Integrating 3D Osteocyte Culture, Microgravity Simulation, and Fluid Flow Reveals Mechanisms of Osteocyte Mechanosensation and Calcium Signaling Altered by Disuse

Osteocytes translate fluid shear stress into biochemical signals critical for bone homeostasis. Here, we combined 3-dimensional (3D) osteocyte culture, microgravity simulation, fluid shear mimicking reloading after disuse, and real-time calcium signaling analysis to elucidate responses of osteocytes under different mechanical environments. Ocy454 cells were seeded onto 3D scaffolds and cultured under static (control) or simulated microgravity (disuse) conditions using a rotating wall vessel bioreactor. Elevated expression levels of <i>Sost</i>, <i>Tnfsf11</i> (Rankl), and <i>Dkk1</i> were detected following disuse, confirming efficacy of the microgravity model. Cell membrane integrity under mechanical challenge was evaluated by subjecting scaffold cultures to fluid shear in medium containing FITC-conjugated dextran (10 kDa). The proportion of dextran-retaining cells, indicative of transient membrane disruption and subsequent repair, was higher in microgravity-exposed osteocytes than controls, suggesting increased susceptibility to membrane damage upon reloading following disuse. Intracellular calcium signaling was assessed under a high but physiological fluid shear stress (30 dynes/cm²). Scaffolds cultured under disuse conditions demonstrated a larger sub-population of osteocytes with high calcium signaling intensity (F/Fo > 10 fold) during fluid shear. The maximum fold change in calcium signaling intensity over baseline and the duration of the peak calcium wave were greater for osteocytes cultured under disuse as compared to static controls, however the bioreactor-cultured osteocytes showed, on average, fewer calcium waves than those cultured under control conditions. Subsequent experiments demonstrated that the sub-population of osteocytes with high calcium signaling intensity following exposure to disuse were those that had experienced a transient membrane disruption event during reloading. Together, these results suggest that simulated microgravity enhances osteocyte susceptibility to formation of transient membrane damage and alters intracellular calcium signaling responses upon reloading. This integrated approach establishes a novel platform for mechanistic studies of osteocyte biology and could inform therapeutic strategies targeting skeletal disorders related to altered mechanical loading.