Effects of RhoA/ROCK inhibitors on chondrogenesis of human periodontal ligament-derived mesenchymal stem cells

Abstract

Several researches reported that the control of cytoskeleton activity via RhoA-ROCK may promote the chondrogenic differentiation of stem cells. Therefore, we determined whether the inhibition of RhoA-ROCK pathway by ROCK inhibitor Y-27632 and actin polymerization inhibitor cytochalasin D can promote chondrogenesis in PDLSCs in the presence or absence of TGF-β3. PDLSCs were isolated and purified from the periodontal ligament of the human third molar teeth. After initiating chondrogenesis with 3D cell cluster formation, the clusters were maintained under 10 ng/mL TGF-β3, 3 μM cytochalasin D, 10 μM Y-27632, TGF-β3+cytochalasin D, TGF-β3+Y-27632 and without treatment as negative control. We analyzed the chondro-clusters by glycosaminoglycan assay (GAG), histology evaluation, safranin O and von Kossa staining and PCR. Immunohistochemistry were performed to measeure the expression for Collagen I, II as well as aggrecan. Clusters treated with TGF-β3 and Y-27632 had the highest level of GAG synthesis. Unlike cytochalasin D, Y-27632 was found to exert a synergistic effect as well. Combination treatment of TGF-β3 and Y-27632 increased the expression of the chodro-related genes, collagen type II and sox9 while decreasing expression of the osteogenic gene, Runx2. On the other hand, treatment with TGF-β3 and cytochalasin D let do the greatest expression of Runx2 and collagen X, a hypertrophic chondrocyte marker gene. Calcium deposits were visualized by von Kossa staining. The least amount of calcium deposition was found in clusters exposed to TGF-β3 with Y-27632. In conclusion, our data suggest that the treatment of TGF-β3 along with RhoA/ROCK inhibition using Y-27632 induced more selective chondrogenesis. Furthermore, PDLSCs and control of the RhoA/ROCK pathway have the potential to be for cartilage tissue repair including the temporomandibular joint disc regeneration.