a normal tendon, reduced the cellularity of the tendons and improved the cartilage thickness. Future studies with a more comprehensive study design, with a larger sample size and longer study duration shall be conducted, based on the findings reported in this study.

343 - High-performance Hydrogel-encapsulated Engineered Exosomes For Supporting Endoplasmic Reticulum Homeostasis And Boosting Diabetic Bone Regeneration

Y. Liu, X. Jiang

Shanghai Jiao Tong University, Shanghai, China

*Purpose/Objectives: Currently, the regeneration of bone defects in diabetic patients still presents significant challenges. Inspired by the discovery that the endoplasmic reticulum is under excessive stress and dysfunction due to hyperglycemia, leading to osteogenesis disorder, a new type of engineered exosomes is proposed to regulate the homeostasis of the endoplasmic reticulum and repair the regenerative function of MSCs. Moreover, the potential therapeutic mechanism of exosomes has been clarified. To enhance the therapeutic effect of the engineered exosomes, a high-performance hydrogel with self-healing, bioadhesive, and exosome-binding properties was developed to encapsulate the engineered exosomes for in vivo application.

*Methodology: The correlation between endoplasmic reticulum dysfunction and osteogenesis disorder was studied using histological staining and immunofluorescence. The appropriate concentration of the small molecule Sep was optimized to regulate endoplasmic reticulum homeostasis under the guidance of osteogenic differentiation effect. The Sep-loaded engineered exosomes were constructed using intermittent ultrasound technology and characterized using TEM, DLS, and Western blot analysis. The therapeutic effect and related mechanism of the engineered exosomes were studied using EdU staining, flow cytometry, RT-PCR, Western blot, and immunofluorescence. The introduction of multifunctional dynamic reversible cross-linking bonds was utilized to fabricate a high-performance hydrogel. The hydrogel was characterized through NMR, FTIR, SEM, rheological experiments, and mechanical tests. Finally, the impact of the engineered exosomes/high-performance hydrogel treatment system on promoting bone regeneration in vivo was evaluated using a femoral defect model in diabetic rats.

*Results: 1. Osteogenesis disorder in a hyperglycemic microenvironment is accompanied by high expression of genes and proteins related to endoplasmic reticulum dysfunction. Small molecule Sep can effectively regulate endoplasmic reticulum homeostasis and promote the osteogenic differentiation of MSCs. 2. The engineered exosomes carrying Sep were successfully developed. Engineered exosomes can enhance cell activity, reduce cell apoptosis, restore endoplasmic reticulum homeostasis, and facilitate cell osteogenic differentiation. MSC-derived exosomes can transfer SHP2 to recipient cells, activate mitophagy, improve mitochondrial quality, reduce cell ROS production, and assist in maintaining endoplasmic reticulum homeostasis. The exosomes with SHP2 knockdown showed impaired therapeutic effects. 3. A high-performance hydrogel has been successfully synthesized, with multiple functions including injectability, self-healing, bioadhesion, and exosomes-binding. 4. Implanting the engineered exosomes/high-performance hydrogel therapy system into the femoral defect model of diabetic rats effectively maintained endoplasmic reticulum homeostasis and significantly promoted the formation of new bone.

*Conclusion/Significance: The homeostasis of the endoplasmic reticulum is closely linked to osteogenic differentiation. The user-friendly therapeutic system, comprising engineered exosomes and a high-performance hydrogel, holds potential for clinical application and may further inspire the development of innovative treatments for diabetic bone diseases. Furthermore, the versatility of this strategy extends beyond diabetic bone regeneration and holds promise for tissue regeneration in other related contexts.

359 - Development Of Hydrogels For Sequential, Affinity-Controlled Delivery Of Angiogenic Growth Factors

J. E. Svendsen, C. Asnes, M. Ford, H. Hochstatter, S. Lester, R. E. Guldberg, M. H. Hettiaratchi

University of Oregon, Eugene, OR

*Purpose/Objectives: Angiogenesis, the growth of new vessels from mature vasculature, requires the temporally regulated presentation of many proteins. Disruptions in this protein sequence caused by injury severity and disease can result in poor healing and impaired tissue function. The objective of this research was to develop tunable biomaterials capable of phased protein delivery to enhance angiogenesis. To achieve this objective, we integrated specific, reversible affinity-based interactions between small protein domains called affibodies and angiogenic proteins to temporally control protein delivery from hydrogels (Fig.1A). We will sequentially deliver three well-characterized angiogenic proteins from our biomaterial: vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and platelet-derived growth factor (PDGF). We expect this approach will recapitulate the natural, staggered presentation of proteins observed during angiogenesis and enhance vascular network formation.

*Methodology: A yeast surface display library of 100 million affibodies underwent magnetic- and fluorescence-activated cell sorting to select for variants with affinity towards VEGF, FGF-2, or PDGF. Yeast displaying protein-specific affibodies were incubated with 10-10,000nM of VEGF, FGF-2, or PDGF to determine dissociation constants (K_D) between affibodies and growth factors (Fig.1B). Affibodies were appended with an N-terminal adjacent hexa-histidine tag and cysteine, cloned into pet28b+ plasmids, and transformed into E. coli. Soluble affibodies were purified by metal affinity and size exclusion chromatography. Affibodies were conjugated into 5% (w/v) polyethylene glycol-maleimide (4-arm PEG-mal, 20kDa) hydrogels and crosslinked with dithiothreitol. Hydrogels were loaded with growth factors at a 500:1 affibody:growth factor ratio. Protein release at 37 °C over 7 days was quantified using enzyme-linked immunosorbent assays. Microvascular fragments (MVFs) were harvested from 6-month-old Lewis rat epididymal fat pads and seeded in 0.3% (w/v) collagen hydrogels at 20,000 fragment/mL in DMEM/F12 media. MVFs received 50nM VEGF, FGF-2, and/or PDGF at day 3 (“codelivery”) or 50nM VEGF, FGF-2, and/or PDGF at day 3, 4, and 5 (“sequential”). After seven days, MVFs were fixed, stained with lectin, and imaged. Each confocal z-stack was 3D median filtered and deconvolved before volumetric segmentation and morphological assessment to measure branching and total network length (Amira). Data were normalized to MVFs without growth factors.

*Results: Three VEGF-, one PDGF-, and three FGF-2-specific affibodies were discovered using yeast surface display, with dissociation constants spanning four orders magnitude (3.07nM-6470nM; Fig.1B). Affibody-conjugated hydrogels encapsulated 85% of loaded growth factors and released less protein as affinity strength increased, demonstrating an inverse relationship between affinity strength and protein release (Fig.1C). Sequential delivery of VEGF followed by FGF-2 increased MVF network length compared to simultaneous codelivery of VEGF and FGF-2 (Fig.1D). Fig.1E depicts an MVF vascular network with branch points highlighted.

*Conclusion/Significance: We have demonstrated that VEGF-, FGF-2-, and PDGF-specific affibodies can tune growth factor release from hydrogels and that sequential protein delivery enhances vascular network formation compared to simultaneous codelivery. In the future, we will investigate additional sequences and timing of protein delivery before tuning our hydrogel to provide this delivery sequence. Overall, our work presents a novel, tunable protein delivery platform with the potential for enhancing angiogenesis.

361 - Beyond Batch Variability: Microfluidic Solutions For Controlled Therapeutic Nano-delivery

S. Gimondi, D. Mendanha, R. L. Reis, H. Ferreira, N. Neves

3B’s Research Group, Barco, Guimarães, Portugal

*Purpose/Objectives: One of the main limitations in the nanomedicine field lies in the lack of control over nanoparticle (NP) synthesis, resulting in the suboptimal regulation of NP features and batch-to-batch variability.The significance of addressing this issue is underscored by the potential impact on the effectiveness of nanoformulations in medical applications as drug delivery carriers. The inability to precisely control NP characteristics compromises the delivery of therapeutic agents. This translates into a substantial roadblock in the development of clinically viable nanoformulations, hindering their ability to progress beyond promising preclinical results and limiting their potential contribution to advancements in medical treatment.

*Methodology: To tackle this critical problem, our approach has been dedicated to achieving controlled NP synthesis. Over the past years, our research efforts have been concentrated on developing a solution enabling the fine-tuning of NP sizes. To realize this objective, we harnessed a micromixer device, demonstrating its efficacy across a spectrum of materials, including polymeric NPs (PLGA-PEG), liposomes, and high molecular weight complexes (Chitosan-Hyaluronic acid).By utilizing the micromixer device, we aimed to bridge the existing gap in achieving reliable and reproducible NP synthesis and consequently drug delivery efficacy. The micromixer chip employed was a passive microfluidic device characterized by a sophisticated herringbone rib channel architecture, enabling rapid mixing of up to three fluid streams on a millisecond scale. At low flow rates, this design induces lamination of the flow streams, diminishing diffusion distances and consequently improving mixing time. Conversely, high flow rates induce swirling in the flow streams, further reducing mixing time. This peculiar feature is strictly related to its geometry, which improves the mixing time and fosters a uniform generation of NPs by enhancing the reactions between building blocks.

*Results: Fine-tuned NPs were successfully obtained and further investigated for their abilities as drug delivery carriers. Our findings reveal that NPs with variations in size as small as 20 nm are internalized through different endocytic pathways, resulting in distinct therapeutic activities. For instance, we observed a size-dependent effect in polymeric NPs when delivering diclofenac during an inflammatory scenario. Size-dependent results were also obtained in the context of anti-cancer treatment, where 50 nm polymeric NPs exhibited higher performances compared to 70 nm NPs. Finally, we have demonstrated the stable and reproducible incorporation of docosahexaenoic acid (DHA) into liposome formulations through microfluidic synthesis. This approach has shown increased efficiency in inducing apoptosis in cancer cells compared to free DHA.

*Conclusion/Significance: Our methodology not only showcases its adaptability across various materials but also highlights its potential for broader applications in the field of nanotechnology and the controlled release of therapeutics.

Through our research, we aspire to provide a transformative solution to the problem of poor control over NP synthesis, contributing to the development of more reliable and effective nano-delivery systems with the potential to revolutionize the clinical translation of nanomedicine.

362 - Effects Of Heparin Hydrogel Sulfation Level On TSG-6 Release And Tissue Response After Supraspinatus Muscle Injury

J. Pearson, J. Mao, J. Temenoff

Georgia Institute of Technology, Atlanta, GA

*Purpose/Objectives: Tumor necrosis factor stimulated gene 6 (TSG-6) is a broadly anti-inflammatory protein that can aid in polarization of macrophages away from pro-inflammatory (M1) and towards anti-inflammatory phenotypes (M2). Its actions can be altered by complexing with glycosaminoglycans. In particular, fully sulfated heparin (Hep) potentiates the anti-plasmin effects of TSG-6 compared to desulfated heparin (Hep-), but effects on polarization have not been explored. Rotator cuff tears can cause chronic muscle inflammation and degeneration that increases over time. A prolonged, potentiated anti-inflammatory treatment may improve outcomes. We hypothesized that a sustained delivery of TSG-6 from a Hep system would enhance the anti-inflammatory response following rotator cuff injury through macrophage polarization, leading to improved muscle regeneration in a rat model.

*Methodology: For sustained release systems, 3.4 kDa acryl-poly(ethylene) glycol (PEG)-succinimidyl valerate was conjugated with a peptide, GGVPMSMRGGGK, cleavable by MMPs found in this rotator cuff tear model. Heparin sodium salt (Hep) was treated as previously described by our lab to remove sulfation forming the desulfated heparin derivative (Hep-). Both heparin derivatives were separately conjugated to form Hep and Hep- methacrylamide. The conjugated PEG (15% w/v) and a heparin derivative (Hep or Hep-) were formed into hydrogels by free radical polymerization (ammonium persulfate and N,N,N′,N′-Tetramethyl ethylenediamine). The hydrogels were fragmented into particles through a 40 µm filter. Hep and Hep- systems were injected into the supraspinatus muscle of Sprague Dawley rats after rotator cuff injury. An in vivo imaging system (IVIS) was used to evaluate release of fluorescently tagged TSG-6 from Hep and Hep- systems for 21 days (n=6/group). For in vivo assessment of treatments, inflammatory cell response through flow cytometry (n=8/group, 3 and 7 days) and myogenic response through immunohistochemistry (IHC, n=5/group, 7 and 14 days) were evaluated for three groups: Saline injection (injured control), Hep+TSG-6 (Hep) and Hep-+TSG-6 (Hep-). Statistics were completed with one or two-way ANOVA with posthoc Tukey’s test (significant difference, p<0.05).

*Results: In vivo, similar rates of controlled release of TSG-6 were found from Hep and Hep- for 14+ days, enabling comparison of heparin biomaterial carriers. Hep showed higher overall and type 2a, anti-inflammatory macrophages on day 7 throughout the muscle (Fig 1A-B) and increased embryonic myosin heavy chain (eMHC) positive fibers on day 7 compared to the saline group (Fig 1D-E). Additionally, on day 14, Hep had significantly increased centrally located nuclei (CLN) compared to all groups and within the Hep group from day 7 to 14 (Fig 1C).

*Conclusion/Significance: Release of TSG-6 from a fully sulfated substrate resulted in increased overall anti-inflammatory cellular response and increased metrics of early muscle regeneration (eMHC and CLN) in this muscle injury model. A potential explanation is that the substrate sulfation amount may play a role in modulating TSG-6 activity in muscle. In addition to providing a strategy for localized, controlled delivery of TSG-6 to muscle, these results suggest that key pairings between biochemistry of the biomaterial substrates and their cargo can be further explored to potentiate cargo bioactivity for a wide variety of applications.

364 - Hydrogel Platform For Localized Sustained Delivery Of Clinically Relevant Costimulation Blocking Biologics For Immunomodulation In Allogeneic Cell Transplantation

O. Marrone Mantovani^1,2, C. M. Li^2,3, P. Buchwald², A. A. Tomei^1,2,3

¹ University of Miami, Coral Gables, FL,

² University of Miami Miller School of Medicine, Miami, FL,

³ University of Miami, Miami, FL

*Purpose/Objectives: Transplantation of allogeneic cells such as beta cell replacement therapy for type 1 diabetes and pluripotent stem cell therapies for regenerative medicine, requires chronic systemic immunosuppression for long-term allograft survival. This causes general immunodeficiency and adverse events, which limits the applicability of these therapies. Therefore, we aim to develop implantable IgG-capturing enzymatically degradable polyethylene glycol (PEG) hydrogels to deliver IgG-based immunomodulatory biologics locally in the cell transplant site in a sustained and prolonged manner (>1 month) to prevent allograft allorejection with negligible systemic adverse effects.

*Methodology: We tested the effect of in vitro polymer concentration, hydrogel degradability, and drug-gel binding on the drug release kinetics. For this, we fabricated hydrogels of different compositions including (i) 5% vs. 10% PEGMAL fully degradable hydrogels with passively loaded FITC-IgG; (ii) fully degradable vs. half degradable vs. non-degradable hydrogels with passively loaded FITC-IgG by utilizing crosslinkers with different sensitivities to cleavage by enzymes (MMPs) and PEG:crosslinker ratios; (iii) drug-binding with AF647-MAL and/or FITC-IgG and IgG-binding peptides vs passively loaded hydrogels. We then incubated the hydrogels in enzyme solutions and quantified the drug release kinetics via fluorescence quantification at selected time points. We then computed cumulative and rate of drug release and fitted mathematical models to understand how drug release kinetics is affected by each parameter. Additionally, we investigated in vivo hydrogel degradation by co-implanting fully degradable fluorescently labelled hydrogels in B6 mice and quantifying graft site fluorescence decrease over time as an indication of hydrogel degradation.

*Results: We confirmed tunability of (i) hydrogel degradation based on crosslinker type and (ii) drug release kinetics depending on hydrogel degradability, PEGMAL concentration, and drug-gel conjugation. We found faster rate of FITC-IgG drug release from 5% PEGMAL hydrogels compared to 10% PEGMAL hydrogels, as well as from fully degradable compared to half degradable hydrogels. Additionally, we found that hydrogels with passively loaded FITC-IgG achieve a biphasic first-order burst drug release kinetics, while drug-binding hydrogels with covalently bound AF647-MAL achieve a zero-order constant drug release kinetics. Lastly, we found that in vivo hydrogel degradation occurs after 70 days post implantation.

*Conclusion/Significance: These results suggest that PEG hydrogel degradation can be tuned by varying polymer concentration, crosslinker type/concentration, and drug-gel conjugation to achieve the desired drug release kinetics. Drug-binding degradable hydrogels achieve sustained and constant drug release kinetics, while passively drug loaded hydrogels achieve a biphasic diffusion-driven burst drug release profile characterized by a decrease in rate of drug release over time. Our enzymatically degradable drug-binding PEGMAL hydrogel biomaterial platform could enable the sustained delivery of clinically effective IgG-based immunomodulatory biologics locally within the transplant site to increase safety and efficacy of allogeneic cell transplant-based regenerative medicine therapies.

365 - High-resolution Mapping Of Oxygen Tension In Tissue-engineered Constructs With Ruthenium-complex Nanomicelle Optical Sensors

K. J. Schilling, C. E. Schutt

Oregon Health & Science University, Portland, OR

*Purpose/Objectives: Characterizing oxygenation within engineered tissue constructs is key to understanding cell behavior and maintaining cell viability for applications in tissue regeneration. In order to report oxygen tension (pO₂) with high spatiotemporal resolution and high sensitivity in biologically-representative tissue culture models, two-photon phosphorescence lifetime microscopy (2P-PLIM) with oxygen-sensing optical probes can be performed. [Ru(dpp)₃]²⁺ (Ru) is an excellent oxygen sensing probe as it is two-photon excitable and possesses a phosphorescence lifetime in the microsecond regime. However, Ru is hydrophobic which limits its potential in biologically-representative tissue culture models. The objective of our study is to create hydrophilic Ru-complex micelles and incorporate these micelle sensors within 3D matrices containing multicellular spheroids to report pO₂ in real-time.

*Methodology: Ru-complex nanomicelles were prepared by complexing [Ru(dpp)₃]Cl₂ with poloxamer-407 via ultrasonication (Fig. 1A) and purified via ultracentrifugation. Characterization of the micelles was performed via nanoparticle tracking analyses (NTA) as well as transmission electron microscopy (TEM). Calibration of the Ru-complex nanomicelles was performed by measuring the phosphorescence lifetimes of the nanomicelles in sealed solutions of known temperature, pH, and oxygen content. To report pO₂ within in vitro tissue models, the water-soluble nanomicelles were incorporated in the media above 1 mm thick collagen hydrogels embedded with breast epithelial (MCF10A) spheroids located in the center of the gel. 2P-PLIM was performed to collect lifetime images of the spheroids and surrounding matrices. Using the established calibration curve, these lifetime images were converted to pO₂ images to assess oxygen distribution.

*Results: Characterization of the Ru-complex micelles showed that the micelles had a mean diameter of 124 nm as determined via NTA and TEM. Ru-complex nanomicelles added to media were visualized within and surrounding spheroids for over 1 week of culture with no perceptible disruption of collagen structure. MCF10A spheroid viability analyses showed that mean viability of about 85% was achieved at effective Ru concentrations of ∼7.5 µM in the media which was used for all spheroid studies. Calibration of the oxygen sensor at physiological and acidic conditions showed a two-phase lag decay fit curve of the data and no significant differences between the curves. Three-dimensional oxygen reporting was performed on embedded spheroids after 1 day of culture and showed heterogenous pO₂ within and surrounding spheroids at various depths. Longitudinal analyses of pO₂ within embedded MCF10A spheroids showed that at 2- and 24-hours post-embedding, the core remained hypoxic with ∼8 mmHg pO₂ present (∼1% O₂), whereas after 72 hours, the pO₂ within the core had equilibrated to similar levels measured in the surrounding matrix. pO₂ image slices of the spheroids showed a gradient of pO₂ along the radial direction of the spheroid (Fig. 1B). pO₂ measurements were further supported by Hypoxyprobe staining.

*Conclusion/Significance: In conclusion, utilizing Ru-complex micelles with 2P-PLIM, we demonstrated the ability to report oxygen tension with high spatiotemporal resolution in 3D matrix-embedded spheroid cultures. This method will allow future developments and expansion of the platform to determine pO₂ gradients and sites of hypoxia within additional 3D tissue-engineered models of multiple cell types.

366 - Noninvasive, Real-time Multimodality Imaging Reveals How Oxygen Releasing Scaffolds Enhance Bone Fracture Healing In Vivo

Y. Ren, N. Sarkar, J. Zhao, A. L. Horenberg, W. L. Grayson, A. P. Pathak

Johns Hopkins University School of Medicine, Baltimore, MD

*Purpose/Objectives: Tissue-engineered oxygen-releasing scaffolds have been shown to facilitate calvarial bone healing [1-2], but our understanding of the underlying mechanism as well as the dynamic, spatiotemporal changes they induce in the bone healing microenvironment has been limited. This has been primarily due to the technical challenges associated with noninvasively acquiring quantitative readouts of angiogenesis and osteogenesis in real-time, in vivo [3]. In this abstract, we demonstrate the feasibility of quantifying the differential effects of 3D-printed control (PCL) or oxygen-releasing scaffolds (PCL-CPO) with multimodality in vivo imaging in a calvarial model of bone fracture healing. Finally, we demonstrate that oxygen-releasing scaffolds enhance bone fracture healing via sustained angiogenesis and osteogenesis in vivo.

*Methodology: Two groups of ten 10-week-old female C57BL/6 mice were anesthetized by intraperitoneal administration of ketamine (80 mg/kg) and xylazine (8 mg/kg). A 4-mm calvarial defect was created on the parietal bone of each mouse. PCL or PCL-CPO scaffolds (4 mm diameter × 0.4 mm thick) were implanted into the calvarial defects, and 5 mm glass coverslips used to seal the defects and permit multimodality in vivo optical imaging consisting of: Intrinsic Optical Signal (IOS) and Laser Speckle Contrast (LSC). These contrast mechanisms enabled the characterizations of structural and functional vascular changes during bone healing at high spatial (5 μm) and temporal (200 ms) resolution and the derivation of intravascular oxygen saturation (SO₂) [4]. Images were acquired weekly, and the vasculature was digitally segmented and processed using customized ImageJ and MATLAB^® algorithms. On post-surgery day 56, animals were euthanized and their skulls harvested. 3D micro-CT images of the skulls were then acquired at 15 μm spatial resolution.

*Results: All cranial windows maintained their clarity over eight weeks of in vivo imaging. We found that angiogenic vessels became visible during the 2^nd week of bone healing and tended to wrap around the struts of the PCL scaffolds, while fewer vessels were found in the CPO group at this stage. The CPO group at later healing stages exhibited greater vascular length, volume, and density (Fig. 1e, f, h), while PCL scaffold group exhibited larger diameter (Fig. 1g). Blood flow within angiogenic vessels of the PCL scaffold was higher, which decreased during the first 3 weeks of healing and increased towards Week 7. Lower blood flow was observed in CPO scaffold throughout, while two groups exhibited similar blood flow levels at the end of healing on week 8 (Fig. 1i). Both groups exhibited similar SO₂ through the entire healing cascade (Fig. 1j). Finally, although no other osteoinductive factors or cells were included in the scaffolds, micro-CT images revealed significantly improved bone healing in the CPO implanted defects (41.4%) compared to that in the PCL implanted defects (22.6%) (Fig. 1k-l).

*Conclusion/Significance: Our multimodality in vivo imaging data demonstrated that the oxygen-releasing PCL-CPO scaffold enhanced angiogenesis and osteogenesis compared to PCL scaffold, resulting in better bone fracture healing. This imaging approach enables non-invasive, real-time in vivo assessments of the impact of novel tissue engineering constructs on bone fracture healing.

367 - Design And Development Of An Aptamer-Based Biosensor For Monitoring Cell Seeding In Tissue Engineering Applications

R. Somoza, T. Av, M. A. Fuentes, J. F. Welter

Case Western Reserve university, Cleveland, OH

*Purpose/Objectives: A major goal of our group is to transform artisanal tissue engineering (TE) into tissue manufacturing. To this end, as many steps as possible should be converted to feedback-controlled automated processes. Cell seeding onto scaffolds is a critical step in many TE applications, but it is usually done “open loop” with no immediate feedback on cell coverage of the scaffolds. Destructive tests like histology waste the construct, and are, thus, not useful in this context. Aptamers (single-stranded DNA or RNA oligonucleotides generated by a process termed SELEX (systematic evolution of ligand by exponential enrichment)) are designed to bind to specific target molecules or whole cells with high affinity. Aptamers change conformation after binding to their target, which can be used for real-time non-invasive, imaging-based measurements. In this study we show that appropriate aptamers can be used to provide a cell-seeding feedback signal.

