Mechanosensitive Ca²⁺ signaling and coordination is diminished in osteocytes of aged mice during ex vivo tibial loading

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.