Effect of the gap junction blocker 1-heptanol on chondrogenic differentiation of mouse bone marrow mesenchymal stem cells in vitro

Abstract

Objective To investigate the effect of the gap junction blocker 1-heptanol on the in vitro chondrogenic differentiation of mouse bone marrow mesenchymal stem cells(MSCs) following induction by GDF-5. Methods MSCs were isolated from mouse bone marrow and cultured in vitro. After 3 passages cells were induced to undergo chondrogenic differentiation with recombinant human GDF-5(100 ng/ml), with or without 1-heptanol(2.5μ mol/L). The effect of 1-heptanol on MSCs proliferation was investigated using the MTT assay. Type, collagen mRNA and protein were examined by RT-PCR and immunocytochemistry respectively, and the sulfate glycosaminoglycan was assessed by Alcian blue dye staining. Connexin43(Cx43) protein was examined by western blotting. Results GDF-5 induced proliferation and chondrogenic differentiation of MSCs. While 1-heptanol treatment had no effect on this proliferation, it inhibited the expression of both type, collagen mRNA and protein. The Alcian blue staining revealed that 1-heptanol also inhibited the deposition of the typical cartilage extracellular matrix promoted by recombinant GDF-5. Western blotting demonstrated that 1-heptanol had no effect on the expression of Cx43. Conclusion These results suggest that mouse bone marrow MSCs can be differentiated into a chondrogenic phenotype by GDF-5 administration in vitro. While the gap junction blocker, 1-heptanol, did not reduce gap junction Cx43, these intercellular communication pathways clearly played an important functional role in GDF-5-induced cartilage differentiation.