*Methodology: We designed an aptamer-based molecular beacon specific to MDA-MB231 cancer cells. The molecular beacon contains a 3’ fluorescent FAM tag with a BHQ-1 black-hole quencher in close proximity (in the absence of target), and a biotinTEG for anchorage via streptavidin. Upon cell binding the aptamer converts to its open conformation, where the fluorophore and the quencher do not interact. The recovered fluorescent signal is used for detection of cell binding to the scaffold. Scaffolds were functionalized with the aptamer, 24h later cells were seeded and fluorescence was quantified every 15 minutes for 24 hrs. Cell counts from live/dead assays were compared to the fluorescent signal from the aptamer for validation. A streptavidin coated plate was used as a proof of principle to test the ability of the molecular beacon to produce a signal upon cell binding.

*Results: The molecular beacon aptamer attached to streptavidin coated plates produced fluorescence signal after cells were seeded at different densities. The signal increased with cell number following a power law. Next, we tested the molecular beacon using collagen scaffolds. The cell-specific aptamer provided a quantifiable fluorescent signal upon the addition of cells. Their presence otherwise had no measurable impact on cell viability by live-dead assays.

*Conclusion/Significance: The novel aptamer-based cell seeding biosensors described here will provide the afferent arm of a real-time feedback loop to be used to automate cell-seeding inside bioreactors. Preliminary results suggest that the aptamers have no impact on cell viability, but longer-term comparative validation studies will be performed. Additionally, the development of more compact data acquisition hardware that will interface with our bioreactor systems will be required and is ongoing.

369 - Stretchable Alginate/GelMA Interpenetrating Network (IPN) Hydrogel Microsprings Based On Coaxial Microfluidic Technique For Skeletal Muscle Tissue Engineering

L. Hu

Southern Medical University, Guangzhou, China

*Purpose/Objectives: The emulation of the natural organization and contractile properties of native stretchable tissues have crucial implications for skeletal muscle tissue engineering (SMTE). Helical structures, ubiquitous in nature and endowed with unique mechanical characteristics, have recently gained significant traction in SMTE applications. However, developing hierarchical hydrogel-based scaffolds that both replicate exceptional mechanical properties and control cellular organization remains challenging.

*Methodology: We present an interpenetrating network (IPN) hydrogel synthesized from alginate and gelatin methacryloyl (GelMA) materials, to fabricate stretchable helical hydrogel microsprings via a coaxial microfluidic chip, showcasing superior elasticity attributed to helical structures.

*Results: The construction of micro- and nanoscale patterned groove/ridge helical structures resulted in hydrogel microsprings capable of directing myoblast alignment and elongation along the longitudinal axis of helical structures in vitro. Furthermore, hydrogel microsprings were applied as injectable micro-scaffolds after cutting into appropriate sizes, affording a mechanical microenvironment for promoting cell infiltration and muscle tissue regeneration in situ in rat models.

*Conclusion/Significance: In summary, we successfully fabricated helical hydrogel microsprings via a coaxial capillary microfluidic system, based on an IPN hydrogel made from a combination of alginate and GelMA materials. These AG IPN hydrogel microsprings showed high stretchability and provided a suitable mechanical microenvironment, which simulated the contraction properties of natural skeletal muscle. Especially, these hydrogel microsprings with micropatterning groove/ridge surfaces microscopically and unique helical structures macroscopically induced higher cellular arrangement and elongation in vitro. Furthermore, it showed that hydrogel helical microsprings significantly promoted cell infiltration and myofiber regeneration in rat models, demonstrating their potential applications in skeletal muscle tissue engineering. Besides, we could blend active ingredients that respond to physicochemical stimuli with the hydrogels, or fabricate cultivation molds with functional devices such as electrical/mechanical stretching devices for the construction of functional hydrogel microsprings. Such microsprings are highly versatile for tissue engineering and would be an excellent choice for applications in a variety of areas.

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372 - Brown Adipogenic Differentiation Of Fibro-adipogenic Progenitors Is Associated With Increased Miro1 Gene Expression And The Formation Of Mitochondria-rich Tunneling Nanotubes

C. M. Mendiondo, D. Leyton-Cifuentes, E. Brey, C. Rathbone

The University of Texas at San Antonio, San Antonio, TX

*Purpose/Objectives: Fibro-adipogenic progenitors (FAPs) reside in between muscle fibers and are involved in skeletal muscle homeostasis and regeneration. In response to injury, FAPs secrete a variety of paracrine factors that support satellite cell-mediated muscle regeneration. Interestingly, research on mesenchymal stem cells (MSCs) supports the notion that FAPs, as a muscle-resident MSC, also enhance mitochondrial content and regeneration of skeletal muscle by transferring mitochondria to satellite cells and myofibers. A recent study demonstrated that FAPs beiged with the beta-3 agonist mirabegron transfer mitochondria to both myoblasts in vitro and myofibers in vivo. Intercellular mitochondrial transfer typically occurs via direct cell-to-cell connections or through indirect means such as extracellular vesicles, but the mechanisms by which FAPs donate mitochondria to myoblasts have not been fully resolved. Furthermore, FAP-mediated mitochondrial transfer in 3D culture environments have received little attention and may provide important insight into the physiological process of mitochondrial transfer in vivo. The objective of the present study is to determine the effect of brown adipogenic differentiation on tunneling nanotube (TNT)-mediated FAP intercellular mitochondrial transfer capacity in 2D and 3D culture.

*Methodology: Muscle precursor cells (MPCs) and FAPs were isolated from the hindlimbs of Lewis rats through protease digestion and differential centrifugation. Following a 24-hour preplate, MPCs and FAPs were expanded separately in standard growth media until passage 1 and passage 3, respectively. FAPs were differentiated in brown adipogenic media for 12 days, and mitochondrial transfer gene expression was examined by RT-PCR. MPCs were expanded in Matrigel-coated wells of a 48-well plate until 50% confluent, at which point they were subjected to H2O2-induced oxidative stress and treated with MitoView Fix 640-labeled brown FAPs suspended in growth media or in 3D 100 uL fibrin gels. After 12 hours of co-culture, MPC-FAP co-cultures were fixed in 4% paraformaldehyde and immunostained with BODIPY and DAPI to visualize lipids and nuclei, respectively, with a Leica TCS SP8 confocal microscope.

*Results: Brown-induced FAPs have numerous TNT-like extensions that are rich in mitochondria - this is especially the case in 3D fibrin gels (Figure 1A). Importantly, FAPs co-cultured with H2O2-stressed MPCs in 2D appear to have transferred mitochondria to MPCs via TNTs (Figure 1B, Figure 1C). Preliminary RT-PCR data indicates that FAPs cultured in brown adipogenic media have the potential for increased gene expression of mitochondrial Rho-GTPase 1 (MIRO1), a calcium-sensitive adaptor protein that promotes intercellular mitochondrial transfer via TNTs (Figure 1D).

*Conclusion/Significance: Ongoing and future studies will (1) verify and quantify FAP-MPC mitochondrial transfer in fibrin gels by cell tracing and flow cytometry and (2) examine the effects of FAP-mediated mitochondrial transfer on regenerative outcomes in tissue-engineered skeletal muscle. A mechanistic understanding of FAP mitochondrial transfer and its role in muscle regeneration may be harnessed to develop novel therapies for musculoskeletal injury and disease.

373 - Biosponge-encased Placental Stem Cells Enhance Regeneration And Diminish Fibrosis Following Volumetric Muscle Loss

D. L. Johnson¹, M. Ridolfo¹, J. Tadiwala¹, A. Jain¹, R. Mueller¹, M. Chermack¹, J. Brockhouse¹, J. Robinson¹, S. Shringarpure¹, K. Bertram², K. Garg¹

¹ Saint Louis University, Saint Louis, MO,

² Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC

*Purpose/Objectives: Volumetric muscle loss (VML) causes irreversible mass and function deficits, often resulting in permanent disability. Mesenchymal stromal cells (MSC) and their secretome can improve muscle regeneration. However, MSCs generally have a brief lifespan and do not persist in injured tissues beyond the first week following transplantation. Human placenta-derived stem cells (hPDSCs), which share cell surface markers with MSCs, have better proliferation and differentiation capabilities due to their fetal origin. We previously developed a biosponge (BS) composed of extracellular matrix (ECM) proteins that enhanced muscle regeneration after VML [1, 2]. This work aimed to co-apply BS therapy and hPDSCs to further improve functional muscle regeneration post-VML.

*Methodology: Human PDSCs (hPDSC) were encapsulated in BS and subjected to hypoxic (3% O₂) preconditioning to mitigate low cell survival post-implantation. A VML defect was created by removing ∼20% of the tibialis anterior (TA) muscle’s mass in male Lewis rats unilaterally. A subset of animals underwent no repair (NR). Others were treated with either an acellular BS, normoxia-conditioned BS encapsulated hPDSCs (N-PDSC-BS), or hypoxia-conditioned BS encapsulated hPDSCs (H-PDSC-BS) (n=7-8/group). Peak isometric torque was measured, and the muscle tissue was harvested for histological and biochemical analysis 28 days post-injury. All data were analyzed using one- or two-way ANOVA with Fisher’s LSD post-hoc tests (“**” indicates one-way ANOVA p<0.0001).

*Results: Histological analysis revealed that H-PDSC-BS group retained more cells (Ku80⁺) within the BS compared to N-PDSC-BS** (Fig. 1A). The H-PDSC-BS and BS groups had significantly higher muscle mass compared to the NR group** (Fig. 1B). Unexpectedly, no improvements in torque were observed over the NR group**. The BS implanted without PDSCs showed extensive remodeling and cellular infiltration, while the BS with H- or N-PDSCs remained largely intact. The myofiber size distribution revealed that both H- and N-PDSC groups had higher percentages of smaller (<500 µm²) fibers relative to other experimental groups (Fig. 1C; Interaction: p<0.0001). Whole muscle lysate protein analysis revealed that both H- and N-PDSCs significantly increased early myogenic marker (MyoD⁺) expression relative to the other treatment groups** (Fig. 1D). The H- and N-PDSCs also showed higher protein expression of both pro-inflammatory** (iNOS⁺) and anti-inflammatory** (Arginase⁺) markers, suggesting a heightened immune response. All BS groups resulted in a higher vessel (vWF⁺) percentage area than the NR group, indicative of angiogenesis**. Additionally, both H- and N-PDSCs reduced the fibrotic tissue accumulation (WGA⁺) relative to the other treatment groups (Fig.1E; ANOVA: p=0.0031).

*Conclusion/Significance: Overall, these results suggest that hPDSCs can be transplanted in a rat VML defect via a BS carrier without adverse consequences. Further, these hPDSCs persisted until day 28. Both H- and N-PDSCs enhanced the percentage of small myofibers (<500 µm²), which are indicative of regenerating myofibers. In support, both H- and N-PDSCs also enhanced myogenic protein expression (i.e., MyoD) and reduced fibrosis. BS with or without PDSCs increased vascularity in the VML defect. Taken together, these results suggest that PDSCs augment the regenerative effect of BS. The combination therapy of PDSCs and BS emerges as a promising strategy that is worthy of further investigation.

374 - Pulsed-electromagnetic Field Stimulation And Mechanical Stimulation For Enhancing Bovine Myoblast Myogenesis

C. Deelkens

KU Leuven, Kortrijk, Belgium

*Purpose/Objectives: Several challenges account for the slow progress of cultured meat production. Primary bovine muscle stem cells have limited cell expansion potential, lose their myogenic differentiation capacity to myotubes after prolonged expansion and the resulting myotubes are immature. The finite pool of myoblasts and the absence of well-structured myofibers hinder the efficient and scalable production of cultured meat. To enhance the myogenic differentiation of primary-derived myosatellite cells, we explore the effect of stimulation on cultured bovine myoblasts during cell expansion and myotube formation.

*Methodology: We evaluate the effect of two different stimulation techniques: pulsed-electromagnetic field stimulation and mechanical stimulation on bovine myoblasts during proliferation as well as differentiation. We investigate the impact of stimulation on the cell number, fusion capacity, cell alignment, myotube diameter, polynucleation, metabolic activity and sarcomere component expression during myogenesis in 2D as well as 3D. In addition, for 3D, mechanical characterization is performed.

*Results: Continuous and intermitted pulsed electromagnetic field stimulation is applied to high- and low-fusion performing bovine myoblasts in 2D. The results demonstrate that the stimulated myoblasts have an increased fusion capacity compared to statically cultured cells, with enhanced myotube formation in low-fusion myoblasts due to the stimulation. Moreover, an increased metabolic activity (7 ± 3%) is observed in the 2D cultured myoblasts after stimulation, indicating a positive impact on cellular function. Mechanical stimulation is tested during the differentiation of 3D muscle constructs of myoblast donors with low-fusion characteristics. The stimulation results in a decrease in Young’s modulus (43 ± 4% kPa), indicating that the stimulated tissues are less stiff. Additionally, the stimulation induces an increase in metabolic activity (75 ± 10%) and an elevated fusion capacity, collectively suggesting improved myoblast myogenesis.

*Conclusion/Significance: This research highlights the potential of stimulating bovine myoblasts to enhance myogenesis, thereby increasing or partially restoring the myogenic fusion capacity. These findings contribute toward overcoming the challenges of immature bovine myoblasts, paving the way for the development of muscle constructs with application to cultured meat production.

375 - Mantarray 3D Engineered Muscle Tissue Platform Demonstrates Clinically-Relevant Disease Stratification Of An In Vitro Human Duchenne Muscular Dystrophy Model

S. Luttrell, J. Macadangdang, K. Gray, S. Kharoufeh, C. A. Worthen, G. Luerman, N. Geisse

Curi Bio, Seattle, WA

*Purpose/Objectives: Accurate in vitro modeling of healthy and disease conditions is crucial for assessing the safety and efficacy of novel therapeutics before clinical trials. For cardiac and skeletal muscle diseases, direct evaluation of muscle contraction is a reliable indicator of overall tissue function, however, this often necessitates expensive and low-throughput non-human in vivo models. Animal models may not accurately predict human responses to compound exposure. Conversely, 3D engineered heart tissues (EHTs) from human induced pluripotent stem cells (iPSCs) offer promising opportunities for in vitro disease modeling. However, generating functional, reproducible, and predictive 3D human tissue models is challenging for many investigators.

*Methodology: Here, we have developed a turnkey platform for facile fabrication of 3D muscle organoids that enables label-free, push-button measurements of contractility. The platform ensures highly reproducible tissues from virtually any cell source. Furthermore, it allows for customized stimulation protocols to individual constructs, while simultaneously measuring contractility across 24 tissues in parallel with minimal user input. This approach enables the stratification of healthy and diseased muscle phenotypes and facilitates dose-dependent compound safety and efficacy screening for evaluation of a drug’s effect on contractile output.

*Results: We present a 3D model of Duchenne muscular dystrophy (DMD) that utilizes EHTs formed from an isogenic pair of healthy and diseased cells. DMD tissues display functional deficits consistent with in vivo disease indicators, including reduced contractile force and slowed relaxation kinetics, though beat rate remains unchanged. EHTs remain functional over months in culture and provide a large experimental window to not only study therapeutic effect, but also disease phenotypes that may present at later stages of development and maturity. We show both acute and chronic effects of pharmaceutical compounds in EHTs, including a drug (BMS-986094) that failed clinical trials due to unanticipated cardiotoxicity. This highlights the utility of using 3D engineered heart tissues to identify toxicity and, ultimately, derisk drug development and clinical testing outcomes. Spontaneously beating EHTs were also subjected to in situ stretching, leading to increased levels of cardiac troponin I (cTnI) in the tissue medium, indicating stretch induced injury in the muscle constructs.

*Conclusion/Significance: These data demonstrate a first-and-only commercial platform that integrates individual, fully-customizable, well-based control of electrical stimulation across a 24-well plate of tissues to pace muscle constructs, model force-frequency, and enable exercise regimens or damage protocols. Heart organoids were stretched under load presenting a challenge for therapeutics targeting injury prevention in the myocardium. Stimulation is coupled with automated assessment of 3D muscle contraction, providing an inclusive, medium-throughput platform for studying muscle-related disorders, advancing therapeutic discovery using a modality-agnostic biosystem.

376 - Dissecting Cardiomyocyte-fibroblast Communication During Homeostasis And Cardiac Fibrosis In A Compartmentalized Biomimetic Tissue Model

S. J. DePalma, D. D. Huang, A. C. Vandeven, A. E. Stis, J. Xia, B. M. Baker

University of Michigan, Ann Arbor, MI

*Purpose/Objectives: Cardiac fibroblasts (CFs) maintain cardiac structure and function by synthesizing and remodeling the extracellular matrix (ECM) found throughout the heart [1,2]. Homeostatic maintenance of the ECM required for healthy tissue function depends on CF communication with cardiomyocytes (CMs) [1,2]. During cardiac fibrosis, signals resulting from tissue injury and dysregulated CF-CM communication promote CF differentiation into α-smooth muscle actin expressing (αSMA) myofibroblasts (MFs), which deposit excess ECM that mechanically impairs heart function [3]. Given the challenges involved in parsing the dynamic and multifactorial heterocellular interactions underlying tissue homeostasis versus fibrosis, we engineered a compartmentalized, bilayer cardiac tissues where iPSC-derived CMs and CFs can be cultured on either side of a shared mechanically tunable, synthetic fibrous ECM to study CM-CF communication.

*Methodology: Tissues were fabricated by electrospinning dextran vinyl sulfone (DVS) fibers onto arrays of microfabricated PDMS wells [4]. Matrix stiffness and alignment were controlled by modulating the speed of the collecting surface and amount of photo-crosslinking via UV light exposure, respectively (Fig. 1A) [4,5]. Subsequently, iPSC-CMs were seeded on the top of the fiber matrices and allowed to spread for 3 days. Primary human CFs were then seeded by pipetting the cells into channels below the fiber matrices and inverting the samples for >2 hours such that the CFs adhere to the underside of the matrix (Fig. 1A). Immunostaining for αSMA was conducted to assess CF differentiation to MFs.

*Results: To examine how various fibrous matrix cues impact MF differentiation, CF monocultures were generated on matrices varying in alignment and stiffness and cultured with exogenous TGFβ1 for 7 days (Fig. 1 C). αSMA expression was greatest on stiff (17.3 kPa) aligned matrices compared to soft (0.68 kPa) aligned matrices or both soft/stiff random matrices, suggesting that both matrix alignment and stiffness are important in driving MF differentiation (Fig. 1 D-E). Previous work from our group showed that soft, aligned matrices yield the most functionally mature CM tissues, so we next explored how the presence of CMs on the opposing side of soft, aligned matrices impacts MF differentiation (Fig. 1F) [4,6]. Interestingly, the presence of co-cultured CMs markedly inhibited MF differentiation despite the presence of TGFβ1 (Fig. 1 G-H). MF differentiation was also impaired in CFs cultured on glass below matrix-seeded CMs, suggesting the role of protective paracrine-mediated intercellular communication between CMs to CFs that acts to maintain fibroblast quiescence and tissue homeostasis (Fig. 1I-K).

*Conclusion/Significance: In this work, we developed a microfabricated, biomimetic co-culture platform to examine CM-CF communication underlying tissue homeostasis versus fibrosis. We found that fibrous mechanical cues modulate MF differentiation, in line with previous findings [5,7]. Further, CM-CF paracrine crosstalk inhibited MF differentiation on soft, aligned fibrous matrices that recapitulate the healthy cardiac ECM. Ongoing studies are focused on identifying the operative heterocellular communication pathways via mass spectrometry analysis of the CM-CF secretome.

References: [1] Souders+, Circ Res, 2009. [2] Hall+, J Am Heart Assoc, 2021. [3] Kanisicak O+, Nat Commun, 2016. [4] DePalma+, bioRxiv, 2023. [5] Davidson+, Acta Biomater, 2020. [6] DePalma+, Biomater Sci, 2021. [7] Bugg+, Circ Res, 2020.

378 - ER Stress And Lipid Imbalance Drive Embryonic Cardiomyopathy In A Human Heart Organoid Model Of Pregestational Diabetes

A. Kostina, Y. Lewis-Israeli, M. Abdelhamid, M. Gabalski, A. Kiselev, B. Volmert, H. Lankerd, A. Huang, A. Wasserman, T. Lydic, C. Chan, S. Park, I. Olomu, A. Aguirre

Michigan State University, East Lansing, MI

*Purpose/Objectives: Congenital heart defects (CHD) constitute the most prevalent human birth defect, affecting approximately 1% of all live births. Pregestational diabetes (PGD, defined as diabetes of the mother before and during the first trimester of pregnancy) is one of the substantial non-genetic factors contributing to CHD. Newborns from mothers with PGD have increased risk of CHD (up to 12% versus ∼1% in the healthy population). Our ability to study the etiology of these disorders in humans is greatly limited due to the inaccessibility of human fetal hearts at crucial stages of development. Here, we used an advanced human heart organoid model that recapitulates complex aspects of heart development during the first trimester to model the effects of pregestational diabetes in the human embryonic heart.

*Methodology: Heart organoid culture conditions were modified to accurately reflect reported physiological levels of glucose and insulin in normal and diabetic mothers.

*Results: We observed that heart organoids in diabetic conditions develop pathophysiological hallmarks like those previously reported in mouse and human studies, including ROS-mediated stress and cardiomyocyte hypertrophy, among others. Single-cell RNA-seq analysis revealed cardiac cell type specific-dysfunction affecting cardiomyocyte, epicardial, and proepicardial populations and pointed to potential dysregulation of redox and mitochondrial homeostasis, endoplasmic reticulum function and fatty acid metabolism as essential contributors in the development of PGD-induced CHD. LC-MS metabolomic profiling of PGD heart organoids identified a critical lipid imbalance affecting very long chain fatty acids (VLCFA) indispensable for cardiogenesis. Further study demonstrated increased oxidative stress predominantly localized to the endoplasmic reticulum and associated with non-canonical IRE1-RIDD pathway activation. We found that abnormal IRE1 exoribonuclease activity affected fatty acid desaturase 2 - a key enzyme involved in the biosynthesis of unsaturated fatty acids. This, in turn, affects VLCFA biosynthesis and causes a crucial VLCFA imbalance during early cardiac development. Finally, we demonstrated that the effects of PGD could be reversed to a significant extent using drug interventions targeting either IRE1 or restoring healthy lipid levels within organoids, opening the door to new preventative and therapeutic strategies in humans.

*Conclusion/Significance: In summary, we established the value of a heart organoid model of pregestational diabetes and CHD, and our findings unveiled a novel ROS-induced ER stress mechanism underlying critical VLCFA balance in PGD-CHD. These findings represent a new route for developing future preventative and therapeutic strategies, thus helping to reduce the incidence of CHD across the population and providing proof-of-concept of the utility of organoid models for human disease modeling and drug development.

379 - Instructing Collagen-based Biomaterials And Human Cells To Work Together For Remodeling The Structure Of Vessel Wall For Advanced In Vitro Models

D. Mantovani

Laval University, Quebec, QC, Canada

*Purpose/Objectives: Over the last 50 years, biomaterials, prostheses and implants saved and prolonged the life of millions of humans around the globe. Today, nano-biotechnology, nanomaterials and surface modifications provides a new insight to the current problem of biomaterial complications, and even allows us to envisage strategies for the organ shortage. In this talk, creative strategies for mixing vascular cells and collagen-based materials for physiologically relevant models will be targeted with the overall aim to envisage today how far innovation can bring tomorrow solutions for reparative and regenerative medicine. Collagen gel is a commonly used scaffold in vascular tissue engineering due to its biological properties including a high potential for supporting and guiding vascular cells in the regeneration process. With the aim to regenerate the vascular wall, the approach we deployed consisted in first reproducing the media, which provide the high elastic properties of the vessel wall, thus making it an essential and effective component for blood and nutrients transportation.

*Methodology: Starting from an original method aimed to process collagen and smooth muscle cells (SMCs), we developed an endothelialised two layers collagen cell-based tubular scaffold. The external layer was composed of fibroblasts (FBs) and SMCs seeded within collagen. The middle layer was composed of SMCs seeded within collagen, and endothelial cells (ECs) were culture on the lumen of the construct. The construct was expected to provide vascular tissue remodeling due to cells/cells and cells/matrix interactions and to produce an engineered tissue with hierarchical structure close to that of blood vessel walls. It was also expected to provide a valid in vitro model for further studies of vascular patho-physiology.

*Results: The middle and external layer were mold around a mandrel, directly in the bioreactor chamber. Then, the mandrel was removed and a ECs solution was perfused inside the lumen. The interaction between cells enhanced the matrix remodeling and the properties of the arterial construct resulted strongly improved. This shows that vascular cells tri-culture using collagen gel scaffold is a valid strategy for the regeneration of the vascular tissue.

