Mechanically induced Ca^(2+) 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 (Ca^(2+) ) dynamics. Here, by simultaneously monitoring Ca^(2+) and actin dynamics in single cells exposed to fluid shear flow, we detected actin network contractions immediately upon onset of flow-induced Ca^(2+) 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 Ca^(2+) 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 Ca^(2+) -dependent contractions and enhances the production and release of EVs containing bone regulatory proteins. Further, blocking Ca^(2+) signaling significantly attenuates adaptation to mechanical loading in vivo, suggesting a critical role for Ca^(2+) -mediated signaling in bone adaptation.

How does Mechanical Loading Make Bone Microstructure: Modeling vs. Remodeling

Bone modeling and remodeling are governed by distinct biochemical processes that may hold unique opportunities for optimizing bone mass[1,2].Remodeling refers to the coupled resorption and subsequent formation on the bone surface,while modeling represents uncoupled formation or resorption.Mechanical loading is known to improve bone mass,though whether this occurs through modeling or remodeling(or by some combination)is unclear.Dynamic in vivo morphometry utilizing high resolution micro-CT and image registration has only recently become feasible and thus holds an untapped and expanding potential for understanding bone metabolism by quantifying and localizing formation/resorption and modeling/remodeling events.16-week-old mice were given 2 baseline weekly micro-CT scans of both tibiae prior to the initiation of daily unilateral loading(contralateral limb for nonloaded control).Weekly scanning and daily loading continued for 5 weeks.Registered images for each mouse in a global coordinate system revealed the time course of each voxel,and changes in bone mass were quantified as modeling or remodeling starting at the onset of loading.In cortical bone,after an initial response to loading in both regimes,modeling emerged as the dominant response.Loading effects were largest in areas of mechanical significance.For example,anabolic modeling on the periosteal surface of the half of the tibia in compression under axial load presented a strong effect of loading,whereas the same measure on the endosteal surface in the area in tension showed no difference.Similarly,in trabecular bone anabolic modeling was significantly increased with loading on trabecular plates but not rods(plates have been shown to be the major contributor to overall bone strength).The catabolic modeling response on the endosteal surface showed an interesting transition over time.Loading initially led to a significant suppression of catabolic modeling,but over time increased it to levels significantly beyond that of nonloaded controls.