Muscle degeneration in chronic massive rotator cuff tears of the shoulder: Addressing the real problem using a graphene matrix

Abstract

Massive rotator cuff tears (MRCTs) of the shoulder cause disability and pain among the adult population. In chronic injuries, the tendon retraction and subsequently the loss of mechanical load lead to muscle atrophy, fat accumulation, and fibrosis formation over time. The intrinsic repair mechanism of muscle and the successful repair of the torn tendon cannot reverse the muscle degeneration following MRCTs. To address these limitations, we developed an electroconductive matrix by incorporating graphene nanoplatelets (GnPs) into aligned poly(l-lactic acid) (PLLA) nanofibers. This study aimed to understand 1) the effects of GnP matrices on muscle regeneration and inhibition of fat formation in vitro and 2) the ability of GnP matrices to reverse muscle degenerative changes in vivo following an MRCT. The GnP matrix significantly increased myotube formation, which can be attributed to enhanced intracellular calcium ions in myoblasts. Moreover, the GnP matrix suppressed adipogenesis in adipose-derived stem cells. These results supported the clinical effects of the GnP matrix on reducing fat accumulation and muscle atrophy. The histological evaluation showed the potential of the GnP matrix to reverse muscle atrophy, fat accumulation, and fibrosis in both supraspinatus and infraspinatus muscles at 24 and 32 wk after the chronic MRCTs of the rat shoulder. The pathological evaluation of internal organs confirmed the long-term biocompatibility of the GnP matrix. We found that reversing muscle degenerative changes improved the morphology and tensile properties of the tendon compared with current surgical techniques. The long-term biocompatibility and the ability of the GnP matrix to treat muscle degeneration are promising for the realization of MRCT healing and regeneration.

Keywords: fat accumulation; graphene; muscle degeneration; rotator cuff tears.

Fig. 1.

(A) Schematic illustration of the fabrication process of the GnP matrix. (B) SEM images of the aligned nanofibers. (C) Mean diameter of the electrospun nanofibers (n = 50). The average diameters of the aligned nanofibers were 676.7 ± 149.1 nm, 645.3 ± 91.84 nm, 652.6 ± 110.8 nm, and 610 ± 186.1 nm for PLLA, GnP 0.5, 1.5, and 2, respectively. (D) Surface conductivities of the aligned matrices (**P ≤ 0.01). (E) Live/dead fluorescent images of cell-seeded matrices with aligned orientation after 1, 7, and 14 d (green, viable cells; red, dead cells). Arrows show the main directions of nanofiber alignment.

Fig. 2.

(A) Immunofluorescent images of differentiated myotubes after 7 d on aligned matrices in GM immune-stained for MHC (green) and nucleus (blue). (B) Quantification of myotube length, fusion index, and maturation index of differentiated myotubes after 7 d on aligned matrices in GM. (C) Immunofluorescent images of differentiated myotubes after 7 d on aligned matrices in DM immune-stained for MHC and nucleus. (D) Quantification of myotube length, fusion index, and maturation index of differentiated myotubes after 7 d on aligned matrices in DM. Comparison of (E) myotube length, (F) fusion index, (G) maturation index between PLLA and GnP 1.5 in both GM and DM, (ns = P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).

Fig. 3.

Calcium imaging of C₂C₁₂ myoblasts on the matrices at days 1, 3, and 7 after cell culturing in both GM and DM. Schematic image showing the effects of the GnP matrix on the concentration of intracellular calcium ions.

Fig. 4.

(A) Schematic illustration of the lipid droplets on GnP 1.5 and PLLA matrices after 12 d of adipogenic induction. (B) CCK8 cell viability on both matrices at day 3 in GM and day 12 in DM. (C) Representative images of the stained lipid droplets on the matrices after 12 d of adipogenic induction. (D) Quantification of the stained lipid droplets was conducted by eluting the Oil Red O stain and measuring absorbance at 510 nm. The readings were normalized to the plain matrix (**P ≤ 0.01).

Fig. 5.

(A) Representative histological images of infraspinatus muscle at 24 wk. The H&E and trichrome staining of cross-sections of the infraspinatus muscles used for the quantification of (B) fibrosis formation, (C) fat accumulation, and (D) muscle atrophy evaluation for different groups at 24 wk. (E) Representative histological images of supraspinatus muscle at 24 wk. The H&E and trichrome staining of cross-sections of the supraspinatus muscles used for the quantification of (F) fibrosis formation, (G) fat accumulation, and (H) muscle atrophy evaluation for different groups at 24 wk. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).

Fig. 6.

(A) Representative histological images of infraspinatus muscle at 32 wk. The H&E and trichrome staining of cross-sections of the infraspinatus muscles used for the quantification of (B) fibrosis formation, (C) fat accumulation, and (D) muscle atrophy evaluation for different groups at 32 wk. (E) Representative histological images of supraspinatus muscle at 32 wk. The H&E and trichrome staining of cross-sections of the supraspinatus muscles used for the quantification of (F) fibrosis formation, (G) fat accumulation, and (H) muscle atrophy evaluation for different groups at 32 wk. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).

Fig. 7.

(A) The immunohistochemistry images of MyHC I (slow), II (fast), and (B) the ratio of MyHC I/MyHC II in infraspinatus muscle at 32 wk for all groups. (C) The immunohistochemistry images of MyHC I and II and (D) the ratio of MyHC I/MyHC II in supraspinatus muscle at 32 wk for all groups. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).

Fig. 8.

H&E staining and the congestion grading of the liver, kidney, lung, spleen, and heart of the GnP implanted and nonimplanted groups at 24 and 32 wk.

Fig. 9.

The representative images of H&E (Top and Middle) and Safranin O (Bottom) staining of the tendon–bone insertion for native, no repair, suture, and GnP matrix at 32 wk following the surgery.

Fig. 10.

The results of biomechanical testing, including the tendon cross-sectional area, stiffness, peak load, and Young’s modulus of (A) supraspinatus tendon at 24 wk, (B) infraspinatus tendon at 24 wk, (C) supraspinatus tendon at 32 wk, and (D) infraspinatus tendon at 32 wk following the RCTs. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).