*Conclusion/Significance: The overall take home message of this talk is aimed to show how nanocoatings, degradable metals and 3D pluri-culture of appropriate material/cell/environment represent the today bottleneck in reparative and regenerative medicine and which are few of the strategies that have to be investigated to push forward innovation in the field, for the benefit of patients and Humans.

References: *S Palladino, A Schwab, F Copes, M D’Este, G Candiani, D Mantovani. Development of a hyaluronic acid—collagen bioink for shear-induced fibers and cells alignment. Biomedical Materials 18 (6), 065017, 2023.

*DB Camasão and D Mantovani. The mechanical characterization of blood vessels and their substitutes in the continuous quest for physiological-relevant performances. A critical review. Materials Today Bio, 10, 100106, 2021.

*DB Camasão, M González-Pérez, S Palladino, M Alonso, JC Rodríguez-Cabello, D Mantovani. Elastin-like recombinamers in collagen-based tubular gels improve cell-mediated remodeling and viscoelastic properties. Biomaterials Science, 8, 12, 3536-3548, 2020.

380 - Tailoring Cell Attachment Via Unconventional Use Of Peptide-functionalized Phase-separated Coating Of 3d Printed Microfiber Scaffolds

K. L. O’Neill, A. Kurtz, I. Liashenko, G. Rocha, P. D. Dalton

University of Oregon - Knight Campus for Accelerating Scientific Impact, Eugene, OR

*Purpose/Objectives: Melt electrowriting (MEW), a precision 3D-printing technique, offers a platform for creating custom scaffolds for tissue engineering (TE), predominantly utilizing poly(ε-caprolactone) (PCL). MEW produces highly porous scaffolds (85%-98% porous) with well-defined and programmable mechanical properties, however cell interactions are directed through non-specific protein adsorption. Our objective is to alter the cell-scaffold interface of PCL microfibers by introducing a peptide-functionalized hydrogel coating. The ideal coating should reduce non-specific interactions, while peptide functionalization would instruct specific cell attachment. Surface modification processes should be rapid, low cost and accommodate the high porosity nature of MEW scaffolds. To solve this challenge, we turned to an unconventional coating approach of phase-separated poly(2-hydroxymethyl methacrylate) (pHEMA) onto microfibers using rapid dip-coating, followed by functionalization with RGD.

*Methodology: In a custom-built MEW 3D printer, medical-grade PCL was heated to 75°C and used to make porous scaffolds with different morphologies using 10µm diameter fibers. With square and triangle pore scaffolds as standard substrates, PCL was processed using 2bar, an applied voltage of 4.8kV and collector speed 8.3mm/sec. Similar printing conditions were applied to MEW onto 1mm mandrel to feature a diamond pattern. PCL scaffolds underwent sterilization and etching with sodium hydroxide (0.1M,30mins) before coating with phase-separated pHEMA. A rapid hydrogel formation process utilized ammonium persulfate and tetramethylethylenediamine to initiate the redox reaction. MEW scaffolds were immersed in the HEMA/pHEMA solution for 80 seconds. Scaffolds were washed in water and subsequently immersed in peptide solution (RGD or RGE) at pH8. PCL scaffolds and pHEMA coated scaffolds were imaged under SEM. In addition to metabolic activity (MTT), NIH-3T3 cells were fixed with 4% paraformaldehyde, stained for actin filaments and nuclei, and imaged under confocal microscopy.

*Results: We achieved a hydrogel coating by capturing droplets from a phase-separated pHEMA solution onto PCL microfibers during scaffold addition and removal from pHEMA solution. Polymerized HEMA droplets, when in excess water, formed a webbing structure on PCL microfibers. Excess droplets were removed, and phase separation was quenched by washing in water. The hydrophobic nature of pHEMA, which prevents protein adsorption, reduced non-specific cell attachment. Cysteine-terminated RGD-peptide conjugation to the pHEMA coating promoted cell adhesion onto flat scaffolds when compared to non-biologically active RGE peptide. In parallel, high-resolution MEW tubes with 1-mm diameter were coated with pHEMA. The technique demonstrates a simple yet new approach for coating shapes with hydrogels that are cell-instructive.

*Conclusion/Significance: Our research discovered that a phase-separated pHEMA solution could alter the surface of a MEW PCL scaffold, increasing both surface area and, when functionalized, providing functional signals. This functionalized hydrogel coating, customizable with cell-specific peptides, demonstrated altered morphologies during scaffold immersion into the pHEMA solution at specified timing and duration. Applied to highly resolved MEW tubes, the coating imparts new properties to tubular scaffolds. The investigation into the use of phase-separated solutions to coat and enhance the surface area of MEW scaffolds is widely applicable. This versatile process can be applied to different scaffolds, potentially beyond PCL or even polymers, while the peptide modification gives a spectrum of utility within TE.

381 - The Boron Transporter NaBC1 Mediates Mechanotransduction Via Fibronectin-Binding Integrins

J. Gonzalez-Valdivieso¹, M. Salmeron-Sanchez¹, P. Rico^2,3

¹ University of Glasgow, Glasgow, United Kingdom,

² Universitat Politècnica de València, Valencia, Spain,

³ Biomedical Research Networking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain

*Purpose/Objectives: This study is based on the functional interaction of integrins, the boron (B) transporter NaBC1, and growth factor receptors (GFRs) upon their activation. In our previous research, we demonstrated that active NaBC1 co-localizes with integrins (specifically Fibronectin (FN)-binding integrins) and GFRs, forming a functional cluster that enhances biochemical signals and facilitates crosstalk mechanisms [1]. This synergy accelerates muscle repair following an injury and restores dystrophic phenotypes in vivo, even in muscular dystrophies with different underlying causes. We propose that NaBC1, in addition to its natural role in controlling boron homeostasis, can also function as a mechanotransducer protein, compensating for abnormal cell-extracellular matrix interactions observed in mechanotransduction disorders.

*Methodology: The objective of this study was to demonstrate a novel role for NaBC1 in mechanotransduction mechanisms, particularly in collaboration with FN-binding integrins. To achieve this, we engineered three sets of PAAm hydrogels with varying stiffness ranging from 0.5 to 35 kPa that were functionalized with FN. We examined whether the rigidity of the hydrogel surface influenced the activation of NaBC1-integrin clusters. C2C12 myoblast cells were seeded onto these hydrogels, and we assessed cell behavior, including adhesion, spreading area, myogenic differentiation, and the activation or repression of gene/signaling pathways, after the simultaneous activation of NaBC1 by soluble B and FN-binding integrins by FN.

*Results: Our results demonstrated that soluble B induced a concentration-dependent primed state for cell adhesion and spreading area in cells grown on stiffer substrates. Furthermore, measurements of retrograde actin flow and traction forces exerted by cells on the different substrates significantly increased in response to B concentration when cultured on hydrogels with varying mechanical properties. Additionally, the simultaneous stimulation of NaBC1 and FN-binding integrins promoted myotube formation, which was dependent on substrate stiffness. To validate our findings, we replicated all experimental conditions using laminin-111-functionalized PAAm hydrogels, since laminin-111 cannot activate mechanotransduction mechanisms [2]. The observed effects resulting from NaBC1 activation were abrogated in the absence of FN onto 3D hydrogels and after NaBC1 silencing, demonstrating the causality role of NaBC1 and its cooperation with FN-binding integrins.

*Conclusion/Significance: In conclusion, this study unveils a novel role for the NaBC1 transporter beyond its involvement in controlling B homeostasis, functioning as a mechanoresponsive protein through specific cooperation with FN-binding integrins.

[1] J. Ciriza et al. Borax-loaded injectable alginate hydrogels promote muscle regeneration in vivo after an injury. Mater. Sci. Eng. C 123, 112003 (2021). [2] Z. Kechagia. et al. The laminin-keratin link shields the nucleus from mechanical deformation and signalling. Nat. Mater. (2023).

383 - Innovative Gradient-free Cancer Traps: Mechanically Guided Capture Of Metastatic Cells For Enhanced Therapeutic Efficacy

D. Caballero, R. L. Reis, S. C. Kundu

University of Minho, Barco GMR, Portugal

*Purpose/Objectives: Current treatments for cancer, including chemotherapy, radiotherapy, and surgery, have demonstrated positive outcomes in general; however, their efficacy is hindered by limitations in addressing metastatic tumors originating from infiltrating cells that escape the primary treatment. This shortfall significantly contributes to the rising number of cancer-related deaths and emphasizes the need for innovative therapeutic strategies. Treatment effectiveness can be enhanced by employing bioengineered cancer traps—implantable devices designed to attract infiltrating cancer cells and prevent their uncontrolled spread in vivo, and potentially enable eradication. Traditional cancer traps typically rely on chemoattractant-loaded materials, where the generated gradients may evolve unpredictably triggering adverse effects on other cells, leading to unwanted responses.

*Methodology: In this study, we have engineered innovative gradient-free cancer traps featuring arrays of microstructured channels organized in an annular morphology. Each channel contains asymmetric and interconnected triangular features, with the pointed edge oriented towards the center of the trap. Fabricated through soft lithography using biocompatible materials, this trap design mechanically attracts metastatic cancer cells and guides their migration following a ratchet mechanism for their selective capture over extended periods, notably in the absence of chemoattractants.

*Results: In vitro assays demonstrated a significant trapping efficiency of the ratchet-like traps against highly disseminating MDA-MB-231 breast cancer cells. Leveraging their 3D-like architecture, the traps functioned as asymmetric potential wells, mechanically entrapping cells within short periods when uniformly deposited at their top (Figure 1). Once captured, the mechanical interaction between the cell nuclei and the topographical walls of the trap dictated the preferred direction of migration, mainly towards the pointed edge of the asymmetric triangles (60±5%), elucidating a clear bias and the operation mechanism of the ratchet traps. This process exploited by cells to transition across adjacent topographic features with a high persistence length and time. As a result of this cyclic process, cancer cells accumulated in the inner region of the trap, showing a >100% increase of captured cells compared to control -isotropic- conditions, showcasing the trap bias effect. Importantly, the traps exhibited reduced efficacy when targeting non-metastatic and non-tumorigenic cells, underscoring their suitability for specifically capturing highly invasive cancer cells. Finally, the traps demonstrated the capability to restrict the spread of cancer cells when infiltrating breast cancer micro-spheroids were seeded at their center. This was achieved by reversing the motion of cells, thereby mitigating their invasion.

*Conclusion/Significance: Overall, this original approach holds promising implications for combating cancer and for being employed as a research tool for attracting, capturing, and eliminating disseminating cancer cells, thus potentially providing valuable insights into the biology and behavior of cancer cells. Furthermore, these traps hold the potential for isolating cancer cells for further in-depth analysis, which could enhance our understanding of cancer progression and metastasis.

384 - Improving Hemocompatibility Of Titania Nanotube Surfaces Using An Algae-derived Biopolymer Alternative To Heparin

S. Baghersad¹, L. Y. Madruga¹, A. F. Martins², K. C. Popat³, M. J. Kipper¹

¹ Colorado State University, Fort Collins, CO,

² University of Wisconsin-River Falls, River Falls, WI,

³ George Mason University, Fairfax, VA

*Purpose/Objectives: The study addresses the important need for sustainable alternatives to current antithrombotic coatings in blood-contacting medical devices, particularly focusing on titanium-based surfaces. Traditional options like heparin (HEP), despite their effectiveness, present challenges in sustainability and potential adverse effects. This research proposes an innovative, environmentally friendly solution by utilizing renewable biopolymers, specifically carboxymethyl-kappa-carrageenan (CMKC) and Tanfloc (TAN), for surface modification. CMKC, a modified polysaccharide with structural similarities to heparin, offers comparable antithrombogenic properties without the drawbacks associated with animal-derived heparin. This shift to plant-based sources, like CMKC derived from algae, represents a significant step towards more ethical and sustainable medical material science. This study demonstrates enhanced hemocompatibility via multiple outcomes on CMKC and TAN coated surfaces compared to uncoated controls and surfaces using similar coatings containing heparin. This work aims to reduce thrombosis risks and improve overall patient outcomes while aligning with environmental sustainability goals.

*Methodology: This study proposes a new strategy to enhance the hemocompatibility of titanium nanotube (TiNT) surfaces using biopolymer multilayers. Specifically, we use the polysaccharide-based CMKC and the condensed tannin derivative TAN in creating polyelectrolyte multilayers (PEMs). TAN-CMKC PEMs compositions were compared to, TAN-kappa-carrageenan (KC), TAN-hyaluronan (HA), and TAN-HEP, to assess their impact on blood-material interactions. Titanium sheets were first anodized to create titania nanotube (TiNT) surfaces. Subsequently, PEMs were deposited using a layer-by-layer approach, ensuring precise control over composition and thickness. Hemocompatibility was assessed by determining fibrinogen adsorption, platelet adhesion and activation, and whole blood clotting kinetics. Fibrinogen adsorption was quantified through an enzyme-linked immunosorbent assay (ELISA); platelet adhesion and activation were analyzed through fluorescence microscopy and scanning electron microscopy; the kinetics of blood clotting on different substrates were assessed by measuring the absorbance of free hemoglobin released from lysed red blood cells. By evaluating interactions of protein, cells, and whole blood with surfaces presenting different chemistries the roles of biopolymers in mitigating thrombosis risk can be determined.

*Results: TiNT surfaces modified with CMKC and TAN PEMs have hemocompatibility outcomes superior to other surfaces studied. This was evidenced by a significant decrease in blood protein (fibrinogen) adsorption and reduction in platelet adhesion and activation. These observations are crucial indicators of improved antithrombogenic properties. Further, the kinetics of whole blood clotting were evaluated, with the CMKC and TAN modifications showing a substantial inhibition of clot formation. Notably, comparative analyses between TAN-CMKC PEMs and TAN-HEP PEMs revealed that while both coatings demonstrated antithrombogenic properties, CMKC-containing surfaces showed a comparable, if not enhanced, performance in reducing clot formation. This shows the potential of CMKC as a sustainable and effective heparin alternative. Also, the impact of surface nanotopography in conjunction with biopolymer coatings was observed to play a significant role in maintaining the functional integrity of blood components.

*Conclusion/Significance: This study highlights the potential of CMKC as a sustainable and effective alternative to heparin for enhancing hemocompatibility in blood-contacting medical devices. The combination of CMKC and TAN with TiNT surfaces offers an innovative, eco-friendly approach to bioengineering, providing a safer and more sustainable approach for medical devices.

385 - Preclinical Development Of Osteogrow-C - A Novel Device For Bone Regeneration

N. Stokovic¹, N. Ivanjko¹, M. Pecin², A. Smajlovic², D. Vnuk², Z. Vrbanac², H. Capak², D. Maticic², S. Vukicevic¹

¹ University of Zagreb School of Medicine, Zagreb, Croatia,

² University of Zagreb Faculty of Veterinary Medicine, Zagreb, Croatia

*Purpose/Objectives: Degenerative diseases of the spine and segmental defects of the long bones are among the most complex conditions in clinical medicine. Osteogrow is a novel osteoinductive device comprised of recombinant human Bone Morphogenetic Protein 6 (rhBMP6) delivered in an autologous blood coagulum. However, Osteogrow should be supplemented with a compression-resistant matrix (CRM) for application in indications where compressive forces are present such as large segmental defects and posterolateral spinal fusion (PLF). Synthetic calcium phosphate (CaP) ceramics are available in a broad range of formulations and have been widely used in clinics due to their osteoconductive properties. This series of preclinical studies aimed to evaluate the safety and efficacy of Osteogrow with CaP ceramics (Osteogrow-C) and determine whether the chemical composition and size of ceramics affect the properties of newly induced bone.

*Methodology: In the preclinical development of Osteogrow-C we have conducted extensive animal studies in rat subcutaneous assay, rabbit and sheep PLF model as well as rabbit ulnar segmental defect model. In these studies, we have evaluated Osteogrow-C with CaP ceramics in different sizes (tested size range from 74 to 4000 µm) and different chemical compositions (hydroxyapatite-HA, tricalcium phosphate-TCP and biphasic ceramics-BCP containing both HA and TCP). Moreover, in the aforementioned experiments, we have determined the timeline of BMP-mediated ectopic osteogenesis in different species and evaluated the longevity of induced bone with a follow-up period of up to one year. Animal studies were approved by the National Ethics Committee.

*Results: Bone volume of induced bone by Osteogrow-C implants at rat ectopic site was determined by the size of ceramic particles and it was significantly higher in implants containing smaller than larger particles. Osteogrow-C implants with CaP ceramics induced rapid spinal fusion in rabbit and sheep PLF models as well as rebridgment of ulnar segmental defects without any adverse effects related to tested implants. Newly formed bone was integrated with native transverse processes as confirmed by microCT and histological analyses. Furthermore, the three-point bending test revealed that induced fusion between adjacent transverse processes was biomechanically competent. Importantly, the chemical composition of ceramics determined particle resorbability since the TCP particles were significantly more resorbed than HA. However, the resorbability of TCP was significantly higher in rabbit PLF and ulnar segmental defect model than at rat ectopic site. The sequence of events in BMP-mediated osteogenesis was similar in different species but the process was significantly slower in larger animals (sheep and rabbits) than in smaller animals (rats).

*Conclusion/Significance: Osteogrow-C was proven safe and efficacious in preclinical studies providing hope that it might be a therapeutic solution in clinics for patients with degenerative diseases of the spine and patients with large bone segmental defects. Size and chemical composition are crucial properties of the CaP ceramics that allow tailoring the structure of newly induced bone and the amount of the residual ceramics.

386 - Eggshell-derived Amorphous Calcium Phosphate: Synthesis, Characterization And Bio-functions As Bone Graft Materials In Novel 3d Osteoblastic Spheroidic Model

Q. Ma¹, K. Rubenis², H. J. Haugen¹, T. Hildebrand¹, L. Piaia¹, Ó. E. Sigurjónsson³, J. Locs², D. Loca²

¹ University of Oslo, Oslo, Norway,

² Riga Technical University, Riga, Latvia,

³ Reykjavík University, Reykjavik, Iceland

*Purpose/Objectives: Bone graft materials can be briefly divided into autologous/allogeneic, xenogeneic and synthetic materials. However, the limited supply, donor-site morbidity, and disease transmission limit the large-scale commercialization of autogenous/allogeneic bone grafts. In contrast, semi-synthetic xenogeneic graft materials with relatively sufficient supply have been involved in a large number of surgical applications but with high bio-cost and potential environmental pollution. Therefore, the top priority of bone graft materials development is to obtain safe, modifiable, and environmentally friendly synthetic biomaterials with sufficient supply, low bio-costs and ideal osteogenic activity. With a high content of calcium and several trace elements, chicken eggshells are considered no longer as wastes but as an attractive source of high value-added biomaterials. In this study, chicken eggshells were employed as raw materials to synthesize amorphous calcium phosphate (ACP) bone graft materials.

*Methodology: Two different ACP materials were synthesized from commercial hydroxyapatite (HA) and waste eggshell with an acidic-to-alkaline strategy. The synthesized ACP materials were characterized by FTIR, SEM, XPS, ICP-MS. Etc. Their biosafety and immunocompatibility were tested in different mammalian cells while their osteogenic activity was inspected in a novel MC-3T3-E1 cell-spheroidic research model with ACP particles embedded inside.

*Results: ACP biomaterials can be synthesized from both HA (Control) and eggshell but with different sizes, trace elements composition, and re-crystallization dynamics. Both Control and Eggshell ACP were free of endotoxin contamination as well as cytotoxicity and exhibited ideal immuno-compatibility. The osteogenic differentiation and mineralization of mouse osteoblastic MC-3T3-E1 cells in the 3D spheroid system were strikingly promoted after ACP embedding. More importantly, the confocal microscopic investigation found that the OPN expression was greatly enhanced, and OPN protein was firmly deposited in the extra-cellular matrix (ECM) around ACP particles, which forms an ideal ECM scaffold facilitating bone regeneration and intercellular anchoring. Eggshell ACP showed higher osteogenic activity than control ACP.

*Conclusion/Significance: As the precursor of the mineral phase of Ca-P and the storage “pool” of calcium and phosphorus, ACP could regulate the speed of bone formation and enhance the mechanical properties of bone tissue by releasing Ca²⁺ and PO₄^3- and mediating the growth of bone apatite⁴. This study developed a novel industrial system for converting eggshell waste to high-value-added, bioactive, and safe ACP graft materials with in vitro evidence based on a novel osteoblastic spheroid system. In vivo research is ongoing. ACKNOWLEDGEMENTS: We acknowledge financial support from the Baltic Research Programme project No. EEA-RESEARCH-85 “Waste-to-resource: eggshells as a source for next generation biomaterials for bone regeneration (EGGSHELL)” under the EEA Grant of Iceland, Liechtenstein and Norway No. EEZ/BPP/VIAA/2021/1 and Research Council of Norway, “MISFAITH” Grant No. 331752 and “DEBRIGEL ”Grant no. 332148.

387 - Natural Compounds From The Seafood Waste To Ameliorate Skin Repair

L. Melotti¹, A. Carolo¹, G. Zivelonghi¹, G. Martinelli², M. Roncoroni², S. Marzorati², A. Venerando³, A. Porzionato⁴, V. Vindigni⁴, M. Sugni², M. Patruno¹

¹ University of Padova, Legnaro, Italy,

² University of Milan, Milan, Italy,

³ University of Udine, Udine, Italy,

⁴ University of Padova, Padova, Italy

*Purpose/Objectives: Wound healing is a complex process orchestrated by the dynamic interaction of multiple cell types and structural proteins aiming to restore the lost skin integrity. However, it does not always lead to wound closure, but it rather results in the formation of scar or, in different circumstances, the generation of chronic wounds. For this reason, wound care has been considered a worldwide challenge and an economical burden; thus, the development of new healing devices is widely investigated. Wound dressings cover the lesion protecting it from damage while promoting its healing. Furthermore, the creation of multifunctional wound dressings by adding bioactive molecules might further improve healing outcomes. Herein, we describe the application of a marine collagen-based and eco-friendly wound dressing enriched with antioxidant molecules to treat skin wounds.

*Methodology: Collagen and antioxidant pigments, namely polyhydroxynapthoquinones (PHNQs), were extracted from sea urchin (Paracentrotus lividus) food-waste. First, the biological activity of PHNQs was assessed in vitro. PHNQs were tested for cytotoxicity on dermal fibroblasts, then cells were exposed to non-cytotoxic concentrations in an oxidative stress environment to assess PHNQs antioxidant properties by investigating reactive oxygen species (ROS) concentration, mitochondrial membrane potential (ΔΨm), and protein expression of antioxidants enzymes. Secondly, PHNQs were incorporated into a marine collagen-based membrane (MCDT) and tested in an experimental wound model. Two circular skin lesions were surgically created on the back of 32 rats: one wound was left untreated, and one treated either with MCDT or MCDT enriched with pigments (A-MCDT). Wounds were monitored daily, and at 5- and 10-days post-wounding samples were collected for histopathological and gene expression analysis.

*Results: PHNQs showed no cytotoxicity at low concentrations (1-10 μg/mL) and demonstrated scavenging activity by reducing ROS levels and protecting cells against oxidative stress. Moreover, they were able to maintain proper levels of ΔΨm and to upregulate the protein expression of SOD2. Histologically, at 5 days untread wounds showed a mild inflammatory infiltration whereas treated wounds showed a similar pattern of inflammatory cells but lower in A-MCDT-treated wounds. At 10 days, inflammation levels were similar. Furthermore, treated lesions showed a more abundant presence of granulation tissue (GT) at day 5 compared to untreated ones; nevertheless, at day 10 an opposite trend was observed: treated wounds, showed a higher maturation level of the GT and a more mature dermis, this result might be related to the up-regulated TIMP-2 gene expression, especially in the A-MCDT group. Also, re-epithelialization appeared higher in A-MCDT-treated wounds as they showed a more prominent presence of neo-epithelium on the healing skin. This result is corroborated by the higher gene expression of MMP-9, a proteinase involved in keratinocyte migration.

*Conclusion/Significance: Overall, treated wounds showed a better trend of healing compared to untreated ones. Indeed, these wounds showed a better development of the GT into mature dermis and higher levels of re-epithelialization, both processes supported at the histological and molecular level. The addition of antioxidant pigments allowed an improved skin reparation as lower levels of ROS in the wound might have sustained proper and faster healing.

