Effects of non-thermal ultrasound on a fibroblast monolayer culture: Influence of pulse number and pulse repetition frequency

Despite the use of therapeutic ultrasound in the treatment of soft tissue pathologies, there remains some controversy regarding its efficacy. In order to develop new treatment protocols, it is a common practice to carry out in vitro studies in cell cultures before conducting animal tests. The lack of reproducibility of the experimental results observed in the literature concerning in vitro experiments motivated us to establish a methodology for characterizing the acoustic field in culture plate wells. In this work, such acoustic fields are fully characterized in a real experimental configu-ration, with the transducer being placed in contact with the surface of a standard 12-well culture plate. To study the non-thermal effects of ultrasound on fibroblasts, two different treatment protocols are proposed: long pulse (200 cycles) signals, which give rise to a standing wave in the well with the presence of cavitation (ISPTP max = 19.25 W/cm), and a short pulse (five cycles) of high acoustic pressure, which produces a number of echoes in the cavity (ISPTP = 33.1 W/cm, with Pmax = 1.01 MPa). The influence of the acoustic intensity, the number of pulses, and the pulse repetition frequency was studied. We further analyzed the correlation of these acoustic parameters with cell via-bility, population, occupied surface, and cell morphology. Lytic effects when cavitation was present, as well as mechanotransduction reactions, were observed.This research was funded by Spanish Research Agency, grant number DPI2016-80254-R, and the CICYT, grant number MAT2014-51918-C2-1-