Effects of low-intensity ultrasound on the differentiation of C2C12 cells

Objectives: Ultrasound stimulation is an established and widely used intervention for accelerating bone growth during fracture healing and distraction osteogenesis in the region of orthopedics. However, no studies have investigated the effects of the mechanical stimulation provided by ultrasound treatment on periodontal disease accompanying bone loss. For periodontal tissue, mainly alveolar bone and acellular cementum, regeneration to occur, undifferentiated pluripotential cells in the periodontal ligament must differentiate into the osteogenic lineage. The purpose of this study was to determine the effect of low-intensity pulsed ultrasound stimulation on the differentiation of pluripotential mesenchymal cells. Methods: A typical pluripotential mesenchymal cell line, C2C12, was used to clarify the effect of ultrasound stimulation on cell differentiation. The cells were cultured in 5% serum-containing medium to induce cell differentiation, either with or without the addition of ultrasound stimulation. The ultrasound signal consisted of 1.5 MHz at an intensity of 70 mW/cm² for 20 minutes for all cultures. To verify the cell lineage after ultrasound stimulation, mRNA expression of cellular phenotype-specific markers characterizing osteoblasts (Runx2, Msx2, Dlx5, AJ18), chondroblasts (Sox9), myoblasts (MyoD), and adipocytes (C/EBP, PPARg) was studied using real-time polymerase chain reaction analysis. The protein expression of Runx2 and activated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) were performed using Western blotting. Results: The mRNA expression of Runx2, Msx2, Dlx5, AJ18, and Sox9 was increased markedly by the ultrasound stimulation, whereas the expression of MyoD, C/EBP, and PPARg was drastically decreased. In the Western blot analysis, ultrasound stimulation increased Runx2 protein expression and phosphorylation of ERK1/2. Conclusion: Our study demonstrated that ultrasound stimulation converts the differentiation pathway of C2C12 cells into the osteoblast and/or chondroblast lineage via activated phosphorylation of ERK1/2.