TGF-β3 improves bone mesenchymal stem cells toward chondrogenic differentiation under hypoxia environment

Abstract

Objective: To investigate the impact of TGF-β3 on the chondrogenesis of bone marrow mesenchymal stem cells (BM-MSCs) under hypoxia environment. Methods: BM-MSCs were obtained from SD rat tibias and femora and cultured with whole bone marrow adherent method. Cell surface antigens were analyzed by flow cytometry and the multiple-directional differentiation capabilities were detected with special differentiation agents to affirm the reality of BM-MSCs. Under normoxia or hypoxia condition, BM-MSCs were induced with TGF-β3 or not. Then, alcian blue and immunofluorescence staining were performed to evaluate the expression level of aggrecan, collagen Ⅱ. qRT-PCR analysis were performed to analyze the expression of aggrecan, collagen Ⅱ and collagen Ⅹ. qRT-PCR and Western blot analysis was performed to detect the mRNA and protein level of HIF-1α, collagenⅡ and β-catenin. Results: BM-MSCs were fibroblast-like shape and had ablities of osteogeic, adipogenic and chondrogenic differentiation, with the expression of CD(29, )CD(44) and CD(90) but not CD(45). Alcian blue and immunofluorescence staining showed that BM-MSCs strongly expressed the aggrecan and collagen Ⅱ with the presence of TGF-β3 under hypoxia condition. qRT-PCR analysis showed the mRNA expression levels of collagen Ⅱ, aggrecan and collagen Ⅹ were up-regulated at 2.46, 2.20 and 1.80 folds, comparing with control group (all P<0.05). Western blot analysis showed that the protein levels of HIF-1α, collagenⅡ in BM-MSCs were up-regulated with the presence of TGF-β3 under hypoxia condition, but β-catenin level was down-regulated. Conclusion: TGF-β3 promotes the chondrogenic differentiation ability of BM-MSCs under hypoxia condition, which may be relative with the inhibition of Wnt/β-catenin signaling pathway.