Assessing Hydrogel Microenvironment Density Effects on Mechanotransduction In Vitro and Bone Regeneration In Vivo

For bone regeneration, matrix density is a critical determinant of scaffold performance, influencing both mechanical stiffness and biological accessibility, that is, how readily cells interact with and remodel the matrix. Here, we investigated how collagen matrix densification modulates stem cell mechanosensing in vitro and bone healing outcomes in vivo using density-tunable collagen microgels. Centrifugation-based collagen densification produced a sixfold increase in stiffness and a compact fibrillar architecture that enhanced early mechanosensitive signaling in human mesenchymal stem cells, including YAP1 and Piezo1 upregulation, reaching levels comparable to biochemical osteoinduction. In vivo, low- and high-density microgels increased bone formation relative to untreated defects, though low-density constructs supported more uniform and volumetric regeneration. Despite these differences in bone volume, bone mineral density and histological maturity were comparable across all groups, while complete bridging was observed only with autologous bone grafts. Our findings demonstrate that matrix densification promotes early mechanotransductive priming, but the microgel format did not achieve full regeneration for either gel group, suggesting that effective bone healing likely requires balancing mechanical stimulation with sufficient biological accessibility. Designing scaffolds with controlled density and adaptive mechanical properties may create dynamically permissive environments that support both early mechanosensing and late-stage remodeling for functional bone repair.