Novel strategies in cartilage tissue engineering: Impact of hMSC-ECM interactions and perfusion cultivation

Studies in this Dissertation focused on the impact of hMSC-ECM interactions and perfusion cultivation for optimal cartilage tissue engineering outcomes. Since processes in cartilage development are not entirely understood, in the first chapter, we set out to uncover important mechanisms controlling human mesenchymal stem cell (hMSC) fate determination. Chondrogenic differentiation with hMSCs is associated with TGFBeta induced differentiation as well as other cartilage microenvironmental associated factors. We hypothesized that cell attachment to specific extracellular matrix (ECM) proteins such as cartilage-associated collagens would play a functional role in promoting chondrogenic differentiation of hMSCs in an in vitro model. To test this hypothesis, we cultured hMSCs on native and denatured collagen types I, II, IX and XI in the presence or absence of soluble serum-free, chondrogenic supplements and TGFBeta and assayed for the presence of cartilage-associated definitive, hallmark differentiation markers (including the production of cartilage matrix: aggrecan, collagen type II and sulfated glycosaminoglycan (sGAG) accumulation) over a 14-day time course.We found that hMSCs responded to native collagen types II and XI with robust gene expression outcomes and increases in sGAG accumulation over controls via responses governed by distinct integrin receptors in particular, alpha1beta1 and alpha2beta1. Furthermore, we observed that the greatest degree of hMSC chondrogenic differentiation occurred on native collagen types II and XI and minimal levels of chondrogenesis took place on their denatured collagen counterparts. We conclude that hMSCs in the presence of chondrogenic stimulants (CH), in contact with collagen types II and XI promotes and enhances chondrogenic differentiation to a substantially higher level than other collagens studied in comparison to their respective controls. In addition, silk scaffolds coated with cartilage-associated collagen types II and XI were capable of inducing higher extents of hMSC chondrogenic differentiation in comparison to uncoated silk scaffolds. Therefore this study suggests that cartilage-associated ECM contact is necessary to maximize chondrogenic induction in hMSCs. These results highlight the potential of engineering the scaffold microenvironment to more closely mimic cellular ECM cues necessary to promote enhanced differentiation for the purpose of in vivo relevance.The objective of our study in chapter two was to characterize the ability of a novel perfusion bioreactor to promote chondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded within 3D silk fibroin-based biomaterials. Scaffold incorporated hMSCs were cultivated in the presence or absence of CH in either static or perfused conditions for up to 21 days. DNA analysis demonstrated significant upregulation of cell proliferation in perfused constructs over static controls. Histological analysis of bioreactor cultivated hMSCs maintained in CH revealed the acquisition of chondrocyte-like morphological features as well as a more uniform cellular distribution throughout the 3D scaffold architecture in contrast to static constructs. In addition, perfusion culture conditions in conjunction with CH supported increased levels of aggrecan, collagen types II, IX, and X mRNA transcript levels as well as substantially higher degrees of sGAG over static controls. Collagen type II and Safranin-O immunohistochemical staining demonstrated robust deposition of cartilage-associated extracellular matrix components in scaffolds exposed to perfusion and CH over nonstimulated controls. Analysis of osteogenic differentiation markers such as runx2 expression revealed significant downregulation in perfused constructs cultured with CH in comparison to undifferentiated and respective static controls.These studies provide a "proof of principle" on (1) the ability of collagen type and structural conformation to serve as a significant control point in hMSC chondrogenesis through the modulation of integrin-mediated ECM interactions. (2) a novel perfusion bioreactor to promote stable and persistent chondrogenic differentiation of hMSCs and thus may be applicable for the production of constructs for in vivo cartilage replacement.