Role of Reactive Oxygen Species during Low-Intensity Pulsed Ultrasound Application in MC-3 T3 E1 Pre-osteoblast Cell Culture

We evaluated the activation of mitogen-activated protein kinase (MAPK) activation through reactive oxygen species (ROS) by application of low-intensity ultrasound (LIPUS) to MC-3 T3 E1 pre-osteoblasts. The cells were subjected to one LIPUS application for either 10 or 20 min, and the control group was exposed to a sham transducer. For ROS inhibition, 10 μM diphenylene iodonium (DPI) was added to the cells an hour before LIPUS application. Samples were collected 1, 3, 6, 12 and 24 h after LIPUS application, and cells were evaluated for ROS generation, cell viability, gene expression and MAPK activation by immunoblot analyses. LIPUS caused a significant increase in ROS and cell viability in the non-DPI-treated group. Expression of RUNX2, OCN and OPN mRNA was higher in the LIPUS-treated groups at 1 h in both the DPI-treated and non-DPI-treated groups; RUNX2 and OCN mRNA levels increased at 6 h. ERK1/2 activation was increased in the LIPUS-treated groups. These results indicate that LIPUS activates MAPK by ROS generation in MC-3 T3 E1 pre-osteoblasts.