Direct extrusion of multifascicle prevascularized human skeletal muscle for volumetric muscle loss surgery

Volumetric skeletal muscle injuries are prevalent, highlighting the imperative need for scaffolds to facilitate the healing process of such wounds. Human skeletal muscle is composed of multiple fascicles, which are parallel bundles of muscle fibres surrounded by a layer of connective tissue that contains blood vessels and nerves. Replicating these structures presents a considerable challenge. Here, we developed a method to fabricate multifascicle human skeletal muscle scaffolds that mimic the natural structure of human skeletal muscle bundles using a seven-barrel nozzle. To form the core material to generate the fascicle structure, human skeletal myoblasts were encapsulated in Matrigel with calcium chloride. Meanwhile, to create the shell that plays a role as the connective tissue structure, human fibroblasts and human umbilical vein endothelial cells within a mixture of porcine muscle decellularized extracellular matrix and sodium alginate at a 95:5 ratio was used. We assessed four types of extruded scaffolds monolithic-monoculture (Mo-M), monolithic-coculture (Mo-C), multifascicle-monoculture (Mu-M), and multifascicle-coculture (Mu-C) to determine the structural effect of muscle mimicking scaffold. The Mu-C scaffold demonstrated cell proliferation, differentiation, vascularization, mechanical properties, and functionality that were superior to those of the other scaffolds. Furthermore, in an in vivo mouse model of volumetric muscle loss, the Mu-C scaffold effectively regenerated the tibialis anterior muscle defect, demonstrating its potential for volumetric muscle transplantation. The multibarrel nozzle device was applied to create functional Mu-C muscle scaffolds that structurally mimicked human skeletal muscle. Our nozzle will be further used to produce other volumetric functional tissues, such as tendons and peripheral nerves. ### Competing Interest Statement The authors have declared no competing interest.