Mechanically induced Ca²⁺ oscillations in osteocytes release extracellular vesicles and enhance bone formation

The vast osteocytic network is believed to orchestrate bone metabolic activity in response to mechanical stimuli through production of sclerostin, RANKL, and osteoprotegerin (OPG). However, the mechanisms of osteocyte mechanotransduction remain poorly understood. We've previously shown that osteocyte mechanosensitivity is encoded through unique intracellular calcium (Ca²⁺) dynamics. Here, by simultaneously monitoring Ca²⁺ and actin dynamics in single cells exposed to fluid shear flow, we detected actin network contractions immediately upon onset of flow-induced Ca²⁺ transients, which were facilitated by smooth muscle myosin and further confirmed in native osteocytes ex vivo. Actomyosin contractions have been linked to the secretion of extracellular vesicles (EVs), and our studies demonstrate that mechanical stimulation upregulates EV production in osteocytes through immunostaining for the secretory vesicle marker Lysosomal-associated membrane protein 1 (LAMP1) and quantifying EV release in conditioned medium, both of which are blunted when Ca²⁺ signaling was inhibited by neomycin. Axial tibia compression was used to induce anabolic bone formation responses in mice, revealing upregulated LAMP1 and expected downregulation of sclerostin in vivo. This load-related increase in LAMP1 expression was inhibited in neomycin-injected mice compared to vehicle. Micro-computed tomography revealed significant load-related increases in both trabecular bone volume fraction and cortical thickness after two weeks of loading, which were blunted by neomycin treatment. In summary, we found mechanical stimulation of osteocytes activates Ca²⁺-dependent contractions and enhances the production and release of EVs containing bone regulatory proteins. Further, blocking Ca²⁺ signaling significantly attenuates adaptation to mechanical loading in vivo, suggesting a critical role for Ca²⁺-mediated signaling in bone adaptation.