Entwicklung eines dreidimensionalen Fibringelmodells zur In-Vitro-Analyse von Fibrose und Angiogenese Alginat-mikroverkapselter Langerhans-Inseln / Immunoprotection of the pancreatic islets by encapsulation with alginate can potentially provide their transplantation without immunosuppression. The fundamental obstacle to large-scale clinical transplantation of encapsulated islets is limited graft survival. Critical for adequate long-term function of the graft is the absence of fibrotic overgrowth and sufficient supplementation with oxygen and nutrients. The aim of this study was to develop a model to investigate molecular and cellular mechanisms of fibrosis and vascularization of alginate-encapsulated islets. A 3D fibrin-gel model was developed to assess the mitogenic and angiogenic activity of encapsulated islet cells and encapsulation material per se. The 3D fibrin-gel matrix, containing filled or empty capsules, was seeded with fibroblasts and endothelial cells. Their migration and prolifiration were evaluated by phase-contrast microscopy and histology. Viability (FDA/PI) and function (insulin-ELISA) of encapsulated islets were assessed at different time-points. Inflammatory and angiogenic cytokines were tested with ELISA. Embedded into the 3D fibrin-gel, encapsulated islet cells preserved their viability and function to day +14 in culture. Encapsulated islet cells influenced migration, proliferation and cytokine production of fibroblasts when these were seeded into the 3D fibrin gel: fibroblasts migrated directly to encapsulated islets and grew firmly around the capsules, migration to empty capsules was much slower and overgrowth less prominent. TNFa production increased significantly after 10 days incubation of encapsulated islets with fibroblasts, but not with empty fibroblasts. Co-culture of encapsulated islets with endothelial cells induced the formation of vascular-like tubular structures around the capsules . This model was useful to test the mitogenic and angiogenic properties of the encapsulation material (purified versus non-purified alginates). Adhesion of endothelial cells to collagen-layered capsules was much more prominent than to non-layered capsules; endothelial cells strongly proliferated, forming a monolayer on the capsule surface. Moreover, they migrated into the fibrin-gel-matrix, forming new tube-like structures. These processes were accelerated by VEGF. The 3D fibrin-gel model is very useful for studying the mechanisms of fibrosis and vascularization of encapsulated islets in vitro. In vivo conditions can be imitated, and factors involved in these processes can be analyzed separately.

Despite recent advances in surgical technique and the development of numerous therapeutic agents, the formation post surgical adhesions (PSA) continues to cause complications for many patients. In this research, we have employed a rational system to develop a novel treatment to address this clinical need. Based on an understanding of the biochemical events that lead to PSA formation, a series of targeted polymeric biomaterials was designed to interrupt the fibrin gel matrix propagation and suppress PSA formation. Using group transfer polymerization, a series of well controlled block copolymers of polyacrylic acid and poly(ethylene glycol-methacrylate) based materials was synthesized. Subsequent functionalization with the pentapeptide Cys-Arg-Glu-Lys-Ala (CREKA) was employed to target the materials to fibrin as a marker of pro-adhesive sites. While preliminary testing of the untargeted materials verified their ability to suppress non-specific protein adsorption to model surfaces, numerous in vitro tests were conducted to study the ability to inhibit fibrin gel propagation. The ability to inhibit both the rate and quantity of fibrinogen deposition to a fibrin coated surface has been demonstrated. In addition, the rate of fibrin gel propagation and the degree of cellular attachment can modulated. Taking advantage of the systematic variation in structure facilitated by the robust synthetic methodology employed, statistical analysis was used to elucidate the structureproperty relationships governing the performance of these materials. The most important factors that lead to enhanced performance in in vitro tests are the length of PEG chain and number of peptide units conjugated to the polymer: increasing PEG chain length and increasing the number of peptides conjugated to the polymer both improve performance in all tests. The synthetic methods that have been developed, in conjunction with the experimental results, will be used to direct future studies, including cytotoxicity and animal studies.

The toll skeletal muscle injury, resulting in significant muscle mass loss, has on the patient reaches far more than physical and emotional, as the tolls are financial as well. Approximately more than 3 billion dollars is spent on the initial medical costs and on subsequent disability benefits, following a volumetric muscle loss. Skeletal muscle has a robust capacity for self-repair; this propensity for repair is hindered when skeletal muscle loss is larger than 20% of the total mass of the muscle. Previous work in our lab, has shown functional and morphological improvements following the cellular therapy, with mesenchymal stem cells (MSC), as well as with nerve relocation to the extracellular matrix (ECM). To further observe the regenerative properties of the above treatments, a defect weighing approximately 307 ± 3.7 mg wet weight and measuring approximately 1x 1cm² was removed from the lateral gastrocnemius (LGAS) of male Sprague Dawley rats. Additionally, the medial branch of the plantar nerve was then relocated and implanted to the middle of the ECM. Seven days post injury bone-marrow derived mesenchymal stem cells were injected directly into the implant using a PEGylated Fibrin hydrogel (PEG). Following 56 days of recovery, partial functional restoration was observed in the LGAS ECM seeded with MSC and implanted with the plantar nerve. The LGAS produced 86.3 ± 5.8% of the contralateral LGAS, a value that was significantly higher than ECM implantation alone (p <.05). The implanted ECM seeded with MSC and implanted with the plantar nerve showed significant increases in blood vessel density and myofiber content (p <.05). The data suggest that a volumetric injury can be repaired by neurotization of an implanted muscle-derived ECM seeded with MSCs. / text

