In vitro model to study confined osteocyte networks exposed to flow-induced mechanical stimuli.

Osteocytes are considered the primary mechanical sensor in bone tissue and orchestrate the coupled bone remodeling activity of adjacent osteoblast and osteoclast cells. In vivo investigation of mechanically induced signal propagation through networks of interconnected osteocytes is confounded by their confinement within the mineralized bone matrix, which cannot be modeled in conventional culture systems. In this study, we developed a new model that mimics this in vivo confinement using gelatin methacrylate (GelMA) hydrogel or GelMA mineralized using osteoblast-like model cells. This model also enables real-time optical examination of osteocyte calcium (Ca 2+ ) signaling dynamics in response to fluid shear stimuli cultured under confined conditions. Using this system, we discovered several distinct and previously undescribed patterns of Ca 2+ responses that vary across networks of interconnected osteocytes as a function of space, time and connectivity. Heterogeneity in Ca 2+ signaling may provide new insights into bone remodeling in response to mechanical loading. Overall, such a model can be extended to study signaling dynamics within cell networks exposed to flow-induced mechanical stimuli under confined conditions.