Determination of a Critical Size Threshold for Volumetric Muscle Loss in the Mouse Quadriceps

Abstract

Skeletal muscle has a remarkable regenerative capacity; however, after volumetric muscle loss (VML) or a loss of a large portion of the tissue, this regenerative response is diminished and results in chronic functional deficits. The critical size at which muscle will not recover has not yet been established; subsequently, the response of crucial muscle components at the critically sized threshold is unknown. In this study, we set out to determine the threshold for a critically sized muscle defect by creating full-thickness VML injuries of 2, 3, or 4 mm diameter in the mouse quadriceps. The 2, 3, and 4 mm injuries resulted in a defect of 5%, 15%, or 30% of muscle mass, respectively. At 14 and 28 days after injury, histological analyses revealed injury size-dependent differences in myofiber morphology and fibrosis; the number of small myofibers and fibers with centrally located nuclei increased with increasing injury size. The results indicated that the 3 mm injury, with 15% mass loss, was at the critical threshold point, characterized by incomplete bridging of myofibers through the defect site, persistent fibrosis and inflammation, and a temporally sustained increase in myofibers with centrally located nuclei as compared with contralateral control muscle. We further investigated the 3 mm VML for nerve and vascular regeneration. Critically sized injured muscles were accompanied by a drastic increase in denervated neuromuscular junctions (NMJs), while assessment of angiogenesis through micro-CT analysis revealed a significant increase in vascular volume primarily from small diameter vessels after the VML injury. Collectively, these data indicate that the fibrotic response and neuromotor component remain dysregulated in critically sized defects, and therefore could be potential therapeutic targets for regenerative strategies. Impact Statement The goal of this study was to determine the threshold for a critically sized, nonhealing muscle defect by characterizing key components in the balance between fibrosis and regeneration as a function of injury size in the mouse quadriceps. There is currently limited understanding of what leads to a critically sized muscle defect and which muscle regenerative components are functionally impaired. With the substantial increase in preclinical VML models as testbeds for tissue engineering therapeutics, defining the critical threshold for VML injuries will be instrumental in characterizing therapeutic efficacy and potential for subsequent translation.