Role of microfilament depolymerization in osteogenic differentiation of bone marrow mesenchymal stem cells promoted by low intensity pulsed ultrasound

Abstract

Objective To investigate the role of microfilament depolymerization in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) promoted by low intensity pulsed ultrasound (LIPUS). Methods Mouse BMSCs were randomly divided into 4 groups: blank control group, LIPUS group, LIPUS+DMSO group and LIPUS+cytochalasin D (CytoD) group. Alkaline phosphatase (ALP) activity of each group was detected by ALP staining. The expression and cellular localization of runt-related transcription factor 2 (RUNX2) were observed by immunofluorescence staining. Western blotting was performed to determine the protein changes of osteogenic differentiation-related genes, type I collagen (collagen 1), osteopontin (OPN) and runt-related transcription factor 2 (RUNX2), as well as the contents of filamentous actin (F-actin) and globular actin (G-actin) in the cells. The changes of cell morphology and microfilament cytoskeleton structure were observed with confocal laser scanning microscopy. Results As compared with the blank control group, LIPUS irradiation significantly enhanced the activity of ALP and upregulated the protein expression of osteogenic related genes Collagen1 (P < 0.01), OPN (P < 0.05) and RUNX2 (P < 0.01), with about a 1.5-fold increase in RUNX2 level (P < 0.01). Immunofluorescence staining showed that RUNX2 expression was obviously increased in the nucleus after LIPUS irradiation, and the cells shrank slightly with punctate distribution of G-actin in the cytoplasm. Western blotting revealed that the ratio of F-actin/G-actin was remarkably lower in the LIPUS group than the control group (P < 0.05). In addition, in comparison with the LIPUS+DMSO group, the LIPUS+CytoD group had greatly promoted the depolymerization of intracellular microfilaments, reduced ratio of F-actin/G-actin (P < 0.05), enhanced activity of ALP (P < 0.05), upregulated expression of RUNX2 in the nucleus (P < 0.05), and improved expression levels of the osteogenic related genes (P < 0.05). Conclusion LIPUS promotes the osteogenic differentiation of BMSCs, which may be related to LIPUS facilitating the depolymerization of microfilaments.

Keywords

• microfilament depolymerization
• low intensity pulsed ultrasound
• bone marrow mesenchymal stem cells
• osteogenic differentiation