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Purpose: The osteocyte is considered the major mechanosensor in bone, capable of detecting forces at a cellular level to coordinate bone formation and resorption. The pathology of age-related bone loss, a hallmark of osteoporosis, is attributed in part to impaired osteocyte mechanosensing. However, real-time evidence of the effect of aging on osteocyte responses to mechanical load is lacking. Intracellular calcium (Ca2+) oscillations have been characterized as an early mechanosensitive response in osteocytes in systems of multiple scales and thus can serve as a real-time measure of osteocyte mechanosensitivity. Our objective was to utilize an ex vivo model to investigate potentially altered mechanosensing in the osteocyte network with aging. Methods: Tibiae were explanted from young-adult (5 mo) and aged (22 mo) female mice and incubated with Fluo-8 AM to visualize osteocyte intracellular Ca2+. Whole tibiae were cyclically loaded while in situ osteocyte Ca2+ dynamics were simultaneously imaged with confocal microscopy. Responsive osteocyte percentage and Ca2+ peak characteristics were quantified, as well as signaling synchrony between paired cells in the field of view. Results: Fewer osteocytes responded to mechanical loading in aged mice compared to young-adult and did so in a delayed manner. Osteocytes from aged mice also lacked the well-correlated relationship between Ca2+ signaling synchrony and cell-cell distance exhibited by young-adult osteocytes. Conclusions: We have demonstrated, for the first time, real-time evidence of the diminished mechanosensing and lack of signaling coordination in aged osteocyte networks in tibial explants, which may contribute to pathology of age-induced bone loss. [ABSTRACT FROM AUTHOR]

Sclerostin antibody romosozumab (EVENITY™, romosozumab-aqqg) has a dual mechanism of action on bone, increasing bone formation and decreasing bone resorption, leading to increases in bone mass and strength, and a decreased risk of fracture, and has been approved for osteoporosis treatment in patients with high risk of fragility fractures. The bone formation aspect of the response to sclerostin antibody treatment has thus far been best described as having two phases: an immediate and robust phase of anabolic bone formation, followed by a long-term response characterized by attenuated bone accrual. We herein test the hypothesis that following the immediate pharmacologic anabolic response, the changes in bone morphology result in altered (lesser) mechanical stimulation of the resident osteocytes, initiating a negative feedback signal quantifiable by a reduced osteocyte signaling response to load. This potential desensitization of the osteocytic network is probed via a novel ex vivo assessment of intracellular calcium (Ca2+) oscillations in osteocytes below the anteromedial surface of murine tibiae subjected to load after short-term (2 weeks) or long-term (8 weeks) treatment with sclerostin antibody or vehicle control. We found that for both equivalent load levels and equivalent strain levels, osteocyte Ca2+ dynamics are maintained between tibiae from the control mice and the mice that received long-term sclerostin antibody treatment. Furthermore, under matched strain environments, we found that short-term sclerostin antibody treatment results in a reduction of both the number of responsive cells and the speed of their responses, which we attribute largely to the probability that the observed cells in the short-term group are relatively immature osteocytes embedded during initial pharmacologic anabolism. Within this study, we demonstrate that osteocytes embedded following long-term sclerostin antibody treatment exhibit localized Ca2+ signaling akin to those of mature osteocytes from the vehicle group, and thus, systemic attenuation of responses such as circulating P1NP and bone formation rates likely occur as a result of processes downstream of osteocyte Ca2+ signaling. • Osteocyte Ca2+ dynamics under mechanical loading are maintained after long-term sclerostin antibody treatment. • Short-term sclerostin antibody treatment results in reduced osteocyte Ca2+ dynamics. • Immature osteocytes stimulated by sclerostin antibody sense mechanical loading differently. [ABSTRACT FROM AUTHOR]