388 - An Axis Of Wnt And Proinflammatory Signals Underlies Mechanically Driven Osteogenesis

H. Jaffery, U. Dhawan, M. Salmeron-Sanchez, M. J. Dalby

University of Glasgow, Glasgow, United Kingdom

*Purpose/Objectives: Musculoskeletal conditions, affecting approximately 1.71 billion individuals worldwide, stand as the predominant cause of disability globally. Mesenchymal stem cells (MSCs), naturally found in diverse bodily reservoirs, serve as multipotent progenitors for osteoblasts, the cells responsible for bone formation. The process of osteogenic differentiation, leading to the formation of bone, is influenced by a myriad of factors, including the response of cells and the entire organism to biomechanical forces. In our research, we have innovated a nanovibrational bioreactor, a cutting-edge biomechanical approach for directing MSCs towards bone formation. This technology has demonstrated significant efficacy in promoting targeted osteogenesis with high precision in preclinical models. However, the detailed molecular mechanisms underlying this process are yet to be fully elucidated. Our current research explores the impact of nanovibrational stimulation on the osteogenic Wnt signaling pathway, a critical component in bone development.

*Methodology: Adipose-derived human MSCs were cultured on our novel bioreactor, delivering nanovibrational stimulation of 30 nm vertical displacement, at 1000 Hz in basal medium (“nano-kicked”; NK) compared to controls stimulated with the osteogenic metabolites (OGM) L-ascorbic acid, β-glycerophosphate and dexamethasone, or controls kept in basal conditions with no stimulus (Ctrl), for 1 to 21 days. Parsing of various pathways involved incorporating Wnt inhibitors, including LGK974, XAV939, AMBMP hydrochloride, CHIR99021, alongside Rock inhibitor Y-27632 and BCL3 mimetic peptide BDP2. Relative gene transcription and protein expression studies were carried out to determine cell molecular responses.

*Results: (A) An axis of proinflammatory, non-canonical Wnt pathway and osteogenic genes all have higher transcriptional expression in nanovibrated stem cells, compared to metabolite-induced cells. (B) Treating nanovibrated cells with Wnt pathway inhibitors XAV939 (targeting canonical-Wnt via Tankyrase) and LGK974 (targeting pan-Wnt secretion via Porcupine) reveals divergent gene transcription signatures - generally, LGK974 reduces expression of genes in the axis, while XAV939 increases axis gene expression. (C) In the presence of nanovibration, a BCL3 mimetic peptide (inhibitor of NF-κB signalling, associated with non-canonical Wnt) inverted the transcriptional signatures produced by XAV939 and LGK974 inhibition. (D) Further studies inhibiting the Wnt pathway with AMBMP and CHIR99021 recapitulated earlier observations that the non-canonical Wnt pathway is integral to forming the axis of osteogenic differentiation. (E) Transcriptional observations throughout were validated with protein expression and use of Rock inhibitor Y-27632 demonstrated functional reversal of nanovibrational signal. (F) In complement to hMSCs, BCL3 knockout murine monocytes abrogated the increase in axis genes observed in wild-type cells during nanovibration-driven osteoclastogenesis. While nanovibration itself did not increase osteoclast number or size, XAV939 treatment of BCL3 knockout cells had a dramatic increase in osteoclastogenesis - exemplary of the axis of proinflammatory, non-canonical Wnt in bone cell differentiation.

*Conclusion/Significance: Broadly, a cell signalling axis comprised of non-canonical Wnt signalling, proinflammatory cytokine signalling and NF-κB regulation determines stem cell osteogenic and osteoclastogenic differentiation in response to nanovibrational biomechanical force.

389 - Connexin Rich Biomaterials Synchronize Induced Pluripotent Stem Cell-derived Cardiomyocytes Beating

N. Momtahan, J. Stachowiak, J. Zoldan

Biomedical engineering, University of Texas at Austin, Austin, TX

*Purpose/Objectives: Advancements in generating and differentiating human induced pluripotent stem cells (hiPSCs) have made hiPSC-derived cardiomyocytes (hiPSC-CMs) a viable cell source for engineered cardiac tissues. However, these hiPSC-CMs display underdeveloped structure and functionality when compared to adult CMs. Although engineered hiPSC-derived cardiac tissues have demonstrated varying degrees of functionality, we still lack a viable approach for coupling engineered tissues electrically and mechanically to the heart. Here we showed that treating hiPSC-CMs with synthetic cells that are rich in Cx43 improves electrochemical coupling of hiPSC-CMs, evident by their synchronous beating.

*Methodology: Three different hiPSCs cell lines: non-repoter hiPSCs (Thermo Fisher), reporter hiPSCs expressing mEGFP-tagged connexin-43 (hiPSC-Cx43GFP, Allen Institute for Cell Science), and hiPSCs transfected with CMV-GCaMP6f (hiPSC-GCaMP; a gift from Dr. Bruce Conklin) were differentiated into CMs using the Wnt pathway modulation [1]. Donor cells for extraction of Connectosomes, including reporter hiPSC-Cx43GFPs, HeLa-Cx43YFPs, and wild-type HeLa cells, were induced to form Connectosomes using an established protocol [2]. Day 20 hiPSC-GCaMP-CMs were treated with Connectosomes and synchronicity of beating was analyzed. Changes in Cx43 expression in treated cells were monitored on both gene and protein levels.

*Results: We successfully harvested Connectosomes from the plasma membrane of donor hiPSCs. The advantage of using hiPSCs as donor cells is that they are inherently rich in Cx43. We demonstrate that harvested hiPSC-Connectosomes retain the high Cx43 expression on their surfaces and that Cx43 hemichannels on hiPSC-Connectosomes are functional. We found that Connectosomes preferentially locate at the interface between adjacent hiPSC-CMs, causing significant increases in Cx43 expression and protein synthesis of target hiPSC-CMs. Most significantly, we demonstrated that hiPSC-Connectosomes significantly improve the synchronization of differentiated CMs compared to cells that were not treated with hiPSC-Connectosomes. The observed synchronization effects persisted for more than 8 days and are Cx43 dependent (Figure 1).

*Conclusion/Significance: Here, we generated Connexin-rich biomaterials that promote coupling between hiPSC-CMs by reinforce sing the gap junctions and interconnectivity between these cells. A key advantage of this strategy is that it uses the cells’ machinery to accomplish the functional insertion of connexin transmembrane proteins into membranes. This approach has the potential to overcome the key hurdles currently preventing the successful implantation of hiPSC-derived tissue replacements in the heart: the lack of electrochemically coupled beating cells.

Figure 1. Synchronicity analysis of beating hiPSC-CMs.(a) Negative control beating samples were not treated with Connectosomes. (b-d) Beating hiPSC-GCaMP-CMs were treated with Connectosomes harvested from HeLa cells (b), HeLa-Cx43YFPs (c), and hiPSCs (d). Representative day 8 beating charts (left) of red, blue, and green regions of interest from the field of view (middle) show evidence of synchronization. Synchronization analysis (right) based on median average deviation of the time of peak arrival of beating hiPSC-GCaMP-CMs. All scale bars are 200 μm.

References: [1] X. Lian, Nat Protoc 2012, 8(1):162-175. [2] Gadok, AK. J Am Chem Soc 2016, 138(39): 12833-12840.

391 - Tissue Chips For Advanced Multipotent Stromal Cell Manufacturing

I. Jain¹, A. Chan¹, J. Lam², K. Sung², N. F. Huang¹

¹ Stanford University, Palo Alto, CA,

² FDA, Washington DC, DC

*Purpose/Objectives: Human mesenchymal stromal cells (MSCs) are a potentially promising therapeutic cell type that has been tested in clinical trials for treatment of cardiovascular disease. However, a limiting step in the clinical application of MSCs is the establishment of robust methodologies that optimize cell quality. The objective is to develop a tissue chip platform for high-throughput systematic control of extracellular matrix (ECM) composition and stiffness effects on MSC function and cell fate specification. This work is intended to identify microenvironmental conditions that optimize MSC culture and differentiation for MSC manufacturing.

*Methodology: We engineered a 24-well tissue chip platform that features multi-component combinations of ECM proteins (collagens II, III, IV, laminin, and fibronectin), substrate stiffnesses (150-900 kPa), and individualized media reservoirs for secretome analysis. Human MSCs (Lonza) were expanded in MSC growth media (MSCGM, Lonza). Cells were used between passages 5-8. MSCs were induced with adipogenic differentiation media (Lonza) for 3 consecutive cycles over the course of 19 days in total. To visualize adipogenesis, Oil Red O was performed. Quantification of adipogenesis was performed by quantifying the % of Oil Red-O-expressing cells and by the cumulative area of lipid droplets.

*Results: Our quantification of adipogenesis, proliferation and immunomodulation on the tissue chips revealed specific preference of MSCs to stiffness and high order ECM combinations (Fig 1A, B, C). Furthermore, transcriptomic analysis demonstrated enrichment of differential cellular pathways in MSCs on different stiffnesses. Additionally, we were successfully able to rank our ECM-Stiffness combinations based on genome-wide enrichment of MSCs on tissue chips to clinical data. It was found that on an average, MSCs cultured on 150kPa and 234F ECM enriched to bone-marrow derived MSCs found in healthy humans (Fig. 1D, E).

*Conclusion/Significance: Overall, we demonstrated how combinatorial ECMs can affect MSC behavior and found non-intuitive findings. We also showed how this technology can be used to benchmark MSC cell lines and culture conditions against clinical data.

407 - Engineered 3D “MatriSpheres” Recapitulate In Vivo Colorectal Tumor Stroma & Cancer Cell Phenotype

M. J. Buckenmeyer¹, E. A. Brooks¹, M. S. Taylor¹, L. Yang¹, R. J. Holewinski², T. J. Meyer², M. Galloux³, M. Garmendia-Cedillos⁴, T. J. Pohida⁴, B. St. Croix¹, M. T. Wolf¹

¹ National Cancer Institute, Frederick, MD,

² Leidos Biomedical Research, Inc., Frederick, MD,

³ No Affiliation, Marseille, France,

⁴ National Institute of Biomedical Imaging and Bioengineering, Bethesda, MD

*Purpose/Objectives: Solid tumors are heterogenous and variable within and across cancer types, which makes them challenging to model and to treat. Many in vitro tumor models lack crucial in vivo elements that limits their predictive value. One possible reason for tumor model infidelity is the lack of a stromal component, in particular the extracellular matrix (ECM). In this study, we (1) developed the tumor “MatriSphere” method of inducing spontaneous assembly of tumor stroma using orthotopic decellularized small intestine ECM (SIS ECM) in 3D colorectal cancer cells (CRCs) and (2) determined the role of ECM in cancer cell organization and transcriptomic regulation after MatriSphere formation.

*Methodology: Porcine small intestine was decellularized using physical and chemical treatments, and enzymatically digested to obtain a partially solubilized ECM suspension. Mass spectrometry was performed to compare ECM biomaterials (SIS ECM, Commercial Collagen I, Matrigel) protein composition similarity to in vivo MC38 colorectal tumor ECM. 3D MatriSpheres were established by combining mouse (MC38 and CT26) and human (HT-29) colorectal cells with low concentrations of ECM in low adherence conditions. Live cell assays (CellTiter Glo 3D, Live-Dead) and histological staining (H&E, PicroSirius Red (PSR), Immunofluorescence) were performed to assess viability and morphological changes in response to ECM biomaterials. Time lapse imaging was used to monitor spheroid formation kinetics and ECM biomaterials were evaluated using rheology and optical absorbance to determine gelation properties at varied ECM concentrations. Single cell RNA Seq was performed on in vivo MC38 tumors and bulk RNA Seq for evaluating all CRC MatriSpheres with SIS ECM versus cells alone spheroids.

*Results: Out of 101 MC38 tumor matrisome proteins detected, SIS ECM shared the highest percentage of proteins (75%) with 42 proteins found exclusively in SIS. In contrast, Matrigel and Collagen I had a substantially lower number of similar proteins with 38% and 4%, respectively providing rationale for using a decellularized ECM for tumor modeling. 3D MatriSpheres incorporated ECM and established a complex and heterogeneous stroma that is lacking with cells alone. Further, ECM dense regions within SIS ECM MatriSpheres reduce Cadherin expression and indicates cell-ECM dominant adhesions. “Scissor” bioinformatics analysis of single-cell and bulk RNA Seq found that MC38 MatriSpheres showed 35% overlap with in vivo MC38 tumor cells in comparison to 19% for cells alone spheroids. SIS ECM MatriSpheres correlated with cells showing in substantial increase in histone-related genes and a decrease in Col3a1 and Col1a1 gene expression. Ingenuity pathway analysis showed that CRC cancer cells have a robust and variable/cell-line dependent response to SIS ECM with MC38 cells primarily slowing metabolism pathways and CT26 and HT-29 activating IFN signaling pathways.

*Conclusion/Significance: We demonstrate a novel spheroid model with a robust ECM-rich stroma, or 3D MatriSpheres. Intratumoral ECM is self-assembled and guided by cell-ECM interaction to create a unique heterogenous microtissue. Decellularized intestine ECM induces dramatic changes in CRC cell phenotypes, which shares transcriptomic expression patterns with in vivo CRC cells. Thus, this model may provide a high-fidelity tool for in vitro testing to improve predictiveness of drug efficacy.

408 - Establishment Of Patient-Derived 3D Tumouroids And Matched Xenografts: Personalised Medicine Tools For Renal Cancer

K. Bokea¹, T. Azimi¹, K. Stamati¹, W. Braithwaite¹, P. M. Bhamidimarri², B. Yener Ilce², E. Yaghini¹, A. J. MacRobert¹, R. Hamoudi^1,2, U. Cheema¹, M. G. Tran¹, M. Loizidou¹, F. Mumtaz^1,2

¹ University College London, London, United Kingdom,

² University of Sharjah, Sharjah, United Arab Emirates

*Purpose/Objectives: Despite the therapeutic advancements in the management of Renal Cell Carcinoma (RCC), there is unmet clinical need for patient-specific in vitro models that can predict patients’ response to therapy, replace the reliance on animal models and deepen our understanding of this heterogeneous disease. Here, we established biomimetic 3D in vitro models, termed tumouroids, that incorporate patient-derived tumour cells and a complex stroma to mimic the physiological tumour microenvironment. We compared tumouroids with 786-O RCC cell line xenografts in terms of response to therapy and for one patient, we developed matched patient-derived xenografts and tumouroids to allow parallel comparisons of the two models.

*Methodology: Tumour cells were isolated from RCC surgical specimens (n=20) using a mechanical-enzymatic method and two tumouroid types were manufactured: simple tumouroids consisting of patient-derived tumour cells, collagen and matrix proteins, and complex tumouroids that incorporated an additional stromal compartment populated with fibroblasts and endothelial cells. Tumouroids were compressed to mimic in vivo tissue density (3.5KPa). Patient-derived cells and tumouroids were characterised through whole exome sequencing, immunofluorescence and histology to investigate resemblance to original tissues. The response of patient-derived cells to Pazopanib (Votrient™), a tyrosine kinase inhibitor (TKI) used in the treatment of advanced RCC, was tested at different culture conditions: 2D, simple and complex tumouroids. 786-O xenografts were also developed to investigate the response to Pazopanib in vivo. Finally, matched tumouroids and xenografts were established from one patient, to compare tumouroids and mouse xenografts in terms of resemblance to original tissue and response to pazopanib.

*Results: We showed that patient-derived tumouroids retained the expression of renal cancer-associated gene markers, including mutant VHL, and characteristic RCC protein markers: carbonic anydrase IX (CA9), cytokeratins (CK7, CK8&18) and EMT markers (αSMA). The subtype-specific histology of the original tumour, e.g., clear cell, was also preserved in tumouroids. We observed a range of responses to Pazopanib, from none to strong (40%) for individual patient-derived simple tumouroids (n=12). In the complex tumouroids (n=3), a stronger response was observed as the endothelial networks were also disrupted (p<0.001 v simple tumouroids). Similarly, tumour size and vessel density were reduced in vivo after pazopanib treatment of the 786-O xenografts (n=3, p<0.05 v controls). Matched patient-derived tumouroids and xenografts responded similarly to pazopanib as confirmed by immunostaining, reduced metabolic activity and reduced tumour growth. However, for patient-derived xenografts lower engraftment and establishment success rates were reported (70%) compared to tumouroids (95%), with increased variability in tumour progression among individual mice and longer, more expensive and challenging protocols.

*Conclusion/Significance: In summary, patient-derived tumouroids mimicked the original tissue, distinguished between different drug responses and successfully reproduced the signatures of response to TKIs as seen in vivo. In addition, their feasibility to establish compared to patient-derived xenografts, supports their suitability as personalized cancer treatment platforms.

409 - Bromelain As A Potential Cytolytic And Mucolytic Agent For Appendiceal Cancer: Cytotoxic And Mucolytic Effects In Patient-derived Appendiceal Tumor Organoids

N. Wajih¹, P. Shen¹, E. Levine², D. Morris³, S. Soker², K. Votanopoulos²

¹ Wake Forest University School of Medicine, Winston-Salem, NC,

² Wake Forest University School of Medicine, Winston Salem, NC,

³ Wake Forest University School of Medicine, Sydney, Australia

*Purpose/Objectives: Mucin production in appendiceal cancer (AC) acts as a barrier to HIPEC drug delivery and treatment resistance. Currently, there are no approved mucolytic agents for AC treatment. Bromelain, a pineapple stem extract with mucolytic properties, has generated research interest. We explored bromelain's cytotoxic and mucolytic effects on mucinous AC in a patient-derived tumor organoid (PTO) model.

*Methodology: Tumor specimens were obtained from patients with AC who had undergone cytoreductive surgery with HIPEC. PTOs were fabricated using an unsorted tumor cell suspension in a collagen-based hydrogel. PTOs underwent HIPEC mimicry treatment with bromelain, cisplatin, and mitomycin C (MMC) at 37 °C and 42 °C conditions. IHC, ATP viability, and immunoblotting were performed to elucidate the mechanism of bromelain-mediated cytotoxicity of AC PTOs.

*Results: A total of eleven appendiceal cancer specimens were collected from 10 patients with low-grade appendiceal cancer (7/11, 63.6%) and high-grade appendiceal cancer (4/11, 36.4%). Testing was successful for all 11 specimens. The mucin-depleting effects of bromelain (600 ug/ml) were more significant in the presence of N-acetylcysteine (NAC, 3% w/v) than in the presence of bromelain alone (50% residual mucin vs. 85%, p=0.002, n=7) and NAC alone (85% residual mucin, p=0.003, n=7). The cytotoxicity of bromelain increased with time and reached statistical significance only 60 min after treatment (>50% post-treatment viability reduction, p<0.01). Bromelain tumor pretreatment increased the cytotoxicity of both cisplatin and MMC under 42 °C HIPEC conditions compared to cisplatin (70% greater reduction in post-treatment cell viability, p=0.03) and MMC (60% greater reduction in viability, p=0.002) alone. After bromelain treatment, IHC demonstrated reduced Ki67, CK20, and MUC2 expression, indicating a decreased tumor cell population in AC PTOs. Increased expression of annexin V and increased activity of caspase 3/7 in bromelain-treated PTOs compared to untreated controls suggests that the antitumor activity of bromelain induces the programmed cell death pathway in AC PTOs. Furthermore, bromelain treatment decreased the expression of anti-apoptotic and pro-survival proteins Bcl-2 (p<0.009, n=3) and Bcl-xL (p<0.01, n=3), suggesting the inhibition of anti-apoptotic and pro-survival pathways in AC PTOs. Moreover, the expression of cyclin A2, D1, E1, and cyclin H cell cycle regulatory proteins decreased upon bromelain treatment. In addition, we also found changes in the expression profile of some of the autophagosomal marker proteins in bromelain-treated AC PTOs, LC3-A/B I (p<0.03, n=3), LC3-A/B II (p<0.31, n=3), Bacline 1 (p<0.03, n=3), Atg7 (p<0.01, n=3), and Atg 12 (p<0.04, n=3). Based on our results, we predict that the induction of apoptotic signaling cascades and the suppression of anti-apoptotic and pro-survival pathways synergistically diminishes the viability of appendiceal cancer organoids and intensifies the cytotoxic effects in response to bromelain treatment.

*Conclusion/Significance: Bromelain displays mucolytic activity against fresh AC-derived mucin enhanced by the NAC and cytotoxic activity against patient-derived AC organoids. These characteristics make bromelain an intriguing pre-HIPEC treatment option for patients with peritoneal carcinomatosis from AC, as it enhances tumor-drug contact by inducing mucolysis and augments cytotoxicity of current HIPEC agents.

411 - A Developmental Tissue Engineering Approach To Study Breast Cancer Metastasis To The Bone

E. Ji¹, A. Lolli¹, J. Witte-Bouma¹, A. Rouchon², M. Cecchi³, E. Palamà⁴, N. Di Maggio², A. Banfi², S. Scaglione⁴, E. Farrell¹

¹ Department of Oral and Maxillofacial Surgery, Erasmus MC University Medical Centre Rotterdam, Rotterdam, NETHERLANDS,

² Department of Biomedicine, Basel University Hospital, Basel, SWITZERLAND,

³ Department of Neuroscience, Psychology, Drug Research and Child Health, Neurofarba, Pharmacology and, University of Florence, Florence, ITALY,

⁴ React4Life, Genova, ITALY

*Purpose/Objectives: Bone is the third most frequent site of metastasis, leading to severe symptoms and high mortality rate. Unfortunately, little is known about the mechanisms and the vulnerabilities of bone metastasis, and the patients are considered incurable. Importantly, experimental models able to recapitulate the bone metastatic environment could lead to a better understanding of the process and aid the development of targeted drugs. Here, we took inspiration from endochondral ossification (EO), the developmental process through which long bones are formed via vascularised cartilage, to develop an in vitro approach to study bone metastasis. Furthermore, we applied a newly-developed 3D-printed device to recapitulate the migration of metastatic breast cancer cells to the bone constructs under fluid-flow conditions.

*Methodology: Bone marrow-derived human marrow stromal cells (hMSCs) were cultured as pellets (2x10⁵ cells) in chondrogenic medium containing transforming growth factor-β to generate cartilage. To induce cartilage mineralisation in vitro, 10 mM β-glycerophosphate was added to the pellets from day 7 of culture. Mineralisation was confirmed by calcium uptake and histology. MDA-MB-231 breast cancer cells were co-cultured with these pellets, and cancer cell migration and proliferation were assessed by transwell assay and DNA quantification, respectively. To induce the formation of a microvascular network in vitro, human umbilical vein endothelial cells (HUVECs) and adipose-derived cells (ASCs) were embedded in fibrin hydrogels containing mineralised pellets. Finally, we applied a fluidic platform whereby GFP+-MDA-MB-231 were injected and flowed through these fibrin hydrogels. Infiltration of hMSC pellets by cancer cells was analysed by qRT-PCR for GFP.

*Results: In vitro, exposure of chondrogenic pellets to osteogenic stimuli (β-glycerophosphate) induced mineralisation of the cartilage template. When in vitro mineralised pellets were co-cultured with MDA-MB-231, cancer cell migration was strongly enhanced (36-fold increase). Exposure of MDA-MB-231 to conditioned medium from in vitro mineralised pellets enhanced cancer cell proliferation and migration, further indicating that factors secreted from the pellets can potentially promotes invasiveness of breast cancer cells. To generate a microvascular network in vitro, mineralised pellets, HUVECs and ASCs were directly co-cultured in fibrin hydrogels for 10 days. Confocal imaging analyses demonstrated the formation of physiologically differentiated microvascular networks, showing apicobasal polarisation as well as well-formed lumina. The microvascular structures were formed in the proximity of the pellets, with signs of close interaction. When in vitro pellets were cultured in a fluidic device with MDA-MB-231 in flow, homing of cancer cells to the pellets was achieved by day 3 of co-culture, as evidenced by GFP mRNA detection in the pellets.

*Conclusion/Significance: Our data demonstrate the possibility to exploit an in vitro tissue engineering approach inspired by EO and a fluidic platform to mimic specific aspects of the process of cancer metastasis to the bone. In vitro-generated mineralised constructs were successfully co-cultured with breast cancer cells and were able to induce a migratory response. Follow-up studies will target achieving further complexity in the bone component, possibly by including additional cell types. These experimental models may be applied to studying the molecular basis of the bone metastatic process and to developing new targeted drugs.

413 - The Cross-talk Between Microglia And Glioblastoma Organoids And Its Effect On Chemotherapeutic Resistance

H. Salem Muthu Sugavanam Sivakumar, A. Skardal

Ohio State University, Columbus, OH

*Purpose/Objectives: Glioblastoma (GBM) is an extremely aggressive and incurable primary tumor of the brain. The disease’s high mortality has not changed much in twenty years. Immunotherapy, which has been widely successful in other cancers, is ineffective in treating GBM. This failure can be attributed to the poor knowledge of multifactorial immune suppression mechanisms in GBM. This project aims to study the cross-talk between the microglia cells and GBM cells in an organoid culture and its effect on the microglia and the GBM cells.

