Skeletal muscle injury treatment using the Silk Elastin® injection in a rat model

Abstract

Background: Skeletal muscle injury (SMI) is often treated conservatively, although it can lead to scar tissue formation, which impedes muscle function and increases muscle re-injury risk. However, effective interventions for SMIs are yet to be established.

Hypothesis: The administration of Silk Elastin® (SE), a novel artificial protein, to the SMI site can suppress scar formation and promote tissue repair.

Study design: A controlled laboratory study.

Methods: In vitro: Fibroblast migration ability was assessed using a scratch assay. SE solution was added to the culture medium, and the fibroblast migration ability was compared across different concentrations. In vivo: An SMI model was established with Sprague-Dawley rats, which were assigned to three groups based on the material injected to the SMI site: SE gel (SE group; n = 8), atelocollagen gel (Atelo group; n = 8), and phosphate buffer saline (PBS group; n = 8). Histological evaluations were performed at weeks 1 and 4 following the SMI induction. In the 1-week model, we detected the expression of transforming growth factor (TGF)-β1 in the stroma using immunohistological evaluation and real-time polymerase chain reaction analysis. In the 4-week model, we measured tibialis anterior muscle strength upon peroneal nerve stimulation as a functional assessment.

Results: In vitro: The fibroblast migration ability was suppressed by SE added at a concentration of 10⁴ μg/mL in the culture medium. In vivo: In the 1-week model, the SE group exhibited significantly lower TGFβ -1 expression than the PBS group. In the 4-week model, the SE group had a significantly larger regenerated muscle fiber diameter and smaller scar formation area ratio than the other two groups. Moreover, the SE group was superior to the other two groups in terms of regenerative muscle strength.

Conclusion: Injection of SE gel to the SMI site may inhibit tissue scarring by reducing excessive fibroblast migration, thereby enhancing tissue repair.

Clinical relevance: The findings of this study may contribute to the development of an early intervention method for SMIs.

Keywords: Fibrosis; Injury; Material; Skeletal muscle.

Fig. 1

(A) Migration assay of skeletal muscle fibroblasts. Fibroblasts were seeded in 6-well plates, and silk-elastin (SE) gel was added to the medium at concentrations of 0, 10¹, 10¹, 10³, and 10⁴ μg/mL. (B) At 24 and 48 h after scratching the bottom of the wells, the area was photographed under a microscope to assess the cells that had migrated to the acellular field. (C), (D) Graph of migration assay results. At both 24 and 48 h, the ratio of the cell migration area was reduced in the medium with 10⁴ μg/mL SE compared with that of fibroblast migration area in the medium without SE.

Fig. 2

Axial sections of injured skeletal muscle stained with hematoxylin–eosin 1 week post-injury. The number of regenerating muscles with a central core and the diameter of the regenerating muscle fibers per mm² were measured, with no significant differences detected among the three groups. Atelo, atelocollagen gel; PBS, phosphate-buffered saline; SE, silk-elastin gel.

Fig. 3

Sagittal sections of injured skeletal muscle stained with Masson trichrome 1 week post-injury. The area of muscle fibers and scar tissue was measured using ImageJ, and the area ratio of scar tissue to muscle fibers was calculated. The ratio of scar tissue area in the atelocollagen group was significantly greater than that in the other two groups. Atelo, atelocollagen gel; PBS, phosphate-buffered saline; SE, silk-elastin gel.

Fig. 4

Axial sections of injured skeletal muscle stained with hematoxylin–eosin 4 weeks post-injury. The silk-elastin group had significantly fewer regenerating muscles than the other two groups, whereas the diameter of regenerating muscles was significantly greater than that in the other groups. Atelo, atelocollagen gel; PBS, phosphate-buffered saline; SE, silk-elastin gel.

Fig. 5

Sagittal sections of injured skeletal muscle stained with Masson trichrome 4 weeks post-injury. The ratio of scar tissue area in the Atelo group was significantly greater than that in the other two groups. The ratio of scar tissue area in the SE group was significantly smaller than that in the other two groups. Atelo, atelocollagen gel; PBS, phosphate-buffered saline; SE, silk-elastin gel.

Fig. 6

Isometric tensile strength of the tibialis anterior muscle during electronic peroneal nerve stimulation at 1 Hz (twitch) and 50 Hz (tetanus). The strength of the injured muscle was assessed as a ratio to the strength of the muscle on the uninjured side. In both types of electrical stimulation, the Atelo group had a significantly weaker muscle strength than the other two groups, and the SE group showed significantly stronger muscle strength than the other two groups. Atelo, atelocollagen gel; PBS, phosphate-buffered saline; SE, silk-elastin gel.

Fig. 7

(A) Real-time PCR analysis revealed reduced TGF-β1 expression in the SE group. Atelo, atelocollagen gel; PBS, phosphate-buffered saline; SE, silk-elastin gel. (B) Immunostaining for TGF-β1 in coronal section tissues 1 week post-muscle injury. The TGF-β1 positive area was significantly smaller in the SE group than in the PBS group.

Fig. 8

mRNA expression levels of PAX7 and MyoD in the scar tissue at 1 week after muscle injury were measured using real-time PCR and the ΔCT method with the median value of PBS as 1.