Pressure and not spatial average temporal average intensity governs mechanosensitive responses of osteoblast-like cells exposed to low intensity pulsed ultrasound.

Low-intensity pulsed ultrasound (LIPUS) is approved to promote healing in non-union bone fractures in the UK (NICE) and USA (FDA). Despite extensive in vitro, pre-clinical, and clinical data indicating efficacy, patient outcomes remain inconsistent. A deeper understanding of the mechanisms by which ultrasound vibrations influence cellular behaviour is critical to optimising LIPUS for bone repair and to enable greater patient benefit. The literature offers a broad experimental base, but collective insights are hindered by two key issues: inadequate reporting of ultrasound exposure conditions, often overlooking reflections and standing waves, and reliance on spatial average temporal average intensity (ISATA) as the sole metric of ultrasound dose. While ISATA informs safety thresholds (TI, MI), it fails to describe the specific acoustic stimuli cells experience, masking variations in pressure, pulse repetition, and duty cycle. To identify the ultrasound parameters that are most important for eliciting mechano-sensing responses in osteoblast-like cells, we systematically evaluated a 1 MHz pulsed field in a controlled cell culture environment. Immunofluorescence analysis of actin and vinculin were used to assess cytoskeletal changes in response to fully described LIPUS exposures. We identified a pulse repetition frequency (PRF) upper limit of 1 kHz, beyond which LIPUS lost efficacy in enhancing mechano-sensing. Optimal response occurred at 20 % duty cycle, 160 kPa, and 60 mW/cm2 ISATA, challenging the currently accepted standard and parameters used to operate existing clinical devices (1 MHz, 30 mW/cm2 ISATA). Our data demonstrate the necessity to report fully the parameters that describe the ultrasound dose experienced by cells to predict which conditions lead to an upregulation in mechano-sensing and that ISATA alone is not an adequate measure unless all other parameters are known and fixed. Finally, since PRF is determinant of achieving a cellular response, we reaffirm the already accepted understanding that pulsed exposures are critical to a cellular ability to detect and/or respond to ultrasound in a way that is useful for fracture repair.