Macroporosity and Stiffness Tailored Hydrogels Enhance Bone Formation via Inside Out and Outside In Regeneration

Synthetic hydrogels are widely used in bone tissue engineering and regeneration; however, their nanoporous architecture restricts encapsulated cells and limits host cell infiltration after implantation. Here, we present a hydrogel system that forms interconnected macroporous networks with simultaneous cell encapsulation, thereby eliminating the need for in vitro cell seeding and pre-culture of prefabricated scaffolds. These macroporous hydrogels are generated by polymerization-induced phase separation in aqueous mixtures of poly(ethylene glycol) and methyl cellulose during enzymatic cross-linking of the former. The resulting hydrogels exhibit tunable pore architectures and mechanical properties. We demonstrate that control over pore size and local mechanical properties enables precise regulation and enhancement of spreading, migration, and proliferation of encapsulated cells. Ultimately, in vivo implantation of these hydrogels promotes pore size-dependent cell infiltration, leading to enhanced formation of bone ossicles and highlighting their potential for efficient, in-depth tissue remodeling. Together, our findings establish phase-separated macroporous hydrogels as a versatile platform for directing tissue regeneration by coordinating cell-driven (inside-out) and host-driven (outside-in) regenerative processes.