Light-programmable gelatin hydrogels for bidirectional stiffness modulation and phase-specific tissue regeneration.

Abstract

Dynamic mechanical cues orchestrate tissue morphogenesis, yet most hydrogels provide only static or one-way mechanics. Here, we present a fully protein-based gelatin hydrogel (GelMA-NB-DB) in which strain-promoted azide-alkyne cycloaddition (SPAAC) forms the primary network under cytocompatible, initiator-free conditions and enables subsequent bidirectional, light-programmable modulation of stiffness in vivo. Orthogonal o-nitrobenzyl photolysis (softening) and methacryloyl photo-crosslinking (stiffening) are actuated by the same 365/405 nm light, allowing bidirectional and spatiotemporally precise modulation of network density. Multi-scale in situ X-ray scattering characterization reveals coherent light-induced transitions from bond-level rearrangement to mesoscale structural reorganization. In 3D cultures, MSCs and iPSC-derived motor neurons exhibit sequence-dependent responses to repeatable stiffness modulation, including enhanced proliferation, spreading, and neurite extension in softened matrices and suppression upon re-stiffening. In vivo, phase-specific soft→stiff programs stabilized early constructs and were associated with coordinated vasculogenesis, angiogenesis, and host-graft anastomosis. In a 60% volumetric muscle loss model, a single 120-μL injection synchronized scaffold mechanics with immune and regenerative phases, yielding homogeneous tissue integration and improved gait coordination. Together, this cytocompatible, bidirectionally tunable hydrogel establishes light-programmed matrix remodeling as an active design strategy for spatiotemporal regulation of vascularized tissue regeneration.