The inhibition effect of low-intensity pulsed ultrasound on osteoclasts progenitor cells

The healing of bone fracture with low-intensity pulsed ultrasound (LIPUS) associated with bone formation by osteoblasts has been validated in previous studies. Yet, little has been known about the effect of LIPUS on the osteoclasts during the bone remodeling process. To extensively explore this issue, osteoblast precursor cells were insonified by LIPUS in which that the insonification effect was verified by differentiation and other activities of cells. Experiments were performed on the osteoclast precursor cells formed by the differentiation of RAW 264.7 cell line via cultured the cells in the growth medium containing receptor-activated nuclear factor K B ligand (RANKL) for 3 days. The 1 MHz LIPUS, with calibrated 125 mW/cm² SATA intensity, 20% duty cycle of a 1 kHz pulse repetition frequency, was arranged to daily insonify the osteoclast precursor cells in the period between the 3^rd and 6^th day during culturing. The morphology and number of tartrate resistant acid phosphatase (TRAP) positive multinucleated osteoclasts were observed and measured. Gene expression of receptor activator of nuclear factor K B (RANK), cathepsine K, matrix metalloproteinase 9 (MMP9), and TRAP were determined by reverse transcription polymerase chain reaction (RT-PCR). Following the LIPUS insonification, the bone resorptive activity of osteoclast precursor cells tended to be inhibited. The number of TRAP positive multinucleated osteoclasts for the experimental group was lower than that of the control group. The resorption pits on the calcium phosphate film also found to be inhibited by LIPUS insonification, where the pit formation of the experimental group was smaller than that of the control group. RT-PCR results reveled that LIPUS decreased the mRNA levels of RANK, TRAP, cathepsin K, and MMP9 mRNA. The LIPUS insonification suppress osteoclast differentiation by the inhibition of multinucleated osteoclast formation be demonstrated in this study. These results suggest that the inhibition effect of LIPUS on osteoclasts is through the reduction of RANKL induced expression of cathepsin K and MMP9 for modulating bone remodeling process.