Growth and differentiation of osteoblasts regulated by low-intensity pulsed ultrasound of various exposure durations

Several studies have investigated the effect of low-intensity pulsed ultrasound (LIPUS) on fractured bone and bone cells. In general, positive results were reported, but they may vary depending on the LIPUS operational mode. In the present study, the effect of exposure duration associated with LIPUS insonification on osteoblasts was extensively investigated. In experiments, osteoblasts were insonified with LIPUS of 3, 5, 10, and 20 minutes, respectively. The employed LIPUS used a 1 MHz frequency, a 100 mW/cm² spatial-average-temporal-average intensity (I_SATA), and a 20% duty cycle at a 1 kHz pulse repetition frequency. The effect of LIPUS on osteoblasts was assessed in terms of the growth and differentiation of cells corresponding to cell viability, alkaline phosphatase (ALP) activity, mRNA expression of ALP and osteocalcin (OCN), and Alizarin red-S staining. Results demonstrate that the growth and differentiation of insonified osteoblasts increased with increasing exposure duration for durations of up to 10 minutes. Specifically, with 10 minutes of LIPUS insonification, the proliferation and ALP activity of osteoblasts increased to 1.07- and 1.31-fold than those of the control groups, respectively; OCN mRNA expression and the mineralization of osteoblasts increased respectively to 2.23- and 2.5-fold than those of the control groups. When the exposure duration of LIPUS was further increased to 20 minutes, the growth and differentiation of insonified osteoblasts did not increase. This indicates that the effect of LIPUS insonification on osteoblasts is exposure-duration-dependent. Furthermore, the temperature of the medium corresponding to LIPUS insonfication varied within 0.5 °C, verifying that the LIPUS insonification provides a non-thermal effect on the regulation of cellular growth and differentiation. Consequently, with appropriate LIPUS exposure duration, the proliferation, differentiation, and mineralization of insonified osteoblasts can be regulated, which could be beneficial for the treatment of bone-related diseases.