"A quantitative understanding of the complex interactions between cells, soluble factors, and the biological and mechanical properties of biomaterials is required to guide cell remodeling towards regeneration of healthy tissue rather than fibrocontractive tissue. The goal of this thesis was to elucidate the interactions between the boundary stiffness of three-dimensional (3D) matrix and soluble factors on valvular interstitial cell (VIC) phenotype with a quantitative approach. The first part of the work presented in this thesis was to characterize the combined effects of boundary stiffness and transforming growth factor-β1 (TGF-β1) on cell-generated forces and collagen accumulation. We first generated a quantitative map of cell-generated tension in response to these factors by culturing VICs within micro-scale fibrin gels between compliant posts (0.15-1.05 nN/nm) in chemically-defined media with TGF-β1 (0-5 ng/mL). The VICs generated 100 to 3000 nN/cell after one week of culture, and multiple regression modeling demonstrated, for the first time, quantitative interaction (synergy) between these factors in a 3D culture system. We then isolated passive and active components of tension within the micro-tissues and found that cells cultured with high levels of stiffness and TGF-β1 expressed myofibroblast markers and generated substantial residual tension in the matrix yet, surprisingly, were not able to generate additional tension in response to membrane depolarization signifying a state of continual maximal contraction. In contrast, negligible residual tension was stored in the low stiffness and TGF-β1 groups indicating a lower potential for shrinkage upon release. We then studied if ECM could be generated under the low tension environment and found that TGF-β1, but not EGF, increased de novo collagen accumulation in both low and high tension environments roughly equally. Combined, these findings suggest that isometric cell force, passive retraction, and collagen production can be tuned by independently altering boundary stiffness and TGF-β1 concentration. In the second part, by using the quantitative information obtained from the first part, we investigated the effects of dynamic changes in stiffness on cell phenotype in a 3D protein matrix, quantitatively. Our novel method utilizing magnetic force to constrain the motion of one of two flexible posts between which VIC-populated micro-tissues were cultured effectively doubled the boundary stiffness and resulted in a significant increase in cell-generated forces. When the magnetic force was removed, the effective boundary stiffness was halved and the tissue tension dropped to 65-87% of the peak value. Surprisingly, following release the cell-generated forces continued to increase for the next two days rather than reducing down to the homeostatic tension level of the control group with identical (but constant) boundary stiffness. The rapid release of tension with the return to baseline boundary stiffness did not result in a decrease in number of cells with α-SMA positive stress fibers or an increase in apoptosis. When samples were entirely released from the boundaries and cultured free floating (where tension is minimal but cannot be measured), the proportion of apoptotic cells in middle region of the micro-tissues increased more than five-fold to 31%. Together, these data indicate that modest temporary changes in boundary stiffness can have lasting effects on myofibroblast activation and persistence in 3D matrices, and that a large decrease in the ability of the cells to generate tension is required to trigger de-differentiation and apoptosis. "

The purpose of this research was to study the synthesis and remodeling of extracellular matrix (ECM) by fibroblasts with special emphasis on the culture environment (media composition and initial ECM composition) and the resulting mechanical integrity of the ECM. This was investigated by culturing fibroblasts for 3 weeks in a variety of culture conditions consisting of collagen gels, fibrin gels, or media permissive to the self-production of ECM (Cell-Derived Matrix), and quantifying the mechanics of the resulting ECM. The mechanical characteristics were related to the biochemistry of the resulting ECM, notably in terms of collagen accumulation and collagen fibril diameters. The ultimate tensile strength (UTS) of the collagen gels and fibrin gels at the end of the 3-week period was 168.5 ± 43.1 kPa and 133.2 ± 10.6 kPa, respectively. The ultimate tensile strength of the cell-derived matrices was 223.2 ± 9 kPa, and up to 697.1 ± 36.1 kPa when cultured in a chemically-defined medium that was developed for the rapid growth of matrix in a more defined environment. Normalizing the strength to collagen density resulted in a UTS / Collagen Density in these groups of 6.4 ± 1.9 kPa/mg/cm3, 25.9 ± 2.4 kPa/mg/cm3, 14.5 ± 1.1 kPa/mg/cm3, and 40.0 ± 1.9 kPa/mg/cm3, respectively. Cells were synthetically more active when they produced their own matrix than when they were placed within gels. The resulting matrix was also significantly stronger when it was self-produced than when the cells rearranged the matrix within gels that corresponded to a significantly larger fraction of non-acid and pepsin extractable collagen. These studies indicate that cell-derived matrices have potential both as in vitro wound healing models and as soft connective tissue substitutes.