*Methodology: The human microglia cell line (HMC3) was cultured in a 3% gelatin methacrylate and 0.1% thiolated Heparasil to fabricate microglia organoids. GBM organoids were fabricated with U373 or A172 glioblastoma cell line in a hyaluronic-based hydrogel. The glioblastoma organoids were co-cultured with microglia organoids for three days and then the glioblastoma organoids’ response to temozolomide was studied. Quantitative polymerase chain reaction(qPCR) was used to evaluate the change in genetic expression of the microglia organoids in response to the co-culture with GBM organoids. The media collected from the co-cultures and single cultures were analyzed for their cytokine secretion.

*Results: The glioblastoma organoids cultured with microglia organoids exhibited resistance to temozolomide treatment. Microglia organoids exhibited higher levels of activation when co-cultured with A172 organoids compared to the U373 organoids. The media from the co-cultures exhibited different secretome profiles compared to the single cultures.

*Conclusion/Significance: Herein, we demonstrate the ability of our hydrogel model to evaluate the cross-talk between microglia organoids and GBM organoids. We have previously established patient-derived GBM organoids that have shown fidelity to the patient tumors corresponding to genotype and drug response. In the future, we hope to use the microglia organoids along with the patient-derived organoids to study the tumor-immune interaction.

414 - Development Of Complex Melanoma Spheroids Through An Assembloid-based Approach

D. B. Rodrigues^1,2, H. R. Moreira^1,2, M. Jarnalo³, R. Horta³, A. P. Marques^1,2, R. L. Reis^1,2, R. P. Pirraco^1,2

¹ I3Bs - Research Institute on Biomaterials, Biodegradables and Biomimetics, 3B's Research Group, Barco, Guimarães, PORTUGAL,

² ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, PORTUGAL,

³ Department of Plastic and Reconstructive Surgery, and Burn Unity, Centro Hospitalar de São João, Porto, PORTUGAL

*Purpose/Objectives: Animal welfare issues together with cost-effectiveness concerns and the inability of animal models to emulate the pathophysiologic heterogeneity of human disease have made 3-Dimensional (3D) in vitro models of tumors to be recognized as viable alternatives for basic research and pharma testing. This has increased the demand for tumor models of greater complexity that better mimic native tissue pathophysiology. The ability of these models to faithfully recapitulate biological processes such as intercellular and matrix interactions within the tumor microenvironment is crucial to render these models capable of mimicking the native levels of tumor aggressiveness and invasion. Herein, we outline the development of intricate 3D melanoma models utilizing an assembloid-based approach that allows the better recapitulation of the tumor microenvironment in an in vitro scenario.

*Methodology: Distinct multicellular spheroids representative of cellular interactions observed in solid tumors were generated: an endothelial cell/fibroblast domain (hDMECs/hDFbs), an endothelial/mesenchymal stem cell domain (hDMECs/hASCs), and a tumoral/fibroblast (VMM15/hDFbs) domain. These multicellular spheroids were then combined and allowed to assemble over 13d into higher complexity melanoma-like tissues (i). Histoarchitecture, gene expression, drug response and invasiveness were evaluated and the results were compared with those of traditional spheroid protocols of simpler complexity.

*Results: The method here presented allowed for the formation of 3D melanoma models with self-organizing capabilities, yielding a histoarchitecture similar to that of native melanoma (ii). The melanoma cells within these models presented stem cell-like features such as upregulated pluripotency markers together with a tendency towards higher expression of EMT-related transcription factors (iii). Furthermore, the invasiveness of the assembloid models when embedded within a 3D matrix (iv) was highly dependent on the stromal cell fraction and suggested to be mediated by what is termed “cooperative invasion”. This was evidenced by stromal cells leading the invasive edge of the produced assembloids with GP100⁺ VMM15 tumor cells following behind. Finally, these models were assessed regarding their responsiveness to the anticancer drug doxorubicin.

*Conclusion/Significance: As far as we are aware, this represents the first instance of using the assembloid method to create 3D melanoma models. The findings indicate that, when contrasted with conventional multicellular spheroids, assembloid-based melanoma models exhibit a closer resemblance to native tumor biology. These results lend support to their potential as alternatives to animal models.

415 - 3D-organotypic Co-culture Models For Depiction Of The Head And Neck Cancer Immune Microenvironment

A. Affolter, L. Hendricks, F. Jungbauer, E. Seiz, A. Azhakesan, J. Kern, N. Rotter

Department of Otolaryngology, Head and Neck Surgery, Mannheim University Hospital, Medical Faculty Mannheim of Heidelberg University, Mannheim, GERMANY

*Purpose/Objectives: Despite the therapeutic advantages of immune checkpoint inhibition (ICI) in many cancer entities, for head and neck squamous cell carcinoma (HNSCC) the objective response rate of single agent checkpoint blockade is low. Combined treatment strategies are required, so we set out to accurately characterize the immunologic microenvironment of HNSCC. The TME is supposed to regulate environmentally induced immune responses and eventually impacts on therapeutic efficacy. 3D tumor models including an intact TME are ultimately valuable for predicting responses to immune CPI. We made use of a 3D HNSCC co-culture system mimicking the tumor in its entirety. The inhibitory receptors CTLA-4, TIM-3, TIGIT and PD-1 associated with HNSCC mediate immunosuppression. CD14 is expressed as a surface protein by monocytes and macrophages.

*Methodology: By the use of 3D organotypic co-cultures (OTC) from vital HNSCC tissue samples the expression profiles of CTLA-4, TIM-3, TIGIT, PD-1 and CD14 were investigated. Tumor tissue slices were cultured on scaffolds of nonwoven viscose fibers, a fibrin/ thrombinogen-mixture and human skin fibroblasts for up to 25 days, then transferred to FFPE and analyzed by immunohistochemistry. The results were validated on archived HNSCC patient material.

*Results: The HNSCC-3D-OTCs proved to be viable over time and showed consistent morphology and expression behavior of relevant markers compared to the original tumor. We detected a distinct peritumoral accumulation of immune cells expressing CD14, TIM-3 and TIGIT. CTLA-4 and PD-1 were only observed at low levels, if at all. Interestingly, all samples showed a co-expression of TIM-3 and TIGIT.

*Conclusion/Significance: The peritumoral clusters of CD14-, TIM-3- and TIGIT-positive immune cells are assumed to have an impact on prognosis. In other tumor types, they have been described to be associated with immunosuppressive signaling and shorter survival. The simultaneous expression of TIM-3 and TIGIT could indicate T cell exhaustion. The follow up data of donors are currently being continuously evaluated. As we suppose that combinatory regimens from multiple ICIs might improve treatment response, we will simulate different immunotherapies in HNSCC-3D-OTCs and correlate the resulting expression patterns of interesting TME markers with the clinical outcome of the donor. Further studies on the expression of inhibitory receptors could contribute to new ICI combinations.

Project funding by the Ministry of Rural Affairs, Food and Consumer Protection Baden-Wuerttemberg, Germany (State Budget 2020/2021 Chap. 0802, Tit. Gr. 74).

416 - A 3d, Compartmental Tumoroid Model Of Patient-derived Bone Metastasis

M. Mohseni Garakani¹, M. Weber¹, M. R. Wertheimer², A. Ajji², D. H. Rosenzweig¹

¹ McGill University, Montreal, QC, CANADA,

² Ecole Polytechnique Montreal, Montreal, QC, CANADA

*Purpose/Objectives: Bone or spine is one of the most frequent sites of tumor metastasis originating from different organs. Complexity in bone-tumor interface with a heterogeneous microenvironment along with complex interactions between the metastatic tumor cells, bone cells, and their extracellular matrix affect cellular sensitivity/resistance to chemotherapeutics. Patient-specific therapies are challenged by current standard chemotherapeutics and the development of new anti-cancer therapeutics has a slow emerging rate mainly due to the absence of appropriate physiological in vitro/in vivo cancer models. Therefore, to properly mimic a human bone-tumor microenvironment and subsequently, the development of new therapies, the generation of more complex and physiologically relevant in vitro 3D models is essential.

*Methodology: We developed a 3D bone-tumor interface model for personalized therapeutic screening comprised of i) an electrospun nanofibrous poly lactic acid, PLA, mat treated with electric discharge plasma active species and seeded with human osteoblasts to mimic a bone tissue ii) a layer of an alginate-gelatin hydrogel embedded with either MDA-MB 231 aggressive breast cancer cell line or patient-derived spine-metastasis tumor cells, isolated from bony spinal metastases, which is overlaid, like an organoid, representing the tumor compartment to create a physiologic 3D interface model. We applied this hybrid 3D co-culture model as a migration assay tool to assess and validate the migratory behavior of different patient-derived bone metastasized cells through quantifying cell invasion/metastasis towards stromal comportment by confocal microscopy and then evaluate the impact of two different chemotherapeutics, Doxorubicin and Cisplatin, on migration and metabolic activity of both patient and immortalized cells.

*Results: We observed a quite similar trend of migration and invasion for all bone-metastasized patient cells toward the target bone tissue compartment over the course of 5 days, however, indicating a statistically significant portion, around 2.5 times more after 5 days of culture compared with two other time points, as shown by quantitative data in Figure 1. By screening both patient-derived BMB (bone-metastasized secondary to Breast) cells and their equivalent MDA-MB 231 cell line with chemotherapy drugs, we found that our innovative 3D platform is able to reduce cellular metabolic activities after exposure to varying dosages of either Doxorubicin or Cisplatin as obtained meaningful IC50 values (dose-response curve in Figure 1) that were largely in accordance with those cells’ characteristics, IC50= 0.36µM versus IC50= 1.52µM; however, Doxorubicin indicated higher efficiency or lower IC50, as for BMB patient cells, IC50=1.52µM and 28.37µM was achieved by Doxorubicin and Cisplatin, respectively. Gene expression analysis of the MDA-MB 231 breast cancer cell line that migrated through our 3D hybrid model proved epithelial-mesenchymal transition (EMT) as a critical biological mechanism occurring in cancer metastasis through increased expression of mesenchymal markers (N-cad, Slug, and Sox2) involved in the metastasis process.

*Conclusion/Significance: Our innovative 3D model enables mimicking “natural” multi-cellular interactions in a synthetic 3D environment based on separate compartments that resemble native tissues in the human body. Our data indicate that this 3D hybrid model can mimic tumor migration in vivo and has great potential to be applied as a drug screening tool for personalized medicine.

417 - Visualizing Epithelial-to-mesenchymal Transition-induced Lung Cancer Intravasation In A Microphysiological System Of The Microvasculature

C. Wong^1,2,3, Z. Lee², R. S. Tuan^1,3,4, A. Blocki^1,3,4

¹ Faculty of Medicine, Institute of Tissue Engineering and Regenerative Medicine, Sha Tin, HONG KONG,

² Faculty of Medicine, School of Biomedical Science, Sha Tin, HONG KONG,

³ Center for Neuromusculoskeletal Restorative Medicine, Sha Tin, HONG KONG,

⁴ Department of Orthopaedics & Traumatology, Sha Tin, HONG KONG

*Purpose/Objectives: Lung cancer is the leading cause of cancer-related deaths worldwide. It is often only diagnosed at advanced metastatic stages when treatment options are limited, resulting in 5-year relative survival rate as low as only 9%. For epithelial lung cancer to metastasize, cancer cells from the primary solid tumor usually undergo five key stages, namely local invasion driven by a hallmark process called epithelial to mesenchymal transition (EMT), intravasation, dissemination, extravasation, and finally colonization as a secondary tumor. Among the five stages, EMT and intravasation are the rate limiting steps determining the number of circulating tumor cells with the potential to metastasize. Conceivably, tackling EMT, local invasion and intravasation could prevent the systemic spread of cancer cells at its start and in this way improve the clinical outcome of lung cancer patients. It is thus of high importance to develop physiologically relevant models of human metastatic lung cancer to enable a better study of the underlying pathological processes, identify suitable drug targets and for high throughput drug screening. The objective of this study was thus to create a human microphysiological in vitro model of EMT-driven lung cancer intravasation in a microfluidic device.

*Methodology: To achieve this, we created a robust EMT induction cocktail (EMT-IC), based on a combination of macrophage conditioned medium (Mφ;-CM) and transforming growth factor- β1 (TGF- β1). Subsequently, the EMT-IC was applied to a co-culture system of lung cancer spheroids (microtumor masses) embedded within 3D microvascular networks (MVNs) in microfluidic devices. To visualize and quantify intravasation events, live cell imaging and high resolution microscopy were used.

*Results: The EMT-IC successfully induced EMT in A549 lung cancer cells in both 2 and 3 dimensional in vitro cultures. The introduction of EMT-IC led to a reduction in basement membrane component laminin as well as cellular junction protein VE-cadherin in MVNs. High resolution microscopy allowed the observation of intravasation events, where A549 lung cancer cells migrated out of their tumor masses and invaded into the neighboring microvascular lumen with their pseudopodia. In contrast, under control conditions fewer A549 cells migrated towards bordering microvessels, which maintained a well-defined border. Almost no intravasation events were recorded in the absence of EMT-IC.

*Conclusion/Significance: Here we have developed an in vitro 3-dimensional lung cancer intravasation model-on-a-chip that offers the ability to bridge the gap between 2D models, animal models, and the real-world clinical situation, addressing high attrition rates in drug development and assisting in the transition to personalized medicine. This model has the potential to replicate the characteristics of human tissues and cancer environment more accurately, while total cost and time spent on the drug discovery process could be reduced.

419 - A Tissue-engineered Approach To Model Pancreatic Cancer

V. J. Kast¹, S. Hauser², A. Nadernezhad¹, D. Pette¹, M. F. Maitz¹, K. C. Honselmann³, F. Baenke⁴, D. E. Stange⁴, C. Werner¹, D. Loessner¹

¹ Leibniz Institute for Polymer Research Dresden e.V., Dresden, Germany,

² Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany,

³ University Medical Center Schleswig-Holstein, Lübeck, Germany,

⁴ University Hospital Carl Gustav Carus, Medical Faculty, Technische Universität Dresden, Dresden, Germany

*Purpose/Objectives: Pancreatic cancer is a devastating disease, and therapeutic options are limited. Standard-of-care chemotherapies are insufficient, and immunotherapies have been unsuccessful in clinical trials thus far. The tumor microenvironment (TME) dictates therapeutic success. In the TME, abundant extracellular matrix (ECM) molecules and immune cells infiltrate the tumor tissue, creating a rigid and immunosuppressive barrier for drug delivery. Cell-secreted factors such as kallikrein-related peptidases (KLKs) play an essential role in the TME, driving disease progression and response to therapy. Thus, pivotal 3D preclinical cancer models integrating different elements of the TME are urgently needed to study treatment response. We developed a fully defined, multicellular 3D model of pancreatic cancer using a protease-sensitive star-shaped poly(ethylene glycol) (star-PEG)-heparin hydrogel matrix to explore new therapeutic options and drug targets. We studied the function of the cancer-associated protease KLK6 in pancreatic cancer.

*Methodology: Human pancreatic cancer cells, patient-derived cancer-associated fibroblasts, and immune cells were grown together in ECM-mimicking star-PEG-heparin hydrogels. The multicellular 3D cultures were characterized by shear rheometry and various high-resolution microscopy techniques. Live/dead staining, metabolic activity, and proliferation assays were applied to understand cell responses to chemotherapy and immunotherapy. Harnessing the CRISPR/Cas9 and RNAseq technologies, we determined the role of KLK6 in promoting cancer cell growth and changes in drug responses in 3D cancer and xenograft models.

*Results: The versatility of star-PEG-heparin hydrogels allowed us to precisely tune the mechanical properties of our 3D cancer model, resulting in a full range of tissue stiffness that aligns with measured data for pancreatic cancer tissue, which is approximately 4-15 kPa. Immunomodulatory drug treatment reduced cancer cell viability, and the CRISPR/Cas9-mediated deletion of KLK6 gene expression significantly slowed down cancer cell growth and impacted the tumor-immune landscape.

*Conclusion/Significance: star-PEG-heparin hydrogels are a powerful tool for developing tumour-engineered platforms that enable the screening of new therapeutics and drug targets.

420 - Bioengineering 3D Leukemic Niches To Develop Stem Cell Therapies

I. Tsigkos, M. J. Dalby, M. Salmeron-Sanchez, M. Vassalli, M. Tsimbouri

University of Glasgow, Glasgow, United Kingdom

*Purpose/Objectives: Standard chemotherapy remains the first line therapy against acute myeloid leukaemia (AML). However, most patients will experience disease relapse. Patients that are relapsed or refractory for standard chemotherapy can only receive haematopoietic stem cell transplantation (HSCT); nonetheless, HSCT efficiency is suboptimal due to graft rejection. While novel therapies are urgently needed in the field of AML, mesenchymal stem cells (MSCs) show great potential due to their influence on haematopoietic stem cells (HSCs). Their secretory profile favours the retention of HSCs while their immunosuppressive and anticancer properties can be of great benefit in a damaged leukemic niche. In this project we aim to demonstrate that MSCs are good candidates for establishment of a humanised leukemic model to investigate the leukemic progression as well as to optimise current cellular therapies.

*Methodology: Our laboratory has developed polymers that can chemically induce the fibrillar conformation of extracellular matrix (ECM) proteins such as the fibronectin (FN) and laminin (LAM). The fibrillar conformation of ECM proteins offers a greater availability of cell and growth factor binding sites that can mimic more accurately the interaction of cells with the ECM as well as facilitate cell-to-cell contact. In this project we are developing a cellular therapy via culturing MSCs on tissue culture surfaces coated with poly(ethyl acrylate) via plasma polymerisation, followed by the addition of FN or LAM. To investigate the leukemic progression as well as efficiency of the cellular therapy, we are using soft polyethylene glycol gels to mimic the physical properties of the bone marrow which can be further biofunctionalized with ECM proteins. The MSCs in the gels are incorporated in the form of spheroids. The gels are introduced in the upper compartment of the transwell insert while the leukemic cells are added to the lower compartment allowing the remodelling of the niche via the microporous membrane. After treatment of the niche with chemotherapy, the combined cellular therapy composed of HSCs and the engineered MSCs will be introduced in the damaged niche to investigate potential enhanced HSC engraftment.

*Results: Preliminary data have indicated that the fibrillar conformation of ECM proteins can maintain the morphology and facilitate the proliferation of MSCs, with the latter one being an essential feature for the development of a cellular therapy, since a significant cell number would be needed for a translational approach. Most importantly, an enhancement on the expression of quiescent and stemness associated as well as HSC stemness promoting markers has been observed with the addition of different ECM proteins. Lastly, regarding the leukemic model, the addition of the leukemic cells has demonstrated notable changes on the spheroid phenotype and morphology while differences can also be observed in chemoresistance.

*Conclusion/Significance: Overall, we believe that MSCs grown on fibrillar ECM acquire multiple necessary features required for improving HSCT while the development of the 3D the leukemic model has the potential to assess the efficiency of targeted therapies and serve as a personalised platform.

421 - Modelling The Interface Between Lung Cancer And The Immune System For Early Detection Biomarkers

A. D. Blake¹, E. J. Powell¹, S. B. Knight², K. G. Finegan³, S. H. Cartmell¹

¹ Department of Materials and Henry Royce Institute for Advanced Materials, Faculty of Science and Engineering, The University of Manchester, Manchester, United Kingdom,

² Division of Immunology, Inflammation and Respiratory Medicine, The University of Manchester, Manchester, United Kingdom,

³ Division of Pharmacy and Optometry, The University of Manchester, Manchester, United Kingdom

*Purpose/Objectives: < As a result of inadequate early detection, lung cancer is the largest cause of cancer-related death worldwide. There is a clear requirement for a biomarker of early-stage lung cancer to improve screening, prior to visibility on clinical scans. Given that immune cells extensively sample antigens and rapidly self-amplify in lung tissue, we believe there is a detectable change in the systemic immune system after the first contacts with early lung cancer. To test this hypothesis, we have developed a novel bioreactor for immune biomarker discovery in lung cancer, which is more physiologically relevant and higher throughput than conventional co-culture systems and in vivo cancer models (Fig. 1A). >

*Methodology: < Bioreactor prototypes were designed using SolidWorks and manufactured from liquid photopolymer resins by stereolithography. Watertight design and diffusion between chambers were tested on a scaled 4-well bioreactor using triarylmethane blue food dye in PBS solution. Cancer naïve human peripheral blood mononuclear cells (PBMCs) from whole blood were co-cultured with A549 lung adenocarcinoma cell line, 16hbe human bronchial epithelial cell line, or primary human bronchial epithelial cells for 48 h. From human squamous cell carcinoma resects, cells PBS perfusion, in localised and nodal spread tumours. All PBMC and resect samples were stained with an extracellular antibody panel for profiling by mass cytometry (CyTOF) high-dimensionality single-cell immunophenotyping. >

*Results: < We have designed, manufactured, and assembled a reusable prototype bioreactor which is watertight and permits diffusion between chambers when required (Fig. 1B, C, D). The design is compatible with plate imaging and mechanical stimulus instruments, which will enable co-culture using 2D and 3D methods. Custom multi-directional flow between 24 wells enables physiological recapitulation of lung tissue and systemic circulation (Fig. 1E). Preliminary immune profiling of PBMCs by CyTOF revealed an expansion of dendritic cells (DCs) in co-culture with A549 lung adenocarcinoma cells, but not with 16hbe bronchoepithelial cells. A similar DC population was observed at higher frequency in local tumour resects, compared to nodal spread. >

*Conclusion/Significance: < We have produced a novel bioreactor which will enable immune biomarker discovery in a high-throughput, physiologically relevant model of the interface between lung cancer tissue and the systemic immune system. Our preliminary immune profiling data shows, including DCs, become interaction with lung cancer. Future work will optimise bioreactor setup for high-throughput co-culture experiments to further characterise immune phenotypes responding to early lung cancer. To better model cancer and immune cell interactions at the earliest stages of malignancy, we are optimising a suite of lentiviral vectors for delivery of oncogenic mutations into non-cancerous epithelial cells, for co-culture with cancer naïve PBMCs. >

422 - Sheet-Based Extrusion Bioprinting: A New Multi-Material Paradigm With Mid-Extrusion Control Over Microchannel Patterning

R. Hooper¹, J. Vazquez-Armendariz^1,2, C. Cummings¹, A. Beck¹, C. Rodriguez^1,2, D. Dean¹

¹ The Ohio State University, Columbus, OH,

² Tecnológico de Monterrey, Monterrey, Mexico

*Purpose/Objectives: To reach clinical viability, bioprinting must produce multi-material constructs at high resolutions that accurately mimic complex tissue structures found in the body. One of the most fundamental structures to regenerative medicine is microvasculature. Its continuous hierarchical branching networks bridge surgically manipulatable arteries (∼1-6 mm) to capillary beds (∼10 µm). Microvascular perfusion must be established quickly for autologous, allogeneic, or tissue engineered grafts to survive implantation and heal in place. However, bioprinting techniques have struggled to produce perfusable constructs with hierarchical branching at the resolution of the arterioles (∼100-10 µm) found in microvascular tissues. Additionally, hydrogel-based bioprinted constructs typically have weak mechanical properties that present challenges for surgical handling and suturing. We recently introduced the CEVIC device (Continuously Extruded Variable Internal Channeling), a novel multi-material technology that breaks the current extrusion-based bioprinting paradigm of pushing cell-laden materials through a nozzle as filaments, instead extruding thin, wide sheets. This device employs continuous microchannel pattern control to produce hierarchical branching channels at the resolution required for microvascular vessels. These sheet constructs can also be combined with other biofabrication technologies, such as Melt Electrowriting (MEW), to create a multimodal construct with enhanced, tunable mechanical properties.

*Methodology: The CEVIC device adapts the “chaotic printing” approach (i.e., a bioprinthead design that mixes two or more hydrogels into micro-scale striations), using electric rotary valves and a novel printhead to control the diameter and number of microchannels on-the-fly during extrusion. This strategy produces continuous gradients of material variations across the construct, allowing the microchannel patterning to mimic hierarchical branching of microvascular vessels. These constructs can include fugitive (i.e., sacrificial) ink which vacates to leave perfusable channels. The device can also deposit hydrogel sheets onto polycaprolactone (PCL) MEW fiber scaffolds before hydrogel curing to create constructs with the combined properties of both materials. As a proof-of-concept, two microvascular cell types, endothelial cells (ECs) and pericytes (PCs), were co-cultured for 1 week in microchanneled Gelatin Methacryloyl (GelMA) - Sodium Alginate (SA) hydrogel sheet constructs.

