Bioengineered human pre-vascularized microtissues reconstruct vascular niche and regenerative microenvironment to enhance functional skeletal muscle regeneration.

Skeletal muscle regeneration mediated by muscle satellite cells (MuSCs) is supported by the specific vascular niche containing endothelial cells, pericytes, and mesenchymal stem cells. Volumetric muscle loss (VML) severely disrupts the vascular niche and impairs the ability of MuSCs to regenerate functional skeletal muscle. Until now, it remains a great challenge to reconstruct the vascular niche for muscle regeneration. Here, we successfully developed a specific 3D induction strategy based on collagen matrix and pro-angiogenic culture system to fabricate a multifunctional human pre-vascularized microtissue (h-VM) in vitro. The h-VM featured a robust vascular network formed by endothelial cells, with pericytes and mesenchymal stem cells accompanying. Under a specific induction medium, the h-VM could differentiate into adipogenic and osteogenic tissues, indicating a strong stemness within the microtissue. Of note, the h-VM significantly boosted the myogenic differentiation of MuSCs through paracrine signaling. Transplantation of the h-VM into the volumetric muscle defect not only facilitated early vessel integration with the host vasculature but also maintained muscle structural stability and provided sustained mechanical support to the defect site. Mechanistically, the h-VM effectively induced MuSCs differentiation and myofiber formation, and M2 macrophage polarization, thereby mitigating muscle fibrosis and atrophy post-injury, ultimately achieving structural and functional angio-myogenesis. Our results highlight that the h-VM can effectively re-establish the vascular regenerative microenvironment, which presents an innovative therapeutic approach to promote functional skeletal muscle regeneration following volumetric muscle loss.