Roles Of Gap Junction On Chondrogenic Differentiation Of Human Bone Marrow Mesenchymal Stem Cells In Vitro

Objective: To investigate the roles of gap junction and connexins on chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro.Methods: The hMSCs were isolated from human bone marrow and cultured in vitro. The cells in passage 3 were chosen to induce into chondrogenic differentiation with recombinant human GDF-5(100ng/ml) or gap juctional blocker: carbenoxolone (2μmol/L). The proliferation effect of carbenoxolone on MSCs was investigated by MTT assay. Immunofluorescence staining method was uesd to detect the expression of collagenⅡ, Connexin43 protein was examined with western blotting and the chondroitic proteoglycan was measured by alcian blue. To observe intercellular bridge connection of hMSCs by Confocal Laser Scanning Microscope and draw fluorescent recovery curve.Results: MTT assay showed that carbenoxolone at the concentration of 2μmol/L had no effect on the proliferation of hMSCs; immunocytochemistry and Immunofluorescence staining showed that carbenoxolone could inhibit collagenⅡexpressed by hMSCs with the intervene of GDF-5 and carbenoxolone (p<0.05). Western blotting demonstrated that carbenoxolone played no effect on the expression of connexin43. Alcian blue staining revealed that carbenoxolone could inhibit deposition of typical cartilage extra cellular matrix promoted by recombinant GDF-5(p<0.05).Recovery curve showed carbenoxolone could decrease the ability of intercellular bridge connection Conclusion: These results suggest that human mouse bone marrow mesencymal stem cells can be differentiated into chondrogenic phonotype with the induction of GDF-5 in vitro, and that connexin43 mediated gap junction cellular communication plays an important role in the process.