*Results: Hydrogel sheets were successfully produced containing 8 to 512 microchannels with average widths ranging from 621.5 µm to 11.67 µm, respectively, covering the resolution range of microvascular networks and beyond. Continuous microchannel pattern control allowed for material switching and gradients of hierarchical channel branching across a single sheet, mimicking microvascular network structure. Vessel-like channel perfusion was demonstrated by injecting food-colored water through the constructs. Co-cultured ECs and PCs maintained over 90% viability for 1 week in GelMA-SA hydrogel constructs. Each cell type was observed within microchannels via 3D confocal microscopy using CD31 and PDGFRB antibody tags, respectively.

*Conclusion/Significance: The novel CEVIC device can fabricate constructs containing multiple microvascular cell types within microchannel patterns reminiscent of microvascular networks. Successful combination of CEVIC-produced, microchanneled constructs with MEW fiber scaffolds provides an efficient method to enhance construct strength for surgical handling and suturability. The in vitro work reported here justifies further investigation of these novel biofabrication methods, as well as pre-culture techniques to produce viable microvascular implants that can promote healing in a small animal model.

423 - Self-Enhancing Sono-Inks Enable Deep-Penetration Acoustic Volumetric Printing

X. Kuang¹, J. Yao², Y. Zhang¹

¹ Harvard Medical School & Brigham and Women’s Hospital, CAMBRIDGE, MA,

² Duke University, Durham, NC

*Purpose/Objectives: Three-dimensional (3D) printing is attracting increasing attention by directly fabricating geometry-complex constructs for prototypes, tissue scaffolds, engineered tissues. Conventional printing methods needs build-platform controlled by a linear-translation stage tosupport the step-wise material-solidification, leading to slow speed and poor surface quality. Volumetric (bio)printing, an emerging additive manufacturing technique, builds objects with enhanced printing speed and surface quality by forgoing the step-wise ink-renewal step. Existing volumetric (bio)printing techniques almost exclusively rely on light energy to trigger photo-polymerization in transparent inks. However, the light-attenuation by inks themselves, the presence of functional additives (e.g., photo-absorbers and fillers), or/and already-cured parts have imposed constraints on the material choices and the build sizes by light-based volumetric printing. Therefore, light-based volumetric printing has intrinsic limitations associated with deep-penetrating, digital-manufacturing schemes and, further, in minimally invasive fabrication scenarios.

*Methodology: We propose a novel deep-penetration acoustic volumetric printing (DAVP) based on self-enhancing sono-inks and focused-ultrasound writing technique. DAVP takes advantage of rapid material-solidification by the sono-thermal effect of the FUS focus in a viscoelastic self-enhancing sono-ink, which provides the building voxel to construct 3D objects without the need for a build-platform. The sonicated ink (or sono-inks) resolves the conflict between the acoustic penetration and streaming. The focused-ultrasound printer enables precise acoustic energy deposition. We also used experiments and acoustic modeling to study the frequency and scanning rate-dependent acoustic printing behaviors.

*Results: The viscoelastic self-enhancing sono-ink exhibited shear thinning and heat-triggered phase transition, resolved the acoustic penetration-streaming conflict. The self-enhancing sono-ink and nonlinear acoustic propagation collectively enhanced the sono-thermal heating at theFUS focus for fast and selective material-solidification (in seconds) as the building voxel. The printing resolution influenced by both the FUS frequency and focus scanning rate. Because ultrasound waves can penetration 100 times deeper than light in scattering media. DAVP enables the printing of biocompatible hydrogels and nanocomposites with various shapes regardless of their optical properties. DAVP also allows printing at centimeter-depths through biological tissues (∼17mm). We showcased the translation potentials in minimally invasive medicine, including left-atrial appendage (LAA) closure, bone-tissue reconstructions, and post-ablative drug delivery.

*Conclusion/Significance: Leveraging the deep-penetrating capability of FUS waves, low acoustic streaming, and rapid sono-polymerization of the viscoelastic self-enhancing sono-inks, we have developed a DAVP technique that can volumetrically build constructs with high printing fidelityand resolution in the absence of a build-platform. The deep penetration of FUS waves allows the volumetric fabrication of opaque (nano)composites and printing through centimeter-thick tissues that are not attainable via state-of-the-art light-based printing techniques. The self-enhancing sono-ink design can be generalized for different systems, greatly expanding the materials library for acoustic printing techniques, paving the avenue toward minimally invasive medicine.

424 - 4d Bioprinting Of A Self-folding Scaffold For Trachea Engineering

I. Chiesa¹, A. Esposito^1,2, G. Vozzi^1,2, R. Gottardi^3,4, C. De Maria^1,2

¹ Research Center E. Piaggio, University of Pisa, Pisa, Italy,

² Department of Information Engineering, University of Pisa, Pisa, Italy,

³ The Children's Hospital of Philadelphia, Philadelphia, PA,

⁴ University of Pennsylvania, Philadelphia, PA

*Purpose/Objectives: In this study we exploited the 4D bioprinting approach (i.e., fabrication via additive manufacturing of scaffolds characterized by a programmed change, over time, under a predefined stimulus) to design and develop an active scaffold able to fold in time when hydrated, to be used for trachea engineering.

*Methodology: The folding in time is achieved exploiting the differential swelling properties of bilayer films. The two layers of the scaffold were made of the same bulk material, i.e., (3-Glycidoxypropyl) methyldiethoxysilane crosslinked gelatin, GPTMS-GEL), thus guaranteeing a chemical bond between the layers, and the swelling behavior of the layers was tuned through the modification of the GPTMS and GEL concentrations. The first layer was fabricated by solvent casting. Then, 1 mm wide lines of the second layer were deposited on the first one by Extrusion-Based Bioprinting. Scaffold folding behavior was tested in deionized water and PBS. Then, 10 mm x 5 mm rectangular scaffold were sterilized by 1h of UV light and incubated in cell culture media for 2h. 20k cells were seeded on each scaffold before the actuation and cultured for 10 days in growth medium. Three different cell types were tested: human vocal folds, lung epithelial cells, and human ear chondral progenitor cells (eCPCs). Cell viability (Live/Dead Assay) and cell proliferation (Alamar Blue Assay) were evaluated. Finally, the ability of eCPCs to differentiate towards mature cartilage was evaluated. Briefly, cells were seeded as described before, and cultured for 7 days to allow the complete coverage of the scaffold. Then, medium was switched to chondrogenic medium and cells were cultured for additional 21 days. Histological Assay and Real time PCR were performed to verify the chondral commitment of cells. Monolayer static scaffold made of the same materials were used as control.

*Results: When dipped in a water-based solution the 4D bioprinted scaffold self-folds maintaining its shape in time (Fig. 1A). The presence of precisely oriented lines as second layer of the scaffold provides a complete control over the film folding direction (Fig. 1B). In vitro tests showed the ability of cells to adhere on the scaffold in its flat position and maintain their adhesion during scaffold folding, that occurs 24h after seeding, and confirmed the biocompatibility of the GPTMS-GEL materials, that promotes viability, proliferation, and metabolic activity of all the tested cell types (Fig. 1C). Regarding the cartilage differentiation analysis, Alcian Blue histological analysis confirmed the deposition of cartilaginous matrix in the external layer of the scaffold (Fig. 1D). Moreover, an overexpression of SOX9, ACAN e COLII was observed on the 4D scaffold, when compared with static monolayer films. No differences arise in the expression of PRG4 and COLX, suggesting the establishment of a stable chondral phenotype on the 4D scaffolds.

*Conclusion/Significance: In this study, we exploited the 4D bioprinting approach to fabricate a self-folding bilayer scaffold for trachea engineering. The reported results represent an important milestone for 4D bioprinting, quantitatively showing that shape-morphing scaffolds in the millimeter scale can promote cells activity and differentiation.

425 - Additive Biomanufacturing Of Scaffold/Membrane Designs For In Vitro Skin Models

I. Liashenko¹, F. Girard², A. Ramon³, S. Luposchainsky¹, J. Rosell-Llompart^4,5, A. Cabot^3,5, H. Xu⁶, D. Hutmacher⁷, M. Rielland², P. Dalton¹

¹ Knight Campus for Accelerating Scientific Impact, Eugene, OR,

² L’Oréal Research and Innovation, Aulnay-sous-Bois, France,

³ Catalonia Institute for Energy Research (IREC), Barcelona, Spain,

⁴ Universitat Rovira i Virgili, Tarragona, Spain,

⁵ Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain,

⁶ Kyoto Institute of Technology, Kyoto, Japan,

⁷ Queensland University of Technology, Brisbane, Australia

*Purpose/Objectives: In vitro tissue engineering of microenvironments is greatly limited by its ability to fully recreate the native tissues of organs, especially for complex layered tissues of the organ skin with distinct cells and morphologies. Many different technologies have been proposed and demonstrated for scaffold fabrication, each one with specific advantages and limitations. ‘Hybrid fabrication’ combines manufacturing techniques to permit new and otherwise unreachable designs for hierarchical cell-material constructs. As presented, the hybrid fabrication approach has been applied for in vitro skin models, resulting in clear hierarchical tissues that neither technique can achieve alone. Using a microscopic 3D printing technology, melt electrowriting (MEW), we achieve fine control over the scaffold morphology in terms of fiber diameters, alignment, porosity, and mechanical properties. Combined with a membrane produced by solution electrospinning (SES), we demonstrate the first application of this advanced bilayer scaffold for the successful engineering of a full-thickness human skin model.

*Methodology: Custom-built MEW printers and SES systems were used in additive biomanufacturing of scaffold/membrane designs from medical-grade polycaprolactone. Scaffolds design with 12±1 µm MEW fibers mimic the microenvironment of the human dermis, while SES membrane with 0.3±0.1 µm fibers act as a cell occlusive membrane between the dermis and epidermis compartments, allowing to seed human fibroblasts and keratinocytes simultaneously. To our knowledge, for the first time submicrometer fibers ranging 0.3-0.9 microns were achieved by employing electrostatic deflection of an airborne jet by jet-steering electrodes.

*Results: A bilayer scaffold/membrane was developed for in vitro culturing by seeding cells onto either side, producing a dermal epidermal junction after only 18 days. Neosynthesized extracellular matrices (ECM) formed in locations as expected, including collagen I, fibrillin, elastin, perlecan in the dermal compartment and in epidermis: collagen IV, keratin 10 in the basal layer, keratin 14 in the stratum spinosum, filaggrin and transglutaminase 1 in the stratum granulosum and stratum corneum. Moreover, the design of the MEW component impacted dermis morphology and permitted tailoring of the skin model. Furthermore, breaking the current record of MEW, we produced fibers as thin as 300 nm, approaching the scale of native ECM fibers. Such submicron fibers were generated at 5 fold higher speeds than typical for MEW, and thus could not be accurately printed with mechanical stages. Instead, their deposition was controlled electrostatically by using auxiliary electrodes. Such nanofibers interact with light to produce structural color, and their accurate printing is a new manufacturing technique that, together with structured membranes, embodies a promising strategy for future designs of advanced scaffolds, opening new opportunities for more complex tissue models.

*Conclusion/Significance: We report the first time of developing and characterizing a scaffold/membrane using SES and MEW for skin in vitro models, resulting in a properly stratified and differentiated epidermis and rich neosynthesized ECM in just 18 days. Moreover, the versatility and adaptability inherent to hybrid fabrication of intricate layered scaffolds present unique opportunities and highlight the advantages toward tissue engineering compared to single biofabrication methods, including the use of gels, bioprinting, or scaffolds with limited control over morphology.

428 - Engineered Cocultures Of Induced Pluripotent Cell-derived Atrial Cardiomyocytes And Atrial Fibroblasts For Modeling Atrial Fibrillation

A. Michell, G. Brown, Y. Han, S. Khetani, D. Darbar

University of Illinois Chicago, Chicago, IL

*Purpose/Objectives: Atrial fibrillation is the most prevalent arrythmia affecting >33M individuals, but current drug therapies are not highly effective. Given species-specific differences in cardiac function, in vitro human atrial models are critical for modeling diseases and novel drug discovery. Induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) are a sustainable and patient-specific cell source useful for elucidating genotype-phenotype relationships. However, current protocols yield functionally immature iPSC-aCMs which restricts their use for the above applications. Furthermore, maturation protocols lack the multiwell plate format routinely employed by the pharmaceutical industry for large-scale drug screening. We sought to test the novel hypothesis that combining micropatterned iPSC-aCMs with co-cultured atrial cardiac fibroblasts (ACFs) could significantly improve iPSC-aCM maturity within a multiwell plate format to facilitate high-throughput drug screening and disease modeling.

*Methodology: Micropatterns of fibronectin (FN) were constructed on tissue culture polystyrene (TCPS) plates using soft lithography followed by sequential seeding of iPSC-aCMs and ACFs onto the patterned substrates to form patterned co-cultures (PC). For random monoculture (RM) controls, iPSC-aCMs were seeded onto FN-coated TCPS. After 3 weeks, sarcomeres were visualized via immunofluorescent staining for cTnT and α-actinin. PC and RM were repeated with iPSC-aCMs derived from a patient with an E428K mutation causing atrial fibrillation and with CRISPR-gene-corrected-E428K iPSC-aCMs (Fig. 1D). Electrophysiological signature was analyzed with optical voltage mapping (OVM) using a voltage sensitive dye. Fibroblast RNA was isolated via TRIzol^TM; RNA sequences were aligned using BioJupies and analyzed in R. Fibroblast gene knockdowns were performed with small interfering (si)RNA prior to PC with iPSC-aCMs, and sarcomeres were visualized compared to RM and to PC with non-targeting siRNA knockdowns.

*Results: iPSC-aCMs selectively attached to the FN micropatterns, followed by CF seeding to create PCs (Fig. 1A). The width and spacing of the patterns were optimized for iPSC-aCM maturation (not shown). Immunofluorescence revealed sparse and randomly oriented sarcomeres in RMs but aligned and organized sarcomeres in PCs (Fig. 1B). Sarcomere length in PC was 1.2-fold higher than RM, and close to that of adult primary cardiomyocytes (Fig. 1C). Additionally, the atrial fibrillation affected line showed a fibrillation phenotype via OVM that could be corrected by CRISPR in PC, but not in RM (Fig. 1E). Since ACFs showed increased expression of ephrin signaling pathways compared to VCFs (Fig. 1F), gene knockdowns of ACF EFNB1 were performed. iPSC-aCMs in PCs with ACF EFNB1 knockdowns showed significantly disrupted sarcomeres (Fig. 1G), thereby suggesting the role of this molecule in the induction of iPSC-aCM maturation by the ACFs.

*Conclusion/Significance: We show for the first time that micropatterned co-culture with ACFs significantly improves the structural and functional maturity of human iPSC-aCMs in a multi-well format for high-throughput drug screening and modeling genetic variants associated with atrial fibrillation We also found that ACF expression of ephrin ligand EFNB1 is critical for iPSC-aCM function, thus revealing a novel mechanism underlying iPSC-aCM maturation. Future efforts are focused on incorporating donor-matched iPSC-derived endothelial cells and fibroblasts in this platform to model heterotypic cell-cell signaling in cardiac physiology and atrial fibrillation.

429 - Mimicry Of Haemodynamic Waveforms For Vascular Bioreactors

T. C. Mitchell, S. G. Wise

School of Medical Sciences, The University of Sydney, Sydney, AUSTRALIA

*Purpose/Objectives: Vascular bioreactors are important tools for in vitro modelling, device or biomaterial evaluation, and tissue engineering. Typically, vascular bioreactors are perfused using mechanical pumping systems such as peristaltic or piston pumps. These pumping systems have a limited capacity to replicate a full set of haemodynamic conditions and focus on volumetric flow rate or shear stress without producing physiologically relevant pressure and pulse rate. In this study we demonstrate a blood-vessel-in-a-box system capable of versatile, high-fidelity reproduction of physiologically and clinically relevant haemodynamic conditions in a 3D environment.

*Methodology: A novel pumping system was developed using pneumatically pressurized chambers inspired by the principles of the human heart. Feedback from an array of sensors allows coordination of the pump chambers to regulate pressure, flow, and shear stress in real time. Specific parameters such as pulse rate, systolic pressure, diastolic pressure, peak flow, and average flow can be selected independently and maintained without user intervention. Specific pressure and flow waveform profiles were demonstrated including sinusoidal waveforms, peripheral artery waveforms and coronary artery waveforms. Coronary and peripheral waveforms were generated based on both generic shape profiles and patient-specific profiles.

*Results: We demonstrate that vascular cells such as human coronary artery endothelial cells (hCAECs), smooth muscle cells (SMCs) and human fibroblasts (hFBs) show distinct changes in morphology, gene and cell surface marker expression under physiological conditions (120/80 mmHg, 60 ml/min, 20 dyne/cm²) compared to either static or conventional (peristaltic) pumping conditions.

*Conclusion/Significance: Culturing vascular cells under physiologically relevant haemodynamic conditions led to significant changes compared with static culture controls, highlighting a key role for exposure to these forces in addition to relevant 3D scaffolds and biomaterial properties. The bioreactor is a new platform for studying cell behaviour and their interactions with biomaterials under physiological and pathological conditions.

430 - 3D In Vitro Modelling Of Human Cardiac Fibrotic Tissue Through Bio-hybrid Stretchable Scaffolds

A. Zoso^1,2, F. Tivano^1,2, M. Spedicati^1,2, M. Lavella³, I. Carmagnola^1,2, V. Chiono^1,2

¹ Politecnico di Torino, Torino, Italy,

² Interuniversity Center for the Promotion of the 3Rs Principles in Teaching and Research, Pisa, Italy,

³ Università degli Studi di Bergamo, Dalmine (BG), Italy

*Purpose/Objectives: Human cardiac fibrotic tissue is a pathological condition that arises after myocardial infarction. It is characterized by outnumber fibroblasts activated into myofibroblasts, increased stiffness and passive mechanical stress during heart functionality. New advanced regenerative approaches are currently under investigation to reduce or revert cardiac fibrosis and in vitro;models of human cardiac fibrotic tissue may improve their preclinical investigation. Herein, an in vitro model of human pathological cardiac tissue based on stretchable scaffolds, able to mimic the biophysical and biochemical properties of post-infarct fibrosis was developed.

*Methodology: Scaffolds of polycaprolactone (PCL) with parallel-wavy fibers pattern were designed and fabricated by melt-extrusion additive manufacturing (MEAM). Gelatin methacrylate (GelMA) hydrogels with different concentrations (5, 7, 10% w/v) were prepared using lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP) as photoinitiator. Photo-rheology allowed to define the optimal hydrogel curing protocol. Both PCL scaffolds and GelMA hydrogels were analysed by SEM imaging.PCL scaffold pores were filled with GelMA hydrogels, to mimic extracellular matrix (ECM)-like microenvironment. To improve interfacial adhesion between GelMA hydrogel and scaffold structure, PCL were surface functionalized with poly(4-Dihydroxy-DL-phenylalanine) (polyDOPA). Static and cyclic tensile tests were performed on PCL/GelMA scaffolds. Human Cardiac Fibroblasts (HCFs) were seeded in PCL/GelMA scaffolds (cell density: 5·10⁶ cells/mL) and cultured for up to 2 weeks. HCFs viability was tested through Resazurin assay, while their morphology and distribution were analyzed through F-actin staining in order to identify optimal GelMA concentration for HCFs culture. Then, cellularized PCL/GelMA scaffolds were subjected to cyclic mechanical stimulation for 7 days. Fibroblast activation into myofibroblasts was analyzed through immunofluorescence of α-Smooth Muscle Actin (α-SMA).

*Results: Stretchable PCL scaffolds with high shape fidelity were obtained by optimizing MEAM processing parameters. Analytic and finite element analysis analyses allowed the validation of the experimental results. Mechanical properties of PCL/GelMA scaffolds were tailored by PCL scaffold thickness, mesh geometry, and GelMA hydrogel concentration in order to mimic the stiffness (1-9 MPa) and maximum elastic strain (15-22%) of human cardiac fibrotic tissue. GelMA hydrogel pore sizes increased by reducing GelMA concentration. PCL/GelMA scaffolds preserved stretchability and integrity after both static and cyclic tensile tests. HCFs viability after 7 and 14 days was significantly higher for cells cultured in PCL/GelMA 5% w/v and PCL/GelMA 7% w/v, compared to PCL/GelMA 10% w/v. F-actin staining showed homogeneous cell distribution inside GelMA hydrogels, while an elongated morphology was noted only on HCFs in PCL/GelMA 5%. Finally, HCFs demonstrated α-SMA expression only on samples treated with cyclic mechanical stimulation. Secretion of fibrotic ECM proteins such as Collagen I, Collagen III, and Fibronectin are under investigation.

*Conclusion/Significance: In this work, an in vitro model of human fibrotic cardiac tissue based on stretchable PCL/GelMA scaffolds was developed. The model demonstrated fibrotic hallmarks in a dynamic cyclic culture environment. In the future, this model will be validated for use in preclinical testing of new cardiac regenerative strategies, with the advantage to allow long-term dynamic testing.

Acknowledgment: BIORECAR project has received funding from the European Research Council (ERC) under the H2020 research and innovation program (772168).

431 - DEVELOPMENT OF PHOTOCROSSLINKABLE BIOINKS WITH IMPROVED ELECTROMECHANICAL PROPERTIES FOR 3D BIOPRINTING OF CARDIAC BIORINGS

A. Mousavi, H. Savoji

University of Montreal, Montreal, QC, Canada

*Purpose/Objectives: 3D bioprinting is an advanced fabrication technique to build biomimetic constructs for different biomedical applications. Here, we developed composite bioinks based on photocrosslinkable natural polymers, gelatin methacryloyl (GelMA) and alginate methacrylate (AlgMA), and electroconductive reduced graphene oxide (rGO) nanomaterials. The ring-shaped cardiac constructs were further 3D bioprinted with different cardiac cells for the fabrication of an automated high throughput heart-on-a-chip model.

*Methodology: The bioinks based on GelMA, AlgMA, and rGO were synthesized and characterized, and the bioinks were formulated with different concentrations. The physicochemical, structural, rheological, electromechanical, printability and printing fidelity, swelling and degradation properties, and cytotoxicity of these bioinks were investigated. The bioinks were further 3D bioprinted in a ring-shaped construct (hereafter BioRing), and cell viability, proliferation, migration, spreading, and functionality were demonstrated. Finally, heart-on-a-chip platforms containing micropillars were designed and fabricated to support these Cardiac BioRings for a proof-of-concept automated high throughput heart-on-a-chip application.

*Results: In this study, a 3D bioprinted ring-shaped cardiac tissue model was successfully developed based on photocrosslinkable natural biopolymers (AlgMA and GelMA) and conductive nanomaterials (rGO). All of these three components of the bioinks were synthesized and validated by different characterization techniques. The structural, electromechanical, rheological, printability, and biodegradation properties of 3D biohybrid constructs were highly tunable using varied concentrations of AlgMA and rGO. The optimization process was performed to achieve an ideal bioink composition that mimics the properties of the native extracellular matrix of cardiac tissue. This optimal bioink was composed of 5 % GelMA, 1 % AlgMA, and 0.2 mg/ml rGO. Cellular evaluations (with encapsulated cardiac fibroblasts (CFs)) confirmed its ability to support high levels of cell adhesion, viability, proliferation, and migration. Cardiac BioRings were further created using different cardiac cell types (primary CFs and cardiomyocytes (CMs) as well as HL-1 CMs), and functional characterizations showed a high level of cell growth, alignment, spreading, elongation, alignment, and interconnection between the cardiac cells with phenotypic morphology (spindle-like CFs and rod-shaped CMs) and detectable troponin complex and sarcomeric structures in CMs. The functionality of cardiac BioRings was further confirmed by calcium transients and cellular contractions. As proof of concept, these cardiac BioRings were bioprinted on PDMS chips containing pillars, and the automated high-throughput bioprinting on-chip was confirmed using 24-well plates.

*Conclusion/Significance: These bioprinted cardiac models could create new outlooks in cardiac tissue engineering and heart-on-a-chip platforms towards contractile biological pumps, heart regeneration, high throughput drug discovery and screening, and cardiovascular disease modeling.

