Temporal Changes in Pathologic Fibrosis Following Volumetric Muscle Loss Injury

Abstract

Volumetric muscle loss (VML) is a devastating orthopaedic injury that results in chronic persistent functional deficits, loss of joint range of motion, pathologic fibrotic deposition and lifelong disability. Clinically, two major problems exist in the care of VML injuries: 1) no standard of care to address the loss of function and skeletal muscle mass, and 2) the pathologic fibrotic response impedes muscle healing and regeneration, causing destruction of muscle architecture and altering of the microenvironment of the muscle. However, to date there is only limited mechanistic understanding of the VML‐induced fibrosis. Our recent work indicates that through 21 days post‐VML there is a 22% loss in TA mass, 40% loss in total protein and mitochondrial content, and 45% loss in maximal isometric torque; however, how total collagen content changes over this time is unknown. We examined the temporal changes in the fibrotic deposition early after VML injury to identify a potential window of effective intervention. Adult male Lewis rats (n=42) underwent a full thickness ~20% (~85mg) VML injury to the tibialis anterior (TA) muscle or sham injury and were harvested for genetic and histologic evaluation. First, at 14 and 56 days post‐VML, fibrotic genes probed had biologically significant upregulation compared to control. Collagen I (Col1a1) and collagen III (Col3a1) had a 42‐ and 26‐fold upregulation in expression after 14 days, respectively. The upregulation was dampened at 56 days, yet still significant with Col1a1 and Col3a1 up 10‐ and 5‐fold from baseline. Interestingly, IL‐6 had the strongest upregulation between the 14 and 56‐day time points, from 4‐ to 59‐fold compared to controls. Second, in order to temporally track fibrotic changes more specifically, we evaluated extracellular proteins at 0, 3, 7, 14, 28, and 48 days post VML‐injury both biochemically and histologically. Histologic results indicate that following VML injury that significant fibrotic deposition begins to develop as early as 7 days post‐VML and there is a substantial cellular infiltration into the VML defect area. This suggests there is an initial attempt to follow a normal healing process; however, it is ultimately failed by a vast overcompensation by the immune system, which is supported by the escalating increase in IL‐6 expression. Additional work is needed to precisely probe the crosslinking of collagen fibers and overall makeup of the extracellular matrix across the early time frame after VML injury. Future work will help to identify potential therapeutic targets and assess the efficacy and timing of early rehabilitation, increased physical activity, and pharmacologic approaches to mitigate the fibrotic development after VML injury.Support or Funding InformationNone.