Generation of equine osteochondral constructs using mesenchymal stromal cells

Focal cartilage injuries are a significant cause of lameness in the horse. Current treatments, such as mosaic arthroplasty, come with significant limitations. Tissue engineered cartilage layered on a bone substrate is a possible alternative to using native osteochondral plugs. The purpose of this thesis is to explore the chondrogenic potential of equine induced pluripotent stem cells (iPSCs) and mesenchymal stromal cells (MSCs) for the purpose of generating osteochondral constructs. Equine iPSCs were first differentiated into MSC-like cells to generate an intermediate progenitor population. When assessed for trilineage differentiation, these MSC-like cells underwent adipogenesis and osteogenesis, but failed to undergo chondrogenesis. Thus, equine MSCs were used for further studies in cartilage tissue engineering. Next, cartilage tissues derived from MSCs from two sources were compared to determine which source showed greater chondrogenic potency. 10 unrelated MSC cultures derived from bone marrow (BM-MSCs) and 10 unrelated MSC cultures from umbilical cord blood (CB-MSCs) were differentiated in a membrane culture system. The generated cartilage tissue was assessed at Day 7, 14, and 21. There were large differences in the quality of tissues generated by individual MSC cultures from both sources. Variance component analysis revealed that donor, and not cell source, is the primary contributor to the variance seen in the deposition of matrix components. CB-MSCs are advantageous for allogeneic use as they can be isolated non-invasively, thus, CB-MSCs were used to generate osteochondral constructs. A novel method was developed whereby MSCs were directly seeded on top of a calcium polyphosphate (CPP) bone substitute in the presence of a ROCK inhibitor to prevent cell contraction. It was found that a seeding density of 1.5x106 cells per CPP generated thick, hyaline-like tissue after 3 weeks of culture. There was no significant difference in tissue quality when the culture length was extended to 6 weeks, or when varying the TGFβ3 exposure in the 6 week cultures. These results are the first to examine the chondrogenic capacity of equine iPSCs and the first to outline a one-step direct differentiation method of producing MSC-derived cartilage on a bone substrate. Further optimization of construct generation is needed prior to testing the constructs in vivo.