Abstract
INTRODUCTION: Volumetric muscle loss (VML) remains a major clinical challenge due to the limited capacity of skeletal muscle to regenerate large-scale injuries. Muscle-derived stem cells (MDSCs) represent a promising therapeutic option for tissue regeneration; however, their clinical application is constrained by poor post-transplantation viability and limited engraftment. Alginate hydrogels offer a supportive three-dimensional microenvironment capable of encapsulating cells, promoting their survival, and enhancing paracrine signaling through the sustained release of growth factors. METHODS: In this study, we developed and characterized MDSC spheroids and evaluated their regenerative potential when encapsulated in RGD-modified alginate hydrogels. Co-culture with endothelial cells significantly enhanced spheroid viability, indicating beneficial paracrine interactions. To further refine this strategy, 5% of the MDSCs were preconditioned with vascular endothelial growth factor (VEGF) prior to spheroid formation and encapsulation, integrating a pharmacological preconditioning step into the cell-hydrogel platform. Encapsulated spheroids were implanted into a murine model of VML. RESULTS: After 30 days, animals treated with alginate-encapsulated MDSC spheroids containing a 5% VEGF-preconditioned subfraction exhibited reduced granulation tissue, fewer degenerating myofibers, lower fibrosis, and improved early rota-rod performance compared with untreated and scaffold-only controls. DISCUSSION: Together, these findings highlight a pioneering proof-of-concept platform that combines 3D MDSC spheroids, alginate-based delivery, and VEGF-mediated pharmacological preconditioning for VML repair. As a 100% unconditioned MDSC+alginate group was not included, the present study should not be interpreted as demonstrating in vivo superiority of VEGF preconditioning over unconditioned MDSCs; instead, it provides a rationale for future head-to-head studies explicitly powered to address this question.