Skeletal muscle is a plastic tissue with robust regenerative capacity following minor injury. However, following the traumatic loss of a large portion of tissue, known as volumetric muscle loss (VML), the muscle is unable to recover and will have chronic functional deficits. Regeneration of skeletal muscle requires on the proliferation and differentiation of a population of muscle stem cells (MuSCs), which are present throughout the tissue in a quiescent state through adulthood. Minor muscle injury initiates a coordinated response of key cellular components in the MuSC niche. Immune cells begin the process of removing damaged or necrotic muscle and fibro-adipogenic progenitors (FAPs) lay down a regenerative matrix, each secreting factors which prime the MuSCs differentiation and fusion. Subsequently, damaged blood vessels and motor neurons will regenerate and remodel returning the muscle to a state nearly indistinguishable to that of uninjured muscle. However, VML results in the concurrent loss of extracellular matrix (ECM), MuSCs, FAPs, blood vessels, and motor neurons, eliciting a cellular response which results in chronic fibrosis and inflammation. The dysregulated response which leads to critical VML has not previously been well characterized, and the overall objective of this thesis was to characterize the cellular response which leads to the accumulation of non-functional fibrotic tissue following critically sized VML injury model.

First, we characterized the tissue response to three different sizes of VML injuries to determine the threshold of a critical sized VML in the mouse quadriceps. We defined the critical threshold as the point where regenerating myofibers were unable to bridge the created muscle defect, replaced instead by a persistent fibrosis and macrophage infiltration. We further characterized this critical threshold and saw substantial revascularization through the fibrotic tissue, but persistent denervation both within and distal to the localized injury. To understand the immune environment which led to these later stage outcomes, we used flow cytometry to quantify the dynamics of myeloid and lymphoid cell subtypes at earlier time points in critical compared to subcritical injuries. Critical injuries had distinct immune infiltration dynamics from subcritical injuries, as they exhibited significantly higher concentrations of lymphoid cell infiltration as well as a sustained significant elevation of anti-inflammatory monocytes and macrophages.

We then sought to understand the response of FAPs and MuSCs, two populations key to proper muscle regeneration. FAPs, which are considered to be the main progenitor population in muscle fibrosis, were of particular interest to us in further understanding the pathology of VML. We determined that critical VML induced a FAPs population which was present at significantly elevated concentrations and were most abundant in the VML injury space. FAPs isolated from VML injured tissue had different secretomes, responsiveness to pro-fibrotic stimuli, and differentiation capacity in vitro compared to FAPs from uninjured tissue. Cell surface markers were tracked over time in subcritical and critical VML, revealing a subset of FAPs which highly expressed β1-integrin that are persistently elevated in critical but not subcritical VML. Increased β1-integrin expression in FAPs was associated with increased gene expression of several pro-fibrotic genes and increased sensitivity to differentiate down a fibrogenic lineage in vitro. In addition to TGFβ-1, a known pro-fibrotic signaling molecule, our data indicate TIMP1 may play a role in the fibrotic differentiation of FAPs. Together, these signaling molecules were also inhibitory of myogenesis in vitro.

These data indicated that critical VML results in a cellular response and local environment with sustained immune cell presence and pro-fibrotic FAPs propagating a hostile environment for muscle regeneration. We then assessed whether a structurally aligned, porous, myoblast seeded collagen scaffold would improve muscle function after critical VML. Aligned collagen scaffolds were able to improve maximal isometric torque over time and with the addition of exogenous cells. Long term engraftment of transplanted cells into the surrounding tissue was evident, but within the scaffold blood vessels and immune cells were more abundant than exogenous cells four weeks after transplantation.

Overall, this thesis has contributed a critical VML injury model with well-defined cellular and tissue pathology for use in preclinical studies. We have identified a subpopulation of pro-fibrotic FAPs specific to critical VML which can aid in future development of targeted therapeutics. Finally, we have tested the feasibility of use of a versatile biomaterial scaffold for cell transplantation in critical VML.