Conductive MXene/adECM hydrogel promotes skeletal muscle regeneration and innervation through Ca2+ influx modulation and neuromuscular junction formation

Volumetric muscle loss (VML) leads to severe skeletal muscle dysfunction. While muscle tissue engineering offers a promising strategy, challenges persist due to insufficient neuromuscular innervation and poor reconstruction of neuromuscular junctions (NMJs). Conductive hydrogels can mimic the electrophysiological microenvironment and thus promote structural and functional regeneration, yet commonly used conductive materials still suffer from poor hydrophilicity, non-degradability, and potential cytotoxicity, while their underlying mechanisms remain unclear. Ti 3 C 2 T x MXene, a class of two-dimensional nanomaterials with high conductivity and biocompatibility, shows potential for repairing electroactive tissues. In this study, we developed a novel biomimetic electroactive hydrogel by incorporating Ti 3 C 2 T x MXene nanosheets into adipose-derived decellularized extracellular matrix (adECM). This study aimed to investigate the effects and mechanisms of MXene/adECM hydrogel on muscle regeneration and innervation. MXene/adECM hydrogel demonstrated excellent biocompatibility, biodegradability, and conductivity. Compared to the adECM hydrogel, the incorporation of MXene promoted myogenesis, along with increased expression of Desmin, MyoD1, and Myf5. Furthermore, the MXene/adECM hydrogel at the optimal concentration increased the average neurite length by 47.29 μm ( p < 0.05) relative to the adECM group. Transcriptomic analysis combined with a neuromuscular co-culture system indicated that the MXene/adECM hydrogel promoted the formation of neuromuscular junctions (NMJs). The incorporation of MXene upregulated the expression of specific voltage-gated calcium channels at the motor endplate, with transcript levels of Cacna1a and Cacna1s increased to 2.1-fold and 3.1-fold, respectively. It was further observed that calcium signaling was enhanced in the MXene/adECM group, with the peak calcium signal intensity being 2.40 times that of the adECM group. In vivo rat VML model confirmed that, compared to the adECM hydrogel, the MXene/adECM hydrogel promoted an increase in regenerated muscle fiber area, reduced collagen deposition, and elevated the fluorescence intensity of CD31 and Tuj. The co-localization percentage of presynaptic and postsynaptic NMJ markers increased from 27.85 ± 8.69% to 42.21 ± 15.52%. Gait analysis showed significant improvements in print area, swing/stance ratio, and movement velocity. In the MXene/adECM group, the isometric tetanic force (ITF) upon sciatic nerve stimulation was significantly higher than that of the adECM group (0.082 ± 0.012 N vs. 0.057 ± 0.014 N, p < 0.05), approaching the level of the uninjured group. Together, these findings demonstrate that the incorporation of MXenes into adECM provides a promising strategy that integrates microenvironmental support with endogenous electrical cues to modulate calcium influx and promote NMJ formation, offering a new paradigm for the treatment of VML. Study on the therapeutic strategy and underlying mechanism of conductive MXene/adECM hydrogel for volumetric muscle loss (VML) repair. Ti3C2Tx MXene nanosheets were integrated into an adipose-derived decellularized extracellular matrix (adECM) to fabricate a conductive composite hydrogel. Upon implantation, the MXene/adECM hydrogel promoted structural regeneration and reinnervation of the injured skeletal muscle. The bioactive microenvironment and endogenous electrical conductivity facilitated neuromuscular junction (NMJ) formation, accompanied by enhanced calcium influx