Modular biofabrication of a vascularized skeletal muscle model through endothelialized microvascular seeds

The clinical translation of engineered skeletal muscle (eSM) for volumetric muscle regeneration is hindered by the challenge of establishing a functional vascular network capable of sustaining its high metabolic demand and ensuring graft survival. Here, we present a bottom-up biofabrication strategy to generate a pre-vascularized in vitro eSM model through the modular assembly of independently matured muscle and vascular compartments. C2C12 myoblasts were encapsulated within core-shell fibers using rotary wet-spinning (RoWS), yielding anisotropically aligned, multinucleated, and contractile myofibers expressing myosin heavy chain and sarcomeric -actinin. In parallel, gelatin methacryloyl (GelMA)-based microvascular seeds ({micro}VS), pre-endothelialized with human umbilical vein endothelial cells, were engineered to guide rapid and structurally stable vascular formation while preventing uncontrolled capillary self-organization. Fully endothelialized {micro}VS were incorporated into a pro-angiogenic bioink and processed via RoWS to generate tubular vascular fibers with physiological diameters (100-200 m) and continuous CD31-positive lumens. After independent maturation, muscle and vascular constructs were bioassembled into a hierarchically organized tissue and co-cultured. By decoupling myogenic and angiogenic differentiation, this strategy overcomes medium incompatibility typical of conventional co-cultures, preserving compartment-specific architecture and function and establishing a versatile platform for muscle-vascular modeling and translational muscle repair.