A controlled in vitro study of optimal low intensity pulsed ultrasound fields for stimulation of proliferation in murine osteoblasts

Clinical, in vivo and in vitro studies have established that Low Intensity Pulsed Ultrasound (LIPUS) stimulates healing of fractured bone, but the mechanisms are not well understood. In vitro studies show cell proliferation, migration and many cellular markers are stimulated by LIPUS at frequencies of 1.0 - 1.5 MHz, even down to 45 kHz [1]. However, most trials did not control or measure the acoustic field, so the dose experienced by the cells across such studies cannot be compared. An in vitro ultrasound exposure method was developed to maintain control of the acoustic field. Murine osteoblasts (MC3T3-E1) were exposed to 20-minute LIPUS fields at the frequencies 1 MHz and 45 kHz and Mechanical Index from 0 (control) to 0.2. Cell proliferation was assessed by counting viable cells immediately before and twenty hours after LIPUS exposure in the centre of a custom-designed cell culture vessel. Initial results indicate that LIPUS fields increase cell proliferation at 1 MHz, 0.1 MI and more significantly at 45 kHz, 0.2 MI compared to controls, but has a detrimental or no effect otherwise. Future work will involve further repeats to acquire larger data sets, along with cell counts in other areas of the cell growth surface to increase data obtained from a single repeat. The study demonstrates the efficacy of the method for quantitative in vitro investigation of LPUS mechanisms and will be used in future work to find optimum LIPUS field characteristics, in terms of frequency, Mechanical Index, and pulse characteristics such as pulse repetition rate and pulse width.