The role of RhoA-ROCK activator and inhibitor during chondrogenesis of periodontal derived mesenchymal stem cells

Abstract

Stem cells can perform important role in therapy of the cartilage disease, especially temporomandibular disease(TMD). Recent studies indicated that periodontal ligament stem cells(PDLSCs) can induce chondrogenesis under specialized conditions. And Pasteurella multocida toxin(PMT) has been considered a stimulator of RhoA/ROCK pathway. We demonstrate that activator and inhibitor of RhoA/ROCK pathway, which affect actin cytoskeleton, can influence PDLSCs differentiation. From the impacted mandibular third molar of a patient, PDL cells were gained. For inducing chondrogenesis, a mechanical force by centrifugation formed 3D cell cluster. In 2-D culture stage, one group is cultured with 10 ng/mL PMT and the other group is cultured in defined media for 2 days. In initiation stage, the PDL-derived 3D clusters of PMT-not treated group were differentiated in defined media as negative control and 10 ng/mL TGF-β3-containing media as positive control. The PDL-derived 3D clusters of PMT-treated group were differentiated with 10 ng/mL PMT, 10 ng/mL TGF-β3 and 10 μM Y-27632, respectively. We maintained the chondrogenic differentiation process for 7 d and 14 d. We performed the glycosaminoglycans assay, RT-PCR assay, histology and immunohistochemistry for analyzing a chondrogenesis. GAG contents were increased by TGF-β3 compared to negative control in 7 d and 14 d

7d:183%, 14d:210%. GAG contents also were increased in PMT-pretreated group compared to negative control in 7 d and 14 d. In PMT/defined group, GAG contents were increased by 113% in 7 d and by 115% in 14 d. Also, in PMT/Y-27632 group, GAG contents were increased by 143% in 7 d and by 188% in 14 d. In PMT/TGF-β3 group, the results are similar

173%(7 d), 204%(14 d). RT-PCR results showed that TGF-β3, PMT/Y-27632, PMT/TGF-β3 increased the level of chondrogenesis markers such as SOX 9, collagen II expression compared to control. Especially in 7 days of culture, the PMT decreased the expression of chondrogenic marker gene such as SOX9 and collagen type II when comparing PMT/TGF-β3 group to TGF-β3 group. But in 14 days of culture, PMT increased the expression of those genes. And the expression of collagen type X is the largest in PMT/TGF-β3 group and was increased both 7 days and 14 days group. The expression of RUNX2 and collagen type I could be observed in almost all groups. The histologic and immunohistochemical analysis indicated that chondrogenesis-associated genes and chondrogenic protein abundance were increased in cells with 10 ng/mL TGF-β3 compared to negative control. PMT-pretreatment groups also showed that more intense staining patterns compared to negative control. In this study, we recognized the regulation cytoskeleton activity via RhoA/ROCK signaling pathway can control chondrogenesis of PDLSCs. We also suggest that hPDLSCs can play an significant role in tissue-engineering strategy as an excellent cell source for chondrogenesis.