Regeneration of Equine MSC-derived Cartilage: Efforts Towards Functional Tissue Engineering

Focal cartilage injuries are a significant cause of lameness in the horse and are an important risk factor for osteoarthritis. In vitro generated cartilage and tissue-engineered osteochondral replacements have been proposed as potential treatment options for articular cartilage defects. Mesenchymal stromal cells (MSCs) provide a potential cell source for cartilage regeneration, however, in vitro-derived cartilage is often functionally inferior to native cartilage. The purpose of this thesis was to improve and characterize the mechanical properties of equine cord-blood MSC-derived osteochondral constructs, enhance MSC-derived neocartilage through chemical activation of TRPV4 channel signaling and extract the biomechanical properties of normal articular cartilage from the native femorotibial joint through double-indentation and stress-relaxation. Osteochondral constructs were generated and improved by increasing neocartilage thickness through a double-layering approach and their properties characterized. Biochemical and mechanical properties of osteochondral constructs were lower than native equine joint cartilage, however, stress-strain values at equilibrium was comparable to non-weight bearing regions from the native joint. iii Next, neocartilage sheets were evaluated by effect of TRPV4 cation channel modulation on MSC-derived neocartilage at both mRNA and extracellular matrix levels. The mRNA levels of chondrogenic, hypertrophic and mechanoresponsive genes were analysed in neocartilage exposed to pulses of the TRPV4 agonist (GSK101) at different concentrations. GSK101 treatment (1nM) increased ACAN levels after treatment for 1-hour per day for 3 days. No increase was detected for hypertrophic markers at this concentration. This treatment regimen also increased sGAG content and enhanced compressive properties compared to untreated controls. GSK101 showed no effect on mechanoresponsive genes at the time-point of analysis. After analysis of normal properties of articular cartilage, it was observed that the medial condyle has the highest collagen content and largest range of compressive and shear moduli, while the patellar groove showed the lowest range for these two mechanical parameters, as well as stress-strain (2%-10%) for the donors evaluated. Additional donors and categorization should decrease variability for further analysis. These results constitute the first efforts to develop scaffold-free biphasic osteochondral constructs from equine cord-blood MSCs with a double-layering approach, as well as enhancing equine MSC-derived neocartilage through TRPV4 signaling.