Cartilage extraceullular matrix-derived scaffolds for joint regeneration

Abstract

The goal of this thesis was to address key limitations associated with autologous chondrocyte implantation (ACI) for articular cartilage regeneration, specifically the need for two hospital stays, cell culture and high cost. As an alternative, this thesis explored the combination of freshly isolated stromal cells and a novel chondroinductive scaffold as a putative alternative to ACI. Consequently, the first objective of this thesis was to develop and optimize a chondro-permissive device able to deliver stem cells and other chondrogenic factors. Extracellular matrix (ECM)-derived materials have previously been used to enhance cartilaginous tissue formation and regeneration. Hence, the first step was to develop a scaffold derived from articular cartilage ECM that could be used as a growth factor delivery system to promote chondrogenesis. Porous scaffolds were fabricated using devitalized cartilage, which were then seeded with human infrapatellar fat pad-derived stem cells (FPSCs). It was found that these scaffolds promoted chondrogenesis, especially when stimulated with transforming growth factor (TGF)-β3. The superior chondrogenesis in the presence of exogenously supplied TGF-β3, led to explore whether this scaffold could be used as a growth factor delivery system. When these scaffolds were loaded with TGF-β3, comparable chondrogenesis to continuous adding TGF-β3 to the media was observed. The next step of this thesis was to optimize the scaffold itself and demonstrate that this scaffold could promote chondrogenesis of freshly isolated stromal cells in vivo. By freeze-drying cryomilled cartilage ECM of differing concentrations, it was possible to produce scaffolds with different architectures. Migration, proliferation and differentiation of FPSCs depended on the scaffold concentration/porosity, with greater sGAG accumulation observed with increases in pore size. Next, it was sought to demonstrate that fresh stromal cells, when seeded onto a TGF-β3 eluting ECM-derived scaffold, could promote chondrogenesis in vivo. While a more cartilage-like tissue could be generated using culture expanded FPSCs compared to non-enriched freshly isolated cells, fresh CD44+ stromal cells were capable of producing a tissue in vivo that stained strongly for sGAGs and type II collagen. These findings open up new possibilities for in-theatre cell based therapies for joint regeneration. Therefore, once it was demonstrated that it was possible to deliver growth factor and chondro-potent cells in an optimized ECM-derived scaffold in vitro and in vivo, the next step was to assess the effect of different doses of exogenously supplied TGF-β3 in different FPSCs donors (healthy and diseased). After comparing the different donors in escalating TGF-β3 conditions it was possible to conclude that the high dose enabled higher matrix formation consistently for all donors. No disparity was observed between healthy and diseased donors. ECM-based biomaterials are commonly xenogeneic, which may elicit an adverse immune response. Native human ECM can be used as an alternative to xenogeneic tissue; however, its supply is limited leading to the need for more readily available source of material. Hence, scaffolds were produced using ECM from xenogeneic articular cartilage, and sheets of engineered cartilage using stem cells. Engineered ECM presented some of the features of native cartilage, although it contained lower levels of type II collagen. Scaffolds produced using both engineered and native ECM possessed similar properties. However, engineered ECM-derived scaffolds supported inferior chondrogenesis when seeded with FPSCs. TGF-β3 eluted in engineered ECM-derived scaffolds enhanced their capacity to support chondrogenesis, to levels comparable to the native ECM-derived constructs. Cartilage ECM was then used to further functionalize well known biomaterials, specifically a fibrin hydrogel and an alginate scaffold. This thesis first explored functionalizing an injectable fibrin hydrogel with cartilage ECM particles and TGF-β3 for cartilage regeneration. Even in the presence of such levels of ECM, chondrogenesis of FPSCs within these fibrin constructs was enhanced when additionally stimulated with TGF-β3. ECM particles could also be used to control the delivery of TGF-β3 to FPSC within fibrin hydrogels in vitro, and furthermore, led to higher levels of sGAG and collagen accumulation compared to control constructs loaded with gelatin microspheres. In vivo, freshly isolated stromal cells generated a more cartilage-like tissue within fibrin hydrogels functionalized with cartilage ECM particles. These tissues stained strongly for type II collagen and contained higher levels of sGAGs. Finally, the overall goal of the last part of the thesis was to develop a mechanically stable anisotropic alginate scaffold featuring shape-memory and biomimetic properties to be used in cartilage regeneration. For this end an architectural and an additional collagen functionalization were performed. The architectural change was created using a directional freezing technique. This enabled the creation of an aligned structure, which improved the mechanical properties. The functionalization with type II collagen improved cell recruitment and consequent tissue formation throughout the construct. Incorporating such collagen into the alginate scaffold did not negatively influence the shape-memory properties of the structure. Coating with type II collagen enabled superior chondrogenesis when seeded with human FPSCs. Compared with coating with type I collagen, type II collagen improved cell proliferation, higher sGAG and collagen accumulation, and the development of a stiffer tissue. These findings open up the possibility of using cartilage ECM-derived type II collagen to functionalize anisotropic shape-memory alginate scaffolds in order to enhance their capability to regenerate cartilage. Both the ECM-derived scaffold and other biomaterials (fibrin and alginate) functionalized with ECM, should be considered for cartilage tissue regeneration in man. These devices in combination with stromal cells and growth factors carried by the ECM-derived scaffold or ECM functionalized devices have shown significant promise as therapeutics for driving articular cartilage repair, overcoming current cell-based limitations observed for example in ACI.