TGF-β1-induced chondrogenesis of bone marrow mesenchymal stem cells is promoted by low-intensity pulsed ultrasound through the integrin-mTOR signaling pathway

Background: Low-intensity pulsed ultrasound (LIPUS) is a mechanical stimulus that plays a key role in regulating the differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the way in which it affects the chondrogenic differentiation of BMSCs remains unknown. In this study, we aimed to investigate whether LIPUS is able to influence TGF-beta 1-induced chondrogenesis of BMSCs through the integrin-mechanistic target of the Rapamycin (mTOR) signaling pathway. Methods: BMSCs were isolated from rat bone marrow and cultured in either standard or TGF-beta 1-treated culture medium. BMSCs were then subjected to LIPUS at a frequency of 3 MHz and a duty cycle of 20%, and integrin and mTOR inhibitors added in order to analyze their influence on cell differentiation. BMSCs were phenotypically analyzed by flow cytometry and the degree of chondrogenesis evaluated through toluidine blue staining, immunofluorescence, and immunocytochemistry. Furthermore, expression of COL2, aggrecan, SOX9, and COL1 was assessed by qRT-PCR and western blot analysis. Results: We found that LIPUS promoted TGF-beta 1-induced chondrogenesis of BMSCs, represented by increased expression of COL2, aggrecan and SOX9 genes, and decreased expression of COL1. Notably, these effects were prevented following addition of integrin and mTOR inhibitors. Conclusions: Taken together, these results indicate that mechanical stimulation combined with LIPUS promotes TGF-beta 1-induced chondrogenesis of BMSCs through the integrin-mTOR signaling pathway.