Multifunctional hyaluronic acid hydrogel integrating exosome‐encapsulating gelatin microspheres accelerates skeletal muscle regeneration via TGF‐β1 activation and enhanced myogenic cell viability

Abstract The effective treatment of acute skeletal muscle injuries remains challenging, necessitating therapeutic strategies that concurrently reestablish vascular perfusion and reactivate myogenic processes. Leveraging the favorable biocompatibility of natural macromolecules including hyaluronic acid and gelatin, we designed an in situ injectable hydrogel system for sustained microRNA delivery. Using microfluidic technology, we fabricated citrate‐coated cobalt nanoparticles and gelatin methacrylate/alginate methacrylate microspheres loaded with miR‐206‐3p‐enriched exosomes from BMSCs. These components were subsequently encapsulated within a double‐bonded hyaluronic acid network to form a multifunctional hydrogel (H/G@Co@miR‐206). Both in vitro and in vivo assessments demonstrated excellent cytocompatibility of H/G@Co@miR‐206, alongside enhanced proliferation and migration of C2C12 myoblasts. The sustained release of Co 2+ ions promoted angiogenesis, while the controlled delivery of engineered exosomes facilitated myogenic differentiation. These exosomes upregulated key myogenic regulators (Pax7, MyoD1) and restored balance to profibrotic signaling pathways (TGF‐β1/Smad3). In a cardiotoxin‐induced acute muscle injury model in rats, the treatment promoted restoration of muscle fiber architecture and neuromuscular junction density (evidenced by hematoxylin and eosin and acetylcholine receptor staining), stimulated new blood vessel formation (CD31, α‐SMA), and elevated expression of myogenic markers (MyoG, MYHC, PAX7, MyoD1). These structural recoveries correlated with functional improvements, including increased paw withdrawal threshold and enhanced hindlimb weight‐bearing capacity during follow‐up. Mechanistically, Western blot analysis revealed suppression of the TGF‐β1/Smad3/HDAC4 axis in injured muscle tissues, consistent with a pro‐regenerative shift. In summary, H/G@Co@miR‐206 integrates angiogenic and myogenic cues within a minimally invasive platform, promoting molecular, histological, and functional recovery, thereby highlighting its translational potential for treating acute skeletal muscle injuries.