Low-Intensity Pulsed Ultrasound Stimulation Enhances Heat-Shock Protein 90 and Mineralized Nodule Formation in Mouse Calvaria-Derived Osteoblasts

Low-intensity pulsed ultrasound (LIPUS) has demonstrated its positive effects on osteogenic differentiation of mesenchymal stem cells and the proliferation and differentiation of osteoblasts, negative effects on osteoclast growth, and promotion of angiogenesis, leading to improvement of the tissue perfusion. Heat-shock proteins (HSPs) are initially identified as molecules encouraged and expressed by heat stress or chemical stress to cells and involved in the balance between differentiation and apoptosis of osteoblasts. However, it remains unclear if the effect of LIPUS on osteoblast differentiation could involve HSP expression and contribution. In this study, mouse calvarial osteoblasts were exposed to LIPUS at a frequency of 3.0MHz by 30mW/cm(2) for 15min or to 42 degrees C heat shock for 20min at day 3 of cell culture and examined for osteogenesis with pursuing induction of HSP27, HSP70, and HSP90. LIPUS as well as heat shock initially upregulated HSP90 and phosphorylation of Smad1 and Smad5, encouraging cell viability and proliferation at 24h, enhancing mineralized nodule formation stronger by LIPUS after 10 days. However, HSP27, associated with BMP2-stimulated p38 mitogen-activated protein kinase during osteoblast differentiation, was downregulated by both stimulations at this early time point. Notably, these two stimuli maintained Smad1 phosphorylation with mineralized nodule formation even under BMP2 signal blockage. Therefore, LIPUS might be a novel inducer of osteoblastic differentiation through a noncanonical signal pathway. In conclusion, LIPUS stimulation enhanced cell viability and proliferation as early as 24h after treatment, and HSP90 was upregulated, leading to dense mineralization in the osteoblast cell culture after 10 days.