The role of chondroitin sulfate in regulating hypertrophy during MSC chondrogenesis in a cartilage mimetic hydrogel under dynamic loading

Mesenchymal stem cells (MSCs) are promising for cartilage regeneration, but readily undergo terminal differentiation. The aim of this study was two-fold: a) investigate physiochemical cues from a cartilage-mimetic hydrogel under dynamic compressive loading on MSC chondrogenesis and hypertrophy and b) identify whether Smad signaling and p38 MAPK signaling mediate hypertrophy during MSC chondrogenesis. Human MSCs were encapsulated in photoclickable poly(ethylene glycol) hydrogels containing chondroitin sulfate and RGD, cultured under dynamic compressive loading or free swelling for three weeks, and evaluated by qPCR and immunohistochemistry. Loading inhibited hypertrophy in the cartilage -mimetic hydrogel indicated by a reduction in pSmad 1/5/8, Runx2, and collagen X proteins, while maintaining chondrogenesis by pSmad 2/3 and collagen II proteins. Inhibiting pSmad 1/5/8 under free swelling culture significantly reduced collagen X protein, similar to the loading condition. Chondroitin sulfate was necessary for load -inhibited hypertrophy and correlated with enhanced S100A4 expression, which is downstream of the osmotic responsive transcription factor NFAT5. Inhibiting p38 MAPK under loading reduced S100A4 expression, and upregulated Runx2 and collagen X protein. Findings from this study indicate that chondroitin sulfate with dynamic loading create physiochemical cues that support MSC chondrogenesis and attenuate hypertrophy through Smad 1/5/8 inhibition and p38 MAPK upregulation.