(227j) Graphene Matrix Promotes Muscle Regeneration and Reverses Degeneration in Chronic Massive Rotator Cuff Tears

Background/Purpose
Massive rotator cuff tears (MRCTs) of the shoulder are a major cause of disability and pain in adults. In chronic injuries, tendon retraction initiates a cascade of degenerative muscle changes, including atrophy, fatty expansion, and fibrosis. These changes persist despite surgical tendon repair, as the intrinsic regenerative ability of muscle cells is insufficient to reverse long-standing degeneration. Consequently, patients with MRCTs often face poor outcomes and high retear rates. Current clinical approaches provide mechanical repair but fail to address muscle pathology, highlighting the urgent need for regenerative engineering strategies.
This study sought to develop and evaluate an electroconductive nanofiber matrix composed of poly(l-lactic acid) (PLLA) and graphene nanoplatelets (GnPs). Because skeletal muscle is electrically excitable, graphene’s electroactivity was hypothesized to enhance cell–material interactions, stimulate myogenic differentiation, and suppress adipogenesis. Our aims were twofold: (1) to assess the matrix’s ability to promote muscle regeneration and inhibit fat formation in vitro and (2) to investigate its capacity to reverse muscle degenerative changes in vivo in a rat chronic MRCT model.

Methods
Fabrication: Non-Functionalized Graphene Nanoplatelets (GnPs) were used in this study which consists of multilayers of platelet-liked graphite nanocrystals with an overall thickness of approximately 3-10 nanometers. A mixture of PLLA and GnPs were prepared in HFIP solvent and aligned nanofibers were fabricated using electrospinning technique to create an electroconductive, aligned scaffold.

In vitro studies: we examined the effects of the GnP matrix on C₂C₁₂ myoblasts and adipose-derived stem cells (ADSCs). Myogenic differentiation was evaluated, while intracellular calcium levels were measured to probe mechanistic pathways. Adipogenic differentiation was evaluated in ADSCs to assess the matrix’s anti-adipogenic potential.

In vivo model: a rat model of chronic MRCT was created by incising both the supraspinatus and infraspinatus tendons in 11 weeks old male Sprague Dawley rats. Treatment surgery was performed 16 weeks later to ensure chronicity of the rotator cuff tear.

In vivo Studies: after surgical induction of tears, animals received either standard suture repair or implantation of the GnP matrix. The matrix was placed and sutured on both muscles. At 24 and 32 weeks after the initial injury, supraspinatus and infraspinatus muscles were harvested for histological evaluation of atrophy, fatty expansion, and fibrosis. Tendon morphology and tensile strength were also analyzed. Pathological assessments of internal organs were performed to confirm long-term biocompatibility.

Results
An aligned PLLA/GnP nanofiber was fabricated using electrospinning with uniform distribution of GnPs. In vitro, the GnP matrix significantly enhanced myogenic differentiation compared with PLLA controls and the cells formed aligned layers along the fibers. Further, myotube formation was confirmed by myosin heavy chain (MHC) staining, showing GnP matrix could induce C₂C₁₂ to form multinucleated myotubes. Mechanistically, these effects correlated with elevated intracellular calcium concentrations in myoblasts, suggesting that graphene facilitates calcium-mediated signaling pathways critical for muscle development. In parallel, GnP matrices suppressed adipogenic differentiation in ADSCs, confirming graphene’s dual role in promoting myogenesis while inhibiting fat formation.

In vivo, GnP matrix implantation yielded marked improvements in muscle pathology. The GnP matrix boosted its therapeutic effects by the long-term implantation. At 32 weeks, fatty expansion grading in the infraspinatus muscle decreased from 2.1 ± 0.57 in the suture group to 0.6 ± 0.74 in the GnP matrix group (P ≤ 0.0001). Muscle atrophy and fibrosis were also significantly reduced (P ≤ 0.01 and P ≤ 0.05, respectively). Comparable results were observed in both supraspinatus and infraspinatus muscles, with no significant differences between GnP-treated tissue and native uninjured muscle (P < 0.05). we showed that the majority of muscle fibers in native muscle are MyHC II (fast muscle fiber). The muscle underwent switching from fast (MyHC II) to slow (MyHC I) fiber type after MRCTs in both supraspinatus and infraspinatus muscles. The suture repair did not alter the expression of MyHC I, we observed switching back to the normal (native) muscle following the RC repair only with the GnP matrix treatment. Histological analysis of internal organs revealed no evidence of toxicity or adverse reactions, demonstrating long-term biocompatibility of the GnP matrix.

Importantly, reversing muscle degenerative changes translated to improvements in tendon outcomes. The expression of glycosaminoglycan (red-stained area in the safranin O stained images) at the tendon-bone interface indicated the formation of a new fibrocartilage layer in GnP matrix group after 32 weeks. Compared with standard repair, GnP treatment significantly enhanced tendon morphology and tensile properties, indicating superior tendon-bone healing.

Conclusion/Discussion
This study demonstrates that electroconductive nanofiber matrices incorporating graphene nanoplatelets effectively promote muscle regeneration and suppress fatty expansion in the context of chronic MRCTs. By enhancing intracellular calcium signaling, the GnP matrix drives myogenic differentiation while simultaneously inhibiting adipogenesis. These dual effects address two key pathological hallmarks of MRCTs: atrophy and fatty expansion.
In vivo findings confirm the therapeutic potential of the GnP matrix. Treated muscles exhibited reduced degeneration, improved histological features, and near-restoration to native tissue architecture at 32 weeks. Furthermore, these benefits extended to the tendon, where improved morphology and biomechanics underscore the matrix’s capacity to enhance overall shoulder healing. Critically, long-term safety was validated by the absence of systemic pathology, supporting translational feasibility.

Taken together, these results highlight the promise of graphene-based biomaterials as next-generation therapeutics for rotator cuff injuries. By integrating electrical conductivity and topographical guidance, the GnP matrix provides a regenerative platform capable of reversing chronic muscle degeneration, improving tendon integrity, and addressing limitations of current surgical repair. Future studies should explore optimization of matrix composition, large-animal validation, and eventual clinical translation. Ultimately, this work positions graphene-based electroconductive scaffolds as a novel solution for reducing retear rates and restoring function in patients with MRCTs