The growth of osteoblasts stimulated by various time duration of low intensity pulsed ultrasounds

In this study, osteoblasts (OBs) were insonated with low intensity pulsed ultrasound (LIPUS) of different exposure durations to extensively investigate ultrasound exposure on the growth of cells. OBs were seeded and then were incubated for 24 hours to allow cells to be adhered on the culture plate. Subsequently, LIPUS of a fixed 100 mW/cm2 intensity from a 1 MHz and 20% duty cycle within 1 kHz pulse repetition frequency was arranged to respectively stimulate various groups of passage OBs for 3, 5, 10 and 20 minutes daily. Another experiment was also arranged using the same protocol to insonify groups of differentiation OBs for 14 days. The insonification responses for those OBs were evaluated by cell morphology, viability, alkaline phosphatase (ALP) activity, Alizarin red-S staining, and mRNA expression by reverse transcriptase ploymerase chain reaction (RT-PCR). Results showed that the proliferation of those insonified OBs for 10 minutes tended to increase to around 1.07 fold compared to cells of the control group after the fourth day of experiments. Similar tendency for the ALP activity was observed, in which the largest activity and proliferation rate of OBs were measured to be at the sixth day after insonation. The largest mineralization of OBs enhanced by LIPUS insonification also corresponded to cells insonified for 10 minutes daily. Moreover, the RT-PCR analysis revealed that those cells insonified for 10 minutes daily tended to increase 2 fold mRNA levels of osteocalcin than those of control group. This study also demonstrated that both proliferation and activation of ALP synthesis of OBs may be enhanced by LIPUS stimulation and is exposure duration dependence in which sufficient longer insonification duration brings more effective in promoting the OBs growth.