Temporal Matrix Stiffening Programs Osteogenic Differentiation and Immunomodulation to Enhance Osteogenesis

Dynamic changes in matrix mechanics play critical roles in bone regeneration, yet most biomaterial platforms provide static mechanical environments that fail to reflect the temporal evolution of the native healing niche. Here, we developed a dynamically stiffening GelMA-based hydrogel to investigate how temporally programmed matrix mechanics regulate bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation and macrophage-to-BMSC paracrine signaling. The hydrogel initially provided a soft, permissive microenvironment that supported cell viability and spreading. Following an induced inflammatory challenge, the static-soft and dynamic conditions maintained macrophage phenotype-associated profiles more consistent with inflammation resolution than the static-stiff condition. Compared with continuous static stiffness, delayed matrix stiffening preserved greater macrophage and BMSC viability, maintained more favorable macrophage-derived paracrine signaling, and enhanced overall osteogenic outcomes. Both stiffened conditions promoted YAP nuclear localization, supporting the involvement of YAP-associated mechanotransduction. Collectively, these findings demonstrate that temporal mechanical programming can coordinate cellular adaptation, macrophage-associated inflammatory responses, and osteogenic differentiation, providing a promising design strategy for immunomodulatory biomaterials in bone tissue engineering and regenerative medicine.