UV-programmed gelatin methacryloyl hydrogel microenvironments reveal a bell-shaped vascularization window and enable compartmentalized neuromuscular disease modeling.

Matrix mechanics and microarchitecture jointly regulate tissue morphogenesis and functional maturation; however, defining their combined effects remains challenging when microenvironmental tuning requires changes in material composition. Here, we establish a UV-programmable gelatin methacryloyl (GelMA) hydrogel system in which ultraviolet exposure coordinates changes in construct architecture, pore morphology, and apparent mechanical properties within a constant material formulation. This framework enables systematic mapping of biological responses to UV-programmed architectural-mechanical microenvironments without compositional confounders. In vivo subcutaneous implantation reveals a nonlinear, bell-shaped vascularization response to UV-programmed GelMA hydrogel properties, identifying a narrow microenvironmental window that supports blood-containing vessel formation, human-derived vascular structures, and host-perfused vascular integration. For neuromuscular modeling, a structurally stable UV-defined regime was selected to support long-term compartmentalized co-culture of human induced pluripotent stem cell-derived myoblasts and motor neuron spheroids. Within this same UV-defined compartmentalized neuromuscular microenvironment, amyotrophic lateral sclerosis (ALS)-derived constructs exhibit impaired myogenic maturation, reduced neuromuscular junction (NMJ)-like structural organization, and altered contractile responsiveness compared with gene-corrected Healthy controls. Pharmacological treatment with the FDA-approved drug Riluzole partially restores these disease-associated phenotypes. Together, these findings establish UV-programmed GelMA hydrogels as an adaptable architectural-mechanical platform for identifying vascularization-permissive microenvironments and supporting compartmentalized neuromuscular disease modeling.