432 - 3D Models Of Dental Development For Personalized Medicine And Reduction Of Diagnostic Wandering In Patients With Rare Oro-Dental Structural Disorders

V. Gribova¹, I. Bugueno^1,2, E. Suss¹, G. Caravello^3,4, M. Kawczynski^1,3, Y. Arntz^5,6, A. Bloch-Zupan^1,3,6

¹ Translational Medicine and Neurogenetics, Institut de génétique et de biologie moléculaire et cellulaire (IGBMC), Illkirch-Graffenstaden, FRANCE,

² Orofacial Development & Regeneration Unit, University of Zurich, Zürich, SWITZERLAND,

³ Reference Centre for Orodental Manifestations of Rare Diseases, Hôpitaux Universitaires de Strasbourg, Strasbourg, FRANCE,

⁴ Laboratoires de diagnostic génétique, Institut de Génétique Médicale d’Alsace, Strasbourg, FRANCE,

⁵ Biomaterials and Bioengineering, Inserm UMR_S 1121, Strasbourg, FRANCE,

⁶ Faculté de Chirurgie Dentaire, Université de Strasbourg, Strasbourg, FRANCE

*Purpose/Objectives: Healthcare professionals, researchers, patients, and their families affected by rare diseases face many difficulties during diagnosis. This is the case of developmental dental anomalies like Amelogenesis imperfecta (AI) and Dentinogenesis imperfecta (DI) /Dentin dysplasia (DD), which are rare genetic diseases. A targeted genetic study on rare diseases with oral manifestations, using high-throughput sequencing technologies (GenoDENT) has allowed the identification of numerous pathogenic genetic variants (>60% of diagnostic rendering), but also numerous variants of unknown significance (VUS) (Figure 1A). The potential pathogenicity and functional impact of these VUS remain uncertain, which complicates genetic diagnosis. In order to unravel the pathogenicity of VUS during amelogenesis and dentinogenesis, we are working on innovative 3D in vitro models of tooth development.

*Methodology: The study includes characterization in 2D of murine and human cell lines recapitulating amelogenesis (ameloblast-like cells) and dentinogenesis (dental pulp stem cells), and CRISPR-Cas9 genome editing to introduce patient-specific mutation. To create 3D models, the cells are assembled into bilayered organoids (Figure 1B) using low-attachment plates and characterized. Finally, biomaterials-based approaches such as culture in hydrogels (Figure 1C), 3D printing or coating the cells with a biomimetic artificial matrix (Figure 1D) are evaluated.

*Results: Gene expression, protein secretion and mineralization assays showed that the selected cell lines were appropriate models for in vitro mimicking amelogenesis and dentinogenesis. The first 3D models, organoids, were efficient for murine cells, with a good distinction between the two cell types used and good stability of the models over time (Figure 1B). However, the formation of organoids from human cells was less efficient. To overcome this problem, we started a new 3DBioDENT project aimed to improve 3D models made of human cells using biomaterials. Two approaches are currently being evaluated: cell-accumulation method, consisting in cell coating with fibronectin and gelatin nanofilms (Figure 1D) and collagen 1 hydrogel-based culture (Figure 1C). Both methods show good cell viability inside the 3D constructs, with better stability over time during differentiation process of nanofilm-coated microtissues. Further characterization is ongoing.

*Conclusion/Significance: In conclusion, we developed new in vitro 3D organotypic cell cultures (submitted), using odontoblast and ameloblast-like cells, and we continue working on model improvement by associating the cells with biomaterials. In the next step, mutant cell lines carrying the VUS, created by CRISPR/Cas9 gene editing, will be studied in a 3D environment in order to determine the functional consequences and pathogenicity of the new VUS identified using GenoDENT NGS panel. Personalized tooth development models will play a vital role in genetic diagnosis, improving diagnostic accuracy and reducing diagnostic wandering in patients with rare oro-dental diseases.

433 - Crispra Based Upregulation Of Runx2 Or Sox9 Expression In Goat Bmsc For Regeneration Of The Temporomandibular Joint

J. D. Stover, A. Almarza

University of Pittsburgh, Pittsburgh, PA

*Purpose/Objectives: The CDC estimates that 54.4 million US adults (22.7%) have been diagnosed with some form of arthritis (osteoarthritis, rheumatoid). The temporomandibular joint (TMJ) is also susceptible to OA (TMJ-OA). It has been shown that a third of patients undergoing total replacement of the TMJ had TMJ-OA without comorbid conditions. Currently, there are no FDA approved allograft alternatives to total joint replacement. Recently, CRISPR epigenome editing has been utilized to engineer bone marrow stem cells (BMSCs) with enhanced therapeutic properties. In this study, we examine the potential of utilizing CRISPR upregulation (CRISPRa) of Runx2 or Sox9 expression in goat BMSCs to enhance their therapeutic potential of regeneration of the TMJ.

*Methodology: CRISPR Epigenome Editing Lentiviral Vector Production: We created CRISPR epigenome editing that express 1) dCas9-VPR-PuroR or 2) gRNAs that target the promotor region of Runx2 or Sox9, or a non-target gRNA sequence that does not match the goat genome (Figure 1A). Lentiviral vectors were produced via co-transfection of HEK293T cells with 4μg of VPR or gRNA lentiviral vector, 3μg psPax2, and 1.2 µg of pMD2.G using lipofectamine 2000. Kill Curve Experiments: To determine the puromycin dosage for selection of VPR expressing cells, we performed a kill curve experiment. Goat BMSCs (n=5 donors) were seeded in 96 well plates and cultured in growth media(α-MEM + 10%FBS +1% Pen/Strep) supplemented with puromycin. Cell viability was measured via CCK-8 assay. Verification of Runx2 and Sox9 Gene Upregulation: BMSCs were co-transduced with VPR and gRNA lentiviral vectors with polybrene (8μg/ml). Following transduction, viral media was removed and replaced with puromycin selection media. Gene expression was measured via RT-qPCR 7 days after transduction.

*Results: Growth media supplemented with 1 µg/mL was demonstrated to be the minimum dose of puromycin required to kill BMSCs after 3 days (Figure 1B), and was thus used in all succeeding steps. Transduction efficiency of BMSC with gRNA vectors was high(90%, Figure 1D inset). BMSCs transduced with gRNAs targeting the Runx2 promotor region exhibited significantly higher expression (p<0.05) of Runx2 when compared to nontarget (NT) controls, with guide g3 exhibiting the highest upregulation of Runx2 expression (8.68 ± 0.17 fold) (Figure 1C). Similarly, BMSCs transduced with gRNAs targeting the Sox9 promotor region exhibited significantly higher expression (p<0.05) of Sox9, when compared to NT controls (Figure 1D). BSMCs transduced with guide g4 exhibited the highest upregulation of Sox9 expression(10.27 ± 0.34 fold). Together, these results demonstrate that CRISPR epigenome editing of BMSC can be utilized to upregulate expression of Runx2 and Sox9, key regulators of osteogenic and chondrogenic differentiation, respectively. Future work will focus on evaluating the ability of CRISPR upregulation of Runx2 and Sox9 to drive BMSC differentiation.

*Conclusion/Significance: These results demonstrate that CRISPR epigenome editing can be utilized to upregulate expression of key transcription factors that regulate BMSC osteogenic and chondrogenic differentiation and suggests this approach may be utilized in therapeutic strategies for TMJ regeneration.

434 - Oral-Maxillofacial Bone Regeneration Using 3D-Printed Scaffolds Loaded With Adipose Stromal Vascular Fraction

C. Lau¹, J. Lim², L. Saigo¹, B. Goh¹

¹ National Dental Centre Singapore, Singapore, Singapore,

² Osteopore International Pte Ltd, Singapore, Singapore

*Purpose/Objectives: Oral-maxillofacial bone defects are challenging to restore due to the presence of multi-directional forces during mouth movement and the complex anatomy of the oral region. Current treatments involving bone grafts provide mechanical support to the defect but do not fully address vascularization and immune response. Stem cells can be applied to accelerate bone formation and vascular growth but traditionally-used bone marrow stem cells (BMSCs) have numerous limitations including complicated harvest, low cell yield and lengthy cell expansion ex vivo. On the other hand, adipose stromal vascular fraction (SVF), the cellular component of adipose tissue, is becoming popular for regenerative medicine due to its heterogeneous cell population of mesenchymal stem cells (MSCs), endothelial cells, and various immune cells, which can promote osteogenesis, angiogenesis and immunomodulation. The objective of this study is to evaluate the efficacy of a SVF-loaded scaffold in vitro and in vivo in a porcine alveolar defect model.

*Methodology: Our study consists of 2 phases. In the first phase, the biocompatibility between porcine SVF and 3D-printed polycaprolactone-tricalcium phosphate (PCL-TCP) scaffolds with different structures was investigated in vitro. In the second phase, the optimal PCL-TCP scaffold from the first phase was loaded with autologous porcine SVF. The SVF-loaded scaffold, the blank unloaded scaffold and autologous bone (serving as positive control) were implanted into porcine alveolar defects to evaluate the efficacy of the added SVF in alveolar bone regeneration. After 3 months, the alveolar regions were harvested from the animals, and the the extents of new bone formation, vascularization and immune reaction of the various groups were evaluated via radiographic and histological analysis.

*Results: In the first phase, we concluded that a pore size of 2mm in the 3D-printed polycaprolactone scaffold was optimal for SVF cell proliferation and viability. In the second phase, radiographic analysis revealed that the addition of SVF to the scaffold improved the bone volume in the defect from 18.6% to 28.7% after 3 months of healing. The efficacy ratio (bone volume of test group / bone volume of “autologous bone” group) also increased from 41% to 65% with the addition of SVF. From the histology data, we deduced that the presence of SVF in the PCL-TCP scaffold led to better formation of new bone, better vascularization and less likelihood of fibrous encapsulation of the scaffold.

*Conclusion/Significance: We showed that the addition of SVF improved the clinical outcome of alveolar bone regeneration. SVF is a superior option compared to BMSCs as the harvest of SVF is easier and less invasive. The rapid process of SVF isolation also avoids the need for ex vivo cell expansion and allows the whole process from tissue harvest to scaffold implantation to be done within one surgery session, saving time and costs for patients and the clinic.

435 - RegendoGEL, a Novel Regenerative Endodontic Treatment Product for Vital Pulp Therapy

W. Zhang¹, A. Bartolome², V. Morello¹, A. Hume¹, S. Olson³, L. E. Bertassoni², P. C. Yelick¹

¹ Tufts University, School of Dental Medicine, Boston, MA,

² OHSU School of Dentistry, Portland, OR,

³ RegendoDent, Inc., Ann Arbor, MI

*Purpose/Objectives: Dental caries remains one of the most common diseases found in humans. The2022 WHO Global Oral Health Status Report estimates that globally, ∼2 billionpeople suffer from caries of permanent teeth, and 514 million children sufferfrom caries of primary teeth. Current vital pulp therapies used to preserve thevitality and function of dental pulp, rely mostly on the use of nondegradablesilicate cements that promote reparative dentin bridge formation to protect underlyingvital dental pulp. Here we propose RegendoGEL (RG) as a novel, biodegradable,regenerative vital pulp therapy, which induces dentin bridge formation in 5days, and dental pulp tissue regeneration by 70 days.

*Methodology: RegendoGEL, created by our RegendoDent Inc. team, consists of hydrogelmicroparticle (RG-MP) or membrane (RG-M) forms containing dentin matrix molecules(DMMs) extracted from bovine teeth. We then compared vital pulp therapy treatedteeth with RG-MP or RG-M form factors using an in vivo beagle dog pulpotomymodel. Eight mandibular teeth (left and right, P2-4 and M1 molars) were treatedper dog. Briefly, a defect was created on the occlusal side of treated teeth toexpose the dental pulp. A small portion of dental pulp within the pulp chamberwas removed, and the RG or control pulp capping material was carefully placedand pressed firmly onto the exposed pulp. Six different capping materials were compared,including RG-M with and w/o MTA (mineral trioxide aggregate, n=6 each), RG-MP withand w/o MTA (n=6 each), MTA alone (n=4), and Biodentine, a pulp cappingmaterial commonly used in dental clinic (Biodentine, n=4). All treated teethwere restored using the glass ionomer Ketac Molar. In total, 32 teeth weretreated for each of 2 time points, 5 and 70 days. Prior to harvest, dogs wereperfused with fixative, and treated teeth and jaw bones were harvested andfixed in formalin. Micro-CT analyses were conducted followed bydemineralization, paraffin embedding and sectioning, and histological andimmunohistochemical analyses.

*Results: Micro-CT and histological analyses showed that RG-MP+MTA (5/6), RG-M+MTA (3/6), and Biodentine (4/4) showed the formation of a thin dentin bridge by5 days after treatment. By 70 days, RG-M + MTA (3/6), RG-MP + MTA (5/6), RG-M(2/6), RG-MP (2/6), and Biodentine (4/4) treated teeth showed dentin bridgeformation. Full dentin bridge was only observed in RG-M (2/6), RG-MP (3/6), andBiodentine (3/4) groups, respectively. None of the MTA groups showed any dentinbridge formation. Additional immunohistochemical analysis is ongoing.

*Conclusion/Significance: Our results showed that both RG-MP andRG-M performed well as pulp-capping materials when used in conjunction with MTA,with the key advantage of being fully biodegradable and regenerative, adesirable property that no existing product offers. Furthermore, dental pulp treatedwith RG exhibited minimal signs of inflammation at 5 days which is advantageousfor maintaining pulp vitality, and thicker dentin bridge and significant vascularizeddental pulp tissue regeneration by 70 days. We present RegendoGEL as a potential first regenerative endodontictreatment for vital pulp therapy.

436 - Stimulation Of Gingiva Self-generation Using Bio-sheets Produced During Fibrotic Encapsulation Of Mold

K. Shiraishi¹, T. Harada¹, Y. Huang¹, U. Taguchi¹, R. Narita¹, Y. Ota², Y. Asawa³, H. Komura³, K. Hoshi¹, M. Komura³

¹ Department of Oral-Maxillofacial Surgery and Orthodontics, The University of Tokyo Hospital, Tokyo, JAPAN,

² Department of Pathology, Research Hospital, Institute of Medical Science, The University of Tokyo, Tokyo, JAPAN,

³ Division of Tissue Engineering, The University of Tokyo Hospital, Tokyo, JAPAN

*Purpose/Objectives: Gingival recession occurs due to inappropriate orthodontic forces, occlusal trauma, brushing pressure and periodontal disease. Currently, this is treated via free gingival graft (FGG) and connective tissue graft (CTG) implantation. These grafting techniques involve harvesting the patient's own palatal gingiva and connective tissue via an invasive procedure; these operations lead to complications such as graft rejection, necrosis, and color tone differences. Our group is studying the promotion of tissue self-regeneration with fibrous tissue bodies (BIO-SHEET), which are formed during the encapsulation of a subcutaneously implanted mold by a foreign body reaction. The BIO-SHEET method has been used successfully to induce self-regeneration of diaphragm and trachea cartilage tissue following transplantation into these affected areas. In the current study we investigated the utility of the BIO-SHEET method for promoting gingival regeneration.

*Methodology: The gingiva of the left front tooth of 6-week-old F344/N rat was excised in a 2 x 2 mm section. The whole palatal gingival graft (FGG) group (FGGgroup) consisted of rats with grafts of a 2 x2 mm-sized palatal gingiva taken from the same individual. The connective tissue graft (CTG) group (CTG group) consisted of rats with grafts of a 2 x 2 mm-sized palatal connective tissue taken from the same individual and covered with persistence gingiva in accordance with the original CTG procedure. The peri-mold encapsulated tissue transplantation group (BIO-SHEET group) consisted of 4-week-old rats with subcutaneous transplants of molds from which the tissue encapsulating the peri-mold was removed 2 weeks later. The left front teeth gingiva of the same individuals was excised in a 2 x 2 mm section before transplantation of the peri-mold encrusting tissue. We recorded the gross findings of the grafted site and measured the color difference compared to the normal gingiva for 4 weeks following implantation. Histological comparisons were also made between each group.

*Results: Gross findings: there was more recession at the graft sites in the FGG and CTG groups than at the contralateral normal gingiva, while no gingival regression was observed in the BIO-SHEET group. The color difference (ΔE) was smallest in the BIO-SHEET group (the color of the graft site was most similar to the normal gingiva), whereas the difference was significantly larger in the FGG and CTG groups. Histological findings: The BIO-SHEET group resembled normal gingiva histologically; lamininγ2-positive areas were evident between the enamel and gingival junctional epithelium in this group. Electron microscopic findings showed hemidesmosomal junctional-like structures between the enamel and gingival junctional epithelium in the BIO-SHEET group. No hemidesmosomal binding was observed in the FGG or CTG groups.

*Conclusion/Significance: There was no gingival recession 4 weeks after implantation in the BIO-SHEET group, and the color at the graft site was similar to that of the normal gingiva, with a good esthetic appearance. We infer that physiological connectivity was restored after BIO-SHEET implantation, and suggest that BIO-SHEETs constitute excellent grafting material for treatment in the event of gingival recession.

437

438 - Engineering External Boundaries To Guide Neotissue Growth Within High Cell Density Bioinks

A. S. Karam, G. S. Kronemberger, K. Chattahy, D. J. Kelly

Trinity College Dublin, Dublin, Ireland

*Purpose/Objectives: Osteoarthritis is a painful inflammatory disease characterized by articular cartilage (AC) degeneration, affecting millions of individuals worldwide. The unique arcade-like collagen architecture of AC is fundamental to its biomechanical function. Classical tissue engineering strategies fail to produce zonally organized AC. Emerging 3D bioprinting techniques could potentially be used to address such limitations by providing spatially defined biochemical and biophysical cues to cells as they self-organize into a structurally organized tissue (Fig. 1A). Therefore, the aim of this study was to determine how the physical constraint provided by external hydrogel boundaries of different sizes (specifically different channel widths; see Fig. 1B) influences neotissue growth and organization within rapidly degrading high cell density bioinks.

*Methodology: Positive moulds with a 12mm long, 1mm high, and 250, 500 or 750µm wide cuboid were designed on Autodesk Fusion 360 and printed with high temperature resin using a Form 2 stereolithography printer (Formlabs, Massachusetts, United States). These moulds were used to cast cuboidal channels in molten agarose (4% w/v, 80°C) with a precision error of 17.3, 2.7 and 1.3% for the 250, 500 and 750µm channels, respectively (Fig. 1D). Caprine AC progenitor cells (ACPs) were isolated from full thickness AC shavings through differential adhesion to fibronectin. Passage 3 ACPs were mixed with 4% oxidized alginate at a density of 80 million cells/mL and then cast into the agarose channels. The bioink was crosslinked with CaCl₂ (60mM) for 30 minutes. Next, agarose (2% w/v, 40°C) was poured over the channels forming an upper boundary (Fig. 1B). The constructs were cultured for 3 weeks in chondrogenic media. Chondrogenesis was assessed through histology, immunohistochemistry and biochemical assays.

*Results: All channel spacings supported chondrogenesis, as evident by the development of an extracellular matrix positive for sulfated glycosaminoglycans (sGAG) and collagen, and negative for calcium deposition (Fig. 1C). More intense alcian blue and picrosirius red staining was observed in the 250µm channels. Significantly higher levels of sGAG deposition was seen in the 250 and 500µm channels compared to the 750µm group. Similarly, there was a significantly greater collagen production in the 250µm channel than the larger channels (Fig. 1D). Polarized light microscopy indicated a degree of organization in the 500 and 750µm channels with collagen fibers aligning on the edges against the agarose boundary. A much thicker and uniform level of collagen organization was observed throughout the tissue that formed in the 250µm channels (Fig. 1C). Collagen fibers were preferentially aligned parallel to the long axis of the channel. It is hypothesized that both the boundary geometry and the stiffness difference between the bioink and agarose played a role in directing collagen alignment.

*Conclusion/Significance: The findings of this study reveal that external boundaries can be used to control neotissue collagen alignment within high cell density bioinks. Increased collagen deposition and alignment was observed in the thinner (250µm) channels. Therefore, future work on bioprinting functional AC will incorporate guiding structures with a 250µm spacing to direct collagen organization with a view towards achieving an arcade-like collagen architecture in bioprinted grafts.

439 - Long Term Investigation Of An Improved Strategy Of Autologous Chondrocyte Implantation With Stratified Zonal Chondrocytes Delivery For Repair Of Articular Cartilage

Y. Wu^1,2, Y. Zheng^1,3, S. Wong⁴, C. Tee⁵, E. Lee^1,2, J. Han^5,6, J. Hui^1,3

¹ National University of Singapore, Singapore, Singapore,

² NUS Tissue Engineering Program, National University of Singapore, Singapore, Singapore, Singapore,

³ NUS Tissue Engineering Program, National University of Singapore, Singapore, Singapore, Singapore, Singapore, Singapore,

⁴ 3Diagnostic Radiology, Sengkang General Hospital, Singapore, Singapore, Singapore,

⁵ Critical Analytics for Manufacturing Personalised-Medicine Interdisciplinary Research Group, Singapore-MIT Alliance in Research and Technology, Singapore, Singapore, Singapore,

⁶ Department of Electrical Engineering and Computer Science, Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA

*Purpose/Objectives: The zonal properties of articular cartilage critically contribute to the mechanical support and lubrication of the tissue. Current treatments for articular cartilage have yet to regenerate this zonal architecture, thus compromising the functional efficacy of the repaired tissue and leading to tissue degeneration in the long term. Layer-by-layer stratified implantation of zonal chondrocytes was proposed to recapitulate the zonal properties of articular cartilage. We have previously reported on the use of a spiral microfluidic device for label-free, high-through-put size-based segregation and enrichment of articular cartilage zonal chondrocytes [1], integrated to dynamic microcarrier culture for the generation of expanded zonal chondrocytes [2]. In this study, the repair efficacy of zonal cartilage regeneration through bilayered implantation of expanded autologous zonal chondrocytes was investigated in a clinically relevant porcine chondral defect model over a period of 12 months.

*Methodology: Autologous chondrocytes extracted from articular cartilage from the non-weight bearing trochlea region of the knee were subjected to an expansion-sorting strategy, integrating dynamic microcarrier (dMC) culture and spiral microchannel size-based zonal chondrocyte separation [2]. Zonal chondrocytes were implanted as bilayered fibrin hydrogel construct (dMC-BL, n=6), comprised of superficial zone chondrocytes overlaying middle/deep zone chondrocytes, into defects at the femoral condyles of the porcine knee. Cartilage repair was evaluated at 12 months including morphological, histology, immunohistochemistry, micro-CT and biomechanical measurement. Lived magnetic resonance imaging (MRI) mapping were performed at 1, 12, 24, 36 and 48 weeks to track the zonal cartilage regeneration progress (Figure 1).

*Results: At 12 months, the bilayer-treated defects (n=6) have significantly higher ICRS macroscopic and histological scores than Control, TCP-FT, and dMC-FT treatments. Histomorphometric analysis staining intensities shows significant improvement in the hyaline cartilage quality. Overview of the MRI T2 relaxation time values of the zonal regions of the repaired tissues indicates significant improvement with dMC-BL treatment in both superficial and middle/deep layers as early as 3 months compared to Control, TCP-FT and dMC-FT groups. Notably, dMC-BL treatment attained a superficial zone T2 value similar to that of normal cartilage, starting from 9 months. Micro-CT analysis indicates significant improvement of the subchondral bone repair, compared to other treatment groups. The improved morphological, histological outcomes and zonal development of the Bilayer-treated defects was corroborated with the biomechanical competency of the repair tissues. Both surface lubrication and tissue compressional strength of dMC-BL treatment attained comparable levels as that of healthy cartilage, not achieved by other treatment groups.

*Conclusion/Significance: The proof-of-value 12 months animal studies provide unequivocal evidence on the efficacy of the stratified zonal chondrocytes implantation for enhanced cartilage repair. We speculate that the positioning of the defined layers of superficial zone over middle/deep zone chondrocytes has contributed to the immediate in situ surface lubrication of the implanted constructs, while established the compressive strength in the deeper zones. This would have facilitated the shear-compression induced zonal maturation of the regenerated tissues, providing better weight distribution in both the cartilage and subchondral bone, that could be important for the long term integrity of the regenerated tissue.

441 - Cartilage-penetrating And Lubricating Zwitterionic Sealant For Early-stage Osteoarthritis Treatment

M. Asadikorayem, P. Weber, F. Surman, M. Zenobi-Wong

ETH Zurich, Zurich, Switzerland

*Purpose/Objectives: Osteoarthritis (OA) is a prevalent degenerative joint disease, affecting a large population worldwide. With a lack of effective interventions in the early stages, patients eventually need to undergo total knee replacements. This common surgery not only has a lot of complications for patients but also imposes a significant economic burden on healthcare. Thus, there is an urgent need for a non-invasive treatment to halt disease progression and also induce cartilage tissue regeneration at the early stages of the disease. This study aims to address this gap by developing an injectable sealant. This sealant is designed to penetrate and seal the tissue to halt degradation and provide tissue regeneration by providing lubrication and reinforcement.

*Methodology: The sealant is a linear polymer with a zwitterionic backbone and phenol-containing moieties to provide in-situ crosslinking. The high charge density of the zwitterionic backbone ensures effective tissue penetration and boundary lubrication. Moreover, the anti-fouling nature of the zwitterions protects tissue from the inflammatory joint environment. The phenol-containing moieties enable enzymatic crosslinking of the polymer chains to each other and also to the native cartilage tissue. This promotes prolonged retention of the sealant as well as tissue reinforcement. Ex vivo experiments using bovine cartilage explants were carried out to test the sealant tissue penetration, protection as well as lubrication. In vivo experiments with collagenase-induced osteoarthritic rat models are underway to test the protective and regenerative effects of the sealant as an injectable formulation.

