Mechanobiological modulation of in situ and in vivo osteocyte calcium oscillation by acoustic radiation force

The biological effect of ultrasound on bone regeneration has been well documented, yet the underlying mechanotransduction mechanism is largely unknown. In relation to the mechanobiological modulation of the cytoskeleton and Ca²⁺ influx by short-term focused acoustic radiation force (FARF), the current study aimed to visualize and quantify Ca²⁺ oscillations in real-time of in situ and in vivo osteocytes in response to focused low-intensity pulsed ultrasound (FLIPUS). For in situ studies, fresh mice calvaria were subjected to FLIPUS stimulation at 0.05, 0.2, 0.3, and 0.7 W. For the in vivo study, 3-month-old C57BL/6J Ai38/Dmp1-Cre mice were subjected to FLIPUS at 0.15, 1, and 1.5 W. As observed via real-time confocal imaging, in situ FLIPUS led to more than 80% of cells exhibiting Ca²⁺ oscillations at 0.3–0.7 W and led to a higher number of Ca²⁺ spikes with larger values at >0.3 W. In vivo FLIPUS at 1–1.5 W led to more than 90% of cells exhibiting Ca²⁺ oscillations. Higher FLIPUS energies led to larger Ca²⁺ spike magnitudes. In conclusion, this study provided a pilot study of both in situ and in vivo osteocytic Ca²⁺ oscillations under noninvasive FARF, which aids further exploration of the mechanosensing mechanism of the controlled bone cell motility response to the stimulus.