The objective of this study was to investigate the hypothesis that the application of dynamic compression following transforming growth factor-beta3 (TGF-beta3) induced differentiation will further enhance chondrogenesis of mesenchymal stem cells (MSCs). Porcine MSCs were encapsulated in agarose hydrogels and cultured in a chemically defined medium with TGF-beta3 (10 ng/mL). Dynamic compression (1 Hz, 10% strain, 1 h/day) was initiated at either day 0 or day 21 and continued until day 42 of culture; with TGF-beta3 withdrawn from some groups at day 21. Biochemical and mechanical properties of the MSC-seeded constructs were evaluated up to day 42. The application of dynamic compression from day 0 inhibited chondrogenesis of MSCs. This inhibition of chondrogenesis in response to dynamic compression was not observed if MSC-seeded constructs first underwent 21 days of chondrogenic differentiation in the presence of TGF-beta3. Spatial differences in sGAG accumulation in response to both TGF-beta3 stimulation and dynamic compression were observed within the constructs. sGAG release from the engineered construct into the surrounding culture media was also dependent on TGF-beta3 stimulation, but was not effected by dynamic compression. Continued supplementation with TGF-beta3 appeared to be a more potent chondrogenic stimulus than the application of 1 h of daily dynamic compression following cytokine initiated differentiation. In the context of cartilage tissue engineering, the results of this study suggest that MSC seeded constructs should be first allowed to undergo chondrogenesis in vitro prior to implantation in a load bearing environment.

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An alternative strategy to the use of in vitro expanded cells in regenerative medicine is the use of freshly isolated stromal cells, where a bioactive scaffold is used to provide an environment conductive to proliferation and tissue-specific differentiation in vivo.The objective of this study was to develop a cartilage extracellular matrix (ECM) derived scaffold that could facilitate the rapid proliferation and chondrogenic differentiation of freshly isolated stromal cells. By freeze-drying cryomilled cartilage ECM of differing concentrations, it was possible to produce scaffolds with a range of pore sizes. The migration, proliferation and chondrogenic differentiation of infrapatellar fat pad derived stem cells (FPSCs) depended on the concentration/porosity of these scaffolds, with greater sGAG accumulation observed in scaffolds with larger sized pores. We then sought to determine if freshly isolated fat pad derived stromal cells, 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.3

Chondrogenically primed bone marrow derived mesenchymal stem cells (MSCs) have been shown to become hypertrophic and undergo endochondral ossification when implanted in vivo. Modulating this endochondral phenotype may be an attractive approach to engineering the osseous phase of an osteochondral implant. The objective of this study was to engineer an osteochondral tissue by promoting endochondral ossification in one layer of a bi-layered construct and stable cartilage in the other. The top-half of bi-layered agarose hydrogels were seeded with culture expanded chondrocytes (termed chondral layer) and the bottom half of the bi-layered agarose hydrogels with MSCs (termed osseous layer). Constructs were cultured in a chondrogenic medium for 21 days and thereafter were either maintained in a chondrogenic medium, transferred to a hypertrophic medium, or implanted subcutaneously into nude mice. This structured chondrogenic bi-layered co-culture was found to enhance chondrogenesis in the chondral layer, appearing to help re-establish the chondrogenic phenotype that is lost in chondrocytes during monolayer expansion. Furthermore, the bilayered co-culture appeared to suppress hypertrophy and mineralisation in the osseous layer.The addition of hypertrophic factors to the media was found to induce mineralisation of the osseous layer in vitro. A similar result was observed in vivo where endochondral ossification was restricted to the osseous layer of the construct leading to the development of an osteochondral tissue. This novel approach represents a potential new treatment strategy for the repair of osteochondral defects.