Re-innervation of neuromuscular junctions by a conductive polypyrrole/silk fibroin/GelMA hydrogel facilitated functional skeletal muscle regeneration following volumetric muscle loss

Volumetric muscle loss (VML) is a significant clinical challenge that severely compromises patients' motor function and often results in irreversible disability. While conventional hydrogels have been explored for VML repair, their inability to address peripheral nerve denervation has limited functional recovery. The objective of this study was to develop a conductive double-crosslinking hydrogel (PPY@SF/GelMA) by integrating polypyrrole (PPY) into gelatin methacryloyl (GelMA) and silk fibroin (SF), aiming to simultaneously promote myotube formation and nerve re-innervation. The micro-architecture, compressive strength, rheological properties, swelling behavior, and conductivity of the PPY@SF/GelMA hydrogel were assessed. The influence of the conductive hydrogel on in vitro myogenic differentiation of C2C12 myoblast cells and angiogenic differentiation of endothelial cells was evaluated. The in vivo biodegradation and biocompatibility of the conductive hydrogel were assessed through subcutaneous implantation in the dorsal region of C57BL mice. The regenerative potential of the conductive hydrogel for skeletal muscle and peripheral nerve repair was investigated using a mouse tibialis anterior VML model. Compared to pure GelMA or SF hydrogels, the PPY@SF/GelMA composite exhibited superior mechanical resilience, tunable swelling kinetics, exceptional biocompatibility, and enhanced electrical conductivity. In vitro experiments using C2C12 murine myoblasts demonstrated that the PPY@SF/GelMA hydrogel markedly upregulated myogenic differentiation markers (e.g., Mhc , Myog , and MyoD ) and promoted the formation of multinucleated myotubes. Additionally, the conductive hydrogel exhibited pro-angiogenic potential by enhancing endothelial cell differentiation, as evidenced by new formation of endothelial tubes. In vivo , histopathological analysis showed no signs of toxicity from the implanted conductive hydrogel. PPY@SF/GelMA implantation facilitated the regeneration of aligned muscle fibers, reduced fibrotic collagen deposition, and accelerated neovascularization. Importantly, the conductive hydrogel successfully promoted peripheral nerve re-innervation by restoring neuromuscular junctions. RNA-seq analysis further revealed the involvement of the phosphoinositide 3-kinase (PI3K) signaling pathway in the newly regenerated muscle treated with PPY@SF/GelMA. Our findings demonstrate PPY@SF/GelMA as a promising therapeutic scaffold to facilitate both myogenic and neurogenic regeneration after VML injuries, offering a translatable strategy for complex musculoskeletal repair. The failure of skeletal muscle regeneration following VML injuries is primarily attributed to the loss of peripheral nerve innervation. The present investigation has demonstrated that the conductive PPY@SF/GelMA hydrogel effectively promoted myofiber maturation and restored neuromuscular junctions, thereby promoting the re-innervation of peripheral nerves in newly regenerated skeletal muscle. Our objective is to translate this conductive hydrogel into a viable clinical strategy for patients requiring the repair of severe muscle damage.