Injectable chondroitin sulfate-glycosylated decellularized extracellular matrix microgels activate Wnt/β-Catenin signaling to promote functional muscle regeneration in VML

Volumetric muscle loss (VML) overwhelms the endogenous regenerative capacity of skeletal muscle, leading to serious fibrosis, which further reduces muscle strength and severely affects patients' quality of life. While decellularized extracellular matrix (dECM) scaffolds offer tissue-specific biochemical cues for muscle regeneration, conventional decellularization protocols deplete critical glycosaminoglycans (GAGs), particularly chondroitin sulfate (CS), which are essential for growth factor sequestration and signaling pathway activation. Here, we developed injectable CS-glycosylated dECM microgels via microfluidic technology to restore GAG functionality to promote comprehensive muscle regeneration. We demonstrate that CS functionalization enhances myoblast adhesion, proliferation, migration, and differentiation in vitro. RNA sequencing analysis reveals that CS-mediated enhancement operates principally through activation of canonical Wnt/β-catenin signaling, with coordinated upregulation of downstream myogenic programs. In a rat tibialis anterior VML model, administration with high-CS formulations (H-CS@ECM) greatly promote muscle regeneration, function recovery and vascularization, with the muscle exhibiting enhanced-, blood perfusion, neuromuscular connectivity, and contractile protein expression at 8 weeks. Our findings suggest that CS-glycosylated dECM microgels possess significant translational potential as a minimally invasive and pro-regenerative implant material for VML repair. More broadly, this work establishes GAG modification as a generalizable design principle for enhancing dECM scaffold performance across diverse endogenous tissue regeneration applications, without the addition of exogenous growth factors, cytokines or drugs.