*Results: The zwitterionic sealant exhibited exceptional penetration, quickly reaching the full thickness of both healthy and degraded cartilage (∼3 mm). Crosslinked samples showed a fourfold increase in sealant retention in degraded cartilage compared to non-crosslinked samples, confirming the interpenetrating network formation through enzymatic crosslinking (Figure 1A). The protective effect was demonstrated by inhibiting cartilage degradation in inflammatory media containing interleukin-1 beta (IL-1β) up to 3 weeks of culture, as shown by histological analysis, gene expression, and mechanical characterization (Figure 1B and C). Additionally, the lubricating effect was shown, with a reduced coefficient of friction in degraded cartilage (∼0.6), both instantly and over a 3-week culture period, approaching the friction coefficient of healthy tissue (∼0.2) (Figure 1D).

*Conclusion/Significance: In conclusion, this study introduces and optimizes an injectable zwitterionic sealant as a non-invasive treatment for early-stage OA. The highly charged and anti-fouling zwitterionic polymers facilitate effective lubrication and tissue protection. The biocompatible in-situ crosslinking mechanism, which is also tunable, provides tissue reinforcement and extends sealant retention. This sealant holds significant potential to replace current injectable formulations that offer only short-term pain relief, by providing a substantial intervention in disease progression.

443 - Scarless Vocal Fold Regeneration By Local Delivery Of Decorin From Injectable Microgels

R. M. Friedman^1,2, E. A. Brown^1,2, M. R. Aronson^1,2, K. B. Zur^2,3, R. Gottardi^1,2

¹ University of Pennsyvlania, Philadelphia, PA,

² Children's Hospital of Philadelphia, Philadelphia, PA,

³ University of Pennsylvania, Philadelphia, PA

*Purpose/Objectives: Vocal fold (VF) scarring causes patients to suffer from increased VF tissue stiffness and impaired speech. This is a result of fibroblasts differentiating to contractile myofibroblasts which excessively secrete extracellular matrix. Current therapies are limited to local steroid injections, but they have low clinical efficacy, rapid drug clearance, and poor regenerative capacity. Herein, we identify decorin (DCN) as a novel regulator of human VF myofibroblast function and show that DCN is inversely associated with scar formation in a rat model of VF scarring. Then, we engineer DCN-loaded hyaluronic acid microgels (MGs) for local drug delivery over 21 days with retained anti-fibrotic activity.

*Methodology: DCN is down regulated in VF scarring: Human VF myofibroblasts were compared to VF fibroblasts via bulk RNA sequencing to identify transcriptional signatures. Findings were confirmed by RT-qPCR and immunostaining. Results were confirmed in vivo by the assessment of VF scarring in a rat model via histology and immunostaining at 3-, 7-, and 21-days post-injury. DCN anti-fibrotic effects: Human VF fibroblasts were treated with or without TGF-beta 1 and DCN or bFGF (clinical standard) to determine the ability of DCN to prevent myofibroblast differentiation (prevention/early intervention) as well as to promote myofibroblast de-differentiation (post-injury treatment). Phenotype was determined by RT-qPCR, immunostaining, and contractility assays in 2D and in 3D type I collagen gels. Unconfined compression tests were performed to quantify tangent modulus of collagen gels. Microgels: Methacrylated hyaluronic acid MGs were fabricated via extrusion fragmentation, lyophilized, and loaded with DCN. Size was determined via microscopy. Drug loading and release in PBS and 5U hyaluronidase modeling in vivo conditions were quantified via protein assay. VF myofibroblasts were treated with releasate from DCN-loaded MGs to confirm retained efficacy of DCN.

*Results: Among collagen-binding small leucine rich proteoglycans, DCN was the most transcriptionally downregulated in human VF myofibroblasts (Fig. 1A), and scarred rat VFs showed decreased DCN (Fig. 1B-D). Exogenous treatment of human VF fibroblasts and myofibroblasts with DCN decreased pro-fibrotic gene expression of alpha-smooth muscle actin (α-SMA) and collagen type I. Results were confirmed by decreased protein expression in both 2D and 3D. Furthermore, collagen hydrogel contraction was significantly reduced after DCN treatment (Fig. 1E). Altogether, anti-fibrotic effects of DCN were superior to bFGF (clinical standard). DCN was loaded into MGs and a sustained drug release over 21 days was achieved (Fig. 1F). For both profiles, the daily release concentrations are within the therapeutic range for DCN. Bioactivity was confirmed after delivery (Fig. 1G-H).

*Conclusion/Significance: DCN is downregulated in VF scarring and treatment with exogenous DCN administration results in fibrosis prevention in 2D and 3D. DCN-loaded MGs for the local, sustained delivery of a therapeutic dose of DCN were engineered, show efficacy in vitro, and are currently being tested in vivo.

444 - OviChip: A Next Generation In Vitro Oviduct Model

J. Kim¹, O. Gazeli², V. Vavourakis², L. Moroni¹, P. Wieringa¹

¹ MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht, Netherlands,

² University of Cyprus, Nicosia, Cyprus

*Purpose/Objectives: Recent studies show that the oviduct, specifically the isthmus, serves as an essential site for early embryo epigenetic reprogramming, thereby having significant impact on human health. The oviduct epithelium, composed predominantly of secretory and ciliated cells, exhibits a distinctive spatial distribution within its tubular architecture, which confers functional variation along the oviduct length. Female hormonal fluctuations, notably variations in estrogen and progesterone across the menstrual cycle, dynamically modulate the composition of the oviduct epithelium as well. Moreover, the intricate interplay between the epithelium and the diverse stromal milieu significantly influences protein expression and architectural features. Existing oviduct models, however, predominantly emphasize the epithelium while overlooking other vital elements. This hampers accurate simulation of oviduct’s unique biological and structural characteristics. We show the development of a more representative and replicable full size 3D in vitro oviduct model, integrating multiple critical compartments such as stromal elements and controlled hormonal modulation.

*Methodology: Mouse oviduct epithelial cells were isolated and cultured as organoids, alongside fibroblasts sourced from the oviduct stromal layer. Simultaneously, a channel platform imitating the oviduct isthmus was constructed through fabricating polydimethylsiloxane (PDMS) device with a central chamber incorporating 4.5 mg/ml bovine collagen type I. The isthmus was created by a 19 G needle that is removed after hydrogel crosslinking, forming a 1 mm perfusable channel. Subsequently, cells obtained from mouse oviduct epithelial organoids were seeded into this channel. Perfusable auxiliary channels were also formed within the hydrogel, below and orthogonal to the main channel, as a means of applying hormones along the isthmus length. We optimized the perfusion of these channels by using COMSOL Multiphysics to computationally model hormone diffusion and fluctuation within our perfused system, corroborated by experimental data. Upon achieving a confluent cell layer within the lumen, the female steroid hormone β-Estradiol was introduced either into the culture medium or via auxiliary channels.

*Results: Our findings demonstrated the successful cultivation of mouse oviduct epithelial organoids, validated through both morphological observations and gene expression analysis in response to stimuli (Dibenzazepine). Simultaneously, we developed a channel device with dimensions mimicking the isthmus segment of the oviduct, comprised of bovine collagen type I with a stiffness comparable to that expected for the mouse oviduct in literatures. We achieved the successful long term (>4 weeks) cultivation of oviduct epithelial cells derived from the organoid and fibroblasts as stromal component within the device. Upon exposing the device to female steroid hormones, we observed responsive behaviors in the epithelial cells within the channel, characterized by gene expression analysis and histology. Additionally, utilizing parameters derived from computational modeling, we established the dynamic application of hormones within the device. The preliminary findings from this advanced model have showcased its ability to mimic tubal fluid flow and emulate tubal response to hormonal release.

*Conclusion/Significance: This proposed 3D in vitro oviduct model, encompassing both stromal and hormonal components, promises to unlock novel insights into the dynamics of the oviduct microenvironment. Its potential extends to deciphering complex cellular interactions, understanding disease mechanisms, and advancing our understanding of reproductive biology.

445 - Development of a 3D Bioengineered Human Lung Airway Model for Personalized Disease Modeling

H. Kim¹, S. Yi¹, K. A. Wikenheiser-Brokamp², J. A. Whitsett², A. P. Naren^1,2, K. Mun¹

¹ Cedars Sinai Medical Center, Los Angeles, CA,

² Cincinnati Children’s Hospital Medical Center, Cincinnati, OH

*Purpose/Objectives: Mucociliary clearance (MCC) is critical in maintaining lung health and preventing respiratory infections. MCC is impaired in people with cystic fibrosis (pwCF) due to the accumulation of thick, sticky mucus resulting from defective cystic fibrosis transmembrane conductance regulator (CFTR) channel function. Robust CF in vitro models are critical to defining the mechanisms by which CFTR mutations impair MCC. Current in vitro airway models rely heavily on an epithelial monolayer culture system with trans-well membrane inserts creating an air-liquid interface (ALI) condition to induce cell differentiation. However, this model does not reproduce the spatial morphology present in the in vivo tissue environment. This study aimed to design and develop a novel three-dimensional (3D) in vitro lung airway model to measure patient-specific MCC that more closely mimics in vivo structure and physiology.

*Methodology: Patient-derived primary submucosal gland ductal epithelial cells (SMGECs) were isolated from CF and non-CF patients following lung transplantation and differentiated into ciliary cells and mucus cells in ALI cultures. The cells were morphologically and functionally assessed by H&E staining, immunofluorescence confocal microscopy, and short-circuit current assays. Cells were treated with Trikafta® (elexacaftor, tezacaftor, and ivacaftor/ETI) to determine pharmacologic effects on CFTR function. An in vitro 3D human lung airway model was created by designing and fabricating a multilayered tubular scaffold incorporating a hydrogel-based extracellular matrix (ECM) mixed with human lung fibroblasts and SMGECs utilizing a 3D bioprinting process. The fabricated lung airway was photo-crosslinked to stabilize the printed structure to withstand cell culture conditions. Ciliary beat frequency (CBF) and MCC velocity (MCCV) were measured in real-time on in situ 3D bioprinted airway scaffolds generated from CF and non-CF patients using high-speed video microscopy.

*Results: We successfully isolated and cultured patient-derived SMGECs from CF and non-CF patients and demonstrated restored mutant ΔF508/ΔF508 CFTR function by ETI treatment. Patient-derived SMGECs cultured on the novel 3D printed scaffold were polarized and differentiated into mucus-secreting and ciliated cells, forming an epithelial cell lining along the lumen side within 14 days. CF airway scaffolds (8.5 ± 0.4 Hz) had lower CBF than non-CF airway scaffolds (10.2 ± 0.2 Hz) by quantitative measurement. Moreover, MCCV was reduced in CF airway scaffolds (4.0 ± 2.3 μm/s) compared to non-CF airway scaffolds (14.3 ± 5.1 μm/s). To rescue MCCV in CF, we treated the CF scaffold with ETI and found that the velocity in the ETI-treated CF was 2.5-fold higher than in CF, suggesting a significant improvement of MCC in the ETI-treated CF cells.

*Conclusion/Significance: Our findings demonstrate that this novel model not only enables the fabrication of human lung airway-like structures in vitro that mimic in vivo physiology but also facilitates quantitative measurement of patient-specific MCC and determines pharmacologic effects. Our results suggest that this model is a valuable tool for understanding mechanisms underlying impaired MCC and testing the efficacy of novel therapeutic strategies for the treatment of respiratory diseases such as CF.

446 - Endotracheal Tube Delivered Therapeutics Modulate The Local Microbiome And Inflammatory Tissue Healing Response After Upper Airway Intubation Injury

G. Cervantes-Gonzales¹, S. Miar², R. Malka³, R. Bizios¹, J. L. Ong¹, G. Dion⁴, T. Guda¹

¹ The University of Texas at San Antonio, San Antonio, TX,

² University of Hartford, San Antonio, TX,

³ Brooke Army Medical Center, San Antonio, TX,

⁴ University of Cincinnati Medical Center, Cincinnati, OH

*Purpose/Objectives: Laryngeal injury associated with traumatic or prolonged intubation may lead to complications including dysphonia, dysphagia, and tracheostomy dependence. Beyond mechanical injury, the endotracheal tube (ETT) also serves as a reservoir for microorganisms to adhere to. The composition and matrix of the biofilm formed is variable depending on the local environment therefore its microbial constituents may be impacted by inflammation. Previous investigations have demonstrated these changes in the airway microbiome and shown correlation between species and specific inflammatory insults such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Understanding the relationship between inflammation and microbial population changes caused by intubation could improve clinical outcomes. The objective of this study was to investigate tissue inflammatory response and local microbiome changes with ETT placement and localized delivery of therapeutics.

*Methodology: Yorkshire crossbreed swine underwent direct laryngoscopy and traumatic intubation injury was simulated using a 3/8 in. stainless steel brush. Endotracheal tubes were coated with Roxadustat or valacyclovir-loaded polycaprolactone (PCL) fibers via electrospinning. Regular endotracheal tubes (ETT) or drug-eluting ETTs were placed for 3, 7, or 14 days (n=3 per group/time ETT type). Immunohistochemistry and inflammatory marker profiling investigated the inflammatory response associated with injury and ETT placement. ETT biofilm formation was observed with scanning electron microscopy (SEM) and µCT models. Changes in the airway microbiome were assessed with 16S rRNA sequencing.

*Results: We examined the presence of CD86 and CD206 positive cells, M1 and M2 macrophages respectively (Figure 1A). There was a significant decrease of M1 macrophage in epithelial tissue with roxadustat ETT placement from 3 days to 14 days (p=0.024). Groups with valacyclovir ETT placement had significant decreases from 3 to 7 days (p= 0.0004) and 3 to 14 days (p=0.004). This trend remained the same for M2 macrophage observations with roxadustat-treated epithelium significantly decreasing from 3 to 14 days (p=0.012) and valacyclovir-treated epithelium significantly decreasing from 3 to 7 days (p=0.043). Analysis of composites based on µCT determined higher biofilm volume for the coated ETTs and SEM demonstrated an abundance of bacteria on the coated ETT surface (Figure 1B). Regarding bacterial composition, there were no significant differences in alpha diversity when considering ETT type and duration (Figure 1C). The PCoA based on Bray-Curtis dissimilarity (Figure 1D) showed 25.6% of total variance between samples and PERMANOVA indicated statistically significant differences in the microbial composition with ETT Type (R²=0.10, p<0.05) and duration of placement (R² = 0.13, p< 0.05). Differential abundance analysis identified significant changes in several genera including negative log fold changes in Actinobacillus, Bacteroides, Bergeyella, and Dielma for groups with roxadustat-coated ETT groups in comparison to regular ETT and significant positive log fold changes in Phascolarctobacterium in groups with valacyclovir coated ETT placement.

*Conclusion/Significance: Inflammation caused by laryngeal injury directly modulates the bacterial composition and diversity of the upper respiratory microbiome, the metabolites from which likely impact local tissue healing. Localized delivery of therapeutics further contributes to these changes with unique differences observed depending on the drug administered.

448 - Sustainable Keratin Based Templates For Term

K. Ng

Nanyang Technological University, Singapore, Singapore

*Purpose/Objectives: Sustainability has permeated across the society, especially after the establishment of the 17 Sustainable Development Goals by the United Nations. There is significant potential for health and environment care to complement each other, hence the demand for sustainable biomaterials is expected to intensify. Nature derived biomaterials are not new and have been widely discussed in the literature. Keratins are a more recent example and are one of the very few human-derived materials that are readily available in abundance. Other than human hair, animal keratins are also easily obtained from farming waste streams such as feathers and wool. Keratins have been found to be biocompatible and capable of inducing positive cell responses to support the regeneration of different tissues. Therefore, the focus of our work is to advance the understanding of fabricating and functionality of keratin-based templates.

*Methodology: From a biomaterials development standpoint, keratins are unique because they contain high proportions of functional groups, especially thiols, to support chemical interactions. Together with their inherent propensity to self-assemble into organized structures, keratins have been demonstrated to be easily processable into a variety of physical formats including coatings, fibers, films, gels and sponges. In our group, we used a variety of techniques to leverage the properties of keratins to produce a range of functional templates.

*Results: A cryogelation technique was developed, which allowed 3D keratin-based hydrogels with tunable physical properties and microarchitectures to be produced. Cryogelation was found to improve the storage and compression moduli of these hydrogels. Strain-stiffening behavior can also be induced by crosslinking keratins and dopamine through a similar cryogelation process. We further made use of thiolate interaction with cations to provide the possibility of a rapid gelling system for bioink development. This cationic induced gelation method also allowed the production of a gradient hydrogel through a single-step fabrication process. By using silver ions as the cationic crosslinker, a gradient hydrogel that releases silver ions as an antimicrobial agent, as the gel degrades, is produced. In the latest development, we adopted the technique of Interfacial Polyelectrolyte Complexation (IPC) to produce keratin-based fibers. Hair keratins were pH adjusted to produce polycationic or polyanionic solutions and partnered together or with established polycations such as chitosan and drawn to produce the micrometer-sized fibers. These fibers, formed through a combination of electrostatic interaction, hydrogen bonding and disulfide bonding, were found to contain predominantly β-sheet secondary structures. The fibers exhibited good mechanical properties that were comparable to commercial suture materials. A preliminary in vivo study was conducted to evaluate the suitability of the keratin-based IPC fibers to function as a suture for closing full-thickness cutaneous wounds in mice. The fibers elicited minimal host tissue response over a 3-week period, with onset of degradation after 1 week.

*Conclusion/Significance: Our work has demonstrated that keratin-based templates are versatile, functional and could provide meaningful outcomes in TERM applications. This presentation will summarize and update developments by our group to advance keratin-based templates as alternative sustainable biomaterials platforms.

449 - Plant-powered Cultured Meat: Crafting A Fat Containing Hybrid Hamburger Composed Of Cell-laden Microcarriers

I. Geurs¹, M. Olenic², E. De Vlieghere³, C. Grootaert¹, K. Dewettinck¹, L. Thorrez², S. De Smet¹, S. Van Vlierberghe¹, J. Van Camp¹

¹ Ghent university, Gent, Belgium,

² KU Leuven, Kortrijk, Belgium,

³ Ghent university/KU Leuven, Gent/Kortrijk, Belgium

*Purpose/Objectives: In this research we present a novel approach to create a hybrid cultured hamburger-like product that integrates an edible plant-based microcarrier for cell culture, cross-linked to a plant-based fat phase. The global demand for more sustainable and ethical meat alternatives has driven advancements in cultured meat research. Although a plethora of research is performed in the framework of cultured meat, several hurdles persist. One of these hurdles is upscaling the production process. Currently, the most promising approach is to use a stirred bioreactor containing microcarriers. However, these microcarriers often require a cell-detachment step due to their inedibility and/or contain animal-derived components. To address these two issues, we created edible, plant-based microcarriers which promote cell attachment and growth. Next, the aim was to develop a plant-based fat phase that could crosslink with the cell-laden SPI microcarriers, thereby creating a product that mimics a hamburger.

*Methodology: To develop the microcarriers, several plant-based proteins (including pea, soy and fava bean) were evaluated (i) for their suitability for enzymatic crosslinking, (ii) regarding the mechanical properties of the resulting cross-linked films, and (iii) for their cell-adhesive properties. The latter was tested both in the presence and absence of fetal bovine serum. Cell adhesion was first assessed in 2D by drop seeding 20 000 C2C12 mouse myoblast cells on top of the plant-based film (ø 6mm). Soy protein isolate (SPI) films exhibited the highest cell attachment and were converted into microcarriers (400 - 600 µm). Subsequently, these microcarriers (1500 mg of wet mass) were seeded with one million bovine myoblasts in a stirred-tank bioreactor (100 ml growth medium). Next, an oil-in-water emulsion was formulated, incorporating sunflower oil as the inner phase and SPI in water as the outer phase. The amount of oil, water and protein were optimized as well as the emulsification temperature and procedure. Next, a plant-based gelling agent was added to the water phase to enhance the extrudability of the emulsion. Moreover, the addition of transglutaminase was optimized as well as the emulsion/microcarrier ratio.

*Results: The results showed that bovine myoblasts were able to attach, grow and differentiate into myotubes on the SPI microcarriers. After seeding one million myoblast cells and culturing them during eight days, the microcarriers yielded up to 24 million cells. Furthermore, it was possible to create a stable 50/50 wt% oil-in-water emulsion using 5 wt% SPI in the water phase. By adding a plant-based gelling agent, it was possible to 3D print the emulsion. The microcarriers could be cross-linked with the emulsion through the addition of transglutaminase, linking the SPI present in the microcarriers with the SPI present in the emulsion. The optimal ratio between emulsion and microcarrier was found to be 30/70 wt%, representing the maximum amount of microcarrier that could be effectively bound together by the emulsion, resulting in the formation of a solid, cohesive product.

*Conclusion/Significance: In conclusion, a prototype of a cross-linked hybrid hamburger was developed, comprised of cell-adhesive SPI microcarriers and a stable plant-based fat phase.

450 - Optimising The Mechanical And Immunological Properties Of Biomimetic Nanocellulosic Composite Fibres For Application As A Ligament Graft

M. Graham¹, A. Gümrah Parry¹, M. Rottmar², J. Gough¹, L. Biant¹

¹ University of Manchester, Manchester, United Kingdom,

² EMPA, St. Gallen, Switzerland

*Purpose/Objectives: Anterior Cruciate Ligament (ACL) reconstruction is the one of the most common sporting surgeries, with 100,000 taking place annually in the US alone. Autografts are the current Gold Standard, despite poor long-term outcomes. Due to the prevalence of this surgery, sustainability and cost are paramount when designing improved graft materials. Cellulose is the most abundant biopolymer on Earth. Cellulose fibres present with a hierarchical structure based on cellulose nanofibrils (CNFs), analogous to the structure of collagen fibrils in tendons/ligaments. Their limitless natural resource, stiffness, and collagen-like morphology make CNFs an attractive ‘building-block’ from which cost-effective, load-bearing biomedical materials can be developed. CNFs can be isolated and wet-spun into macroscale fibres with a collagen-mimetic, hierarchical structure. However, these fibres are very stiff and brittle. Moreover, CNFs do not degrade in-vivo, leaving them vulnerable to macrophage-induced foreign body reaction. In this work, we designed all-cellulose composite fibres in which CNFs were added to a methylcellulose (MC) matrix, managing energy dissipation to produce tougher fibres. We also altered macrophage response to CNF through conjugation of the immunomodulatory peptide DGEA.

*Methodology: Bacterial cellulose-nanofibrils (BCNF) were prepared by TEMPO-mediated oxidation. Composite hydrogels were produced by varying the ratio of MC to BCNF. Stability, morphology, and flow properties of the hydrogels were assessed using zeta potential (ZP), AFM, and rheology measurements. Hydrogels were wet-spun into an acetone coagulation bath at low (1 µl/s) and high (3 µl/s) flow-rates. BCNF alignment was characterised via polarised optical microscopy (POM), SEM and fibre diffraction measurements. Fibre mechanics were assessed via tensile tests. TEMPO-BCNF were further functionalised with the peptide DGEA via a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) cross-linking mechanism. Samples were cultured with M1 or M2 macrophages for 1, 2, and 3 days, and characterised by PCR and immuno-fluorescence staining.

*Results: TEMPO-mediated oxidation of BCNF introduced negative carboxylate groups to fibril surfaces (ZP=-47.55 mV). Upon addition of MC, ZP decreased to -20.3 mV, despite no visible deterioration in stability, likely due to strong inter-fibril adhesion (confirmed via AFM). Increasing the concentration of MC produced hydrogels with a lower G’, larger linear viscoelastic region, and improved spinnability. Only the suspensions where MC was in excess could be spun into continuous fibres. Interestingly, POM analysis suggests a low flow-rate increases fibril alignment (figure) and improves mechanical properties, with 1 µl/s and 3 µl/s fibres presenting with an E of 79 MPa and 62 MPa, UTS of 125 MPa and 81 MPa, and strain-at-break of 4% and 3%, respectively. Expression of M1 markers CD197 and CXCL10 decreased over three days on BCNF and DGEA-BCNF. After three days, the M2 marker CD206 was upregulated 24-fold in BCNF-DGEA, compared to a 5-fold in BCNF.

*Conclusion/Significance: In this work, we investigated BCNF as a sustainable, cost-effective material for ACL graft development. We improved the processability and mechanics of BCNF-based fibres through the addition of MC. Spinning at a lower flow rate increased nanoscale alignment and further improved fibre mechanical properties. Finally, peptide modification of BCNF was shown to impact both M1 and M2 macrophages.

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