An Emodin-Depot Microsphere-in-Hydrogel Reprograms the Immuno-myogenic Niche to Enable Volumetric Muscle Loss Repair Revealed by Single-Cell Profiling.

Volumetric muscle loss (VML) causes irreversible loss of contractile tissue and creates a hostile regenerative niche marked by sustained inflammation, oxidative stress, fibrosis, and poor functional recovery. Here, we develop an injectable and photocurable microsphere-in-hydrogel platform that couples structural support with sustained small-molecule immunoregulation. To counteract this hostile microenvironment, we utilized emodin, a natural anthraquinone recognized for its potent anti-inflammatory and reactive-oxygen-species-scavenging properties. Emodin-loaded sodium alginate microspheres were generated via ionic crosslinking and embedded within a gelatin methacryloyl matrix to form E-AMs@GM. The composite hydrogel exhibited defect-conforming moldability, porous microarchitecture, tunable swelling/degradation, and broad interfacial adhesion. In vitro, E-AMs@GM showed excellent cytocompatibility and attenuated intracellular reactive oxygen species in human bone-marrow-derived mesenchymal stem cells under oxidative challenge. In macrophages, E-AMs@GM reduced pro-inflammatory activation while enhancing pro-regenerative programs, accompanied by decreased inflammatory cytokines and increased interleukin-10. E-AMs@GM also promoted C2C12 myogenic differentiation and myotube maturation. In a murine VML model, E-AMs@GM alleviated inflammation and fibrotic remodeling, increased myogenic progenitor activity and myofiber regeneration, and improved locomotor performance by CatWalk analysis. Mechanistically, scRNA sequencing revealed that E-AMs@GM enriches a reparative Mmp12 + macrophage subset and rewires macrophage-muscle satellite cell interactions through SPP1-CD44 and SPP1-integrin signaling, consistent with accelerated transition from inflammatory clearance to tissue reconstruction. Together, this depot-enabled hydrogel provides an instructive biomaterial strategy for functional VML repair.