Abstract
Volumetric muscle loss (VML) injuries after extremity trauma results in an important clinical challenge often associated with impaired healing, significant fibrosis, and long-term pain and functional deficits. While acute muscle injuries typically display a remarkable capacity for regeneration, critically sized VML defects present a dysregulated immune microenvironment which overwhelms innate repair mechanisms leading to chronic inflammation and pro-fibrotic signaling. In this series of studies, we developed an immunomodulatory biomaterial therapy to locally modulate the sphingosine-1-phosphate (S1P) signaling axis and resolve the persistent pro-inflammatory injury niche plaguing a critically sized VML defect. Multiparameter pseudo-temporal 2D projections of single cell cytometry data revealed subtle distinctions in the altered dynamics of specific immune subpopulations infiltrating the defect that were critical to muscle regeneration. We show that S1P receptor modulation via nanofiber delivery of Fingolimod (FTY720) was characterized by increased numbers of pro-regenerative immune subsets and coincided with an enriched pool of muscle stem cells (MuSCs) within the injured tissue. This FTY720-induced priming of the local injury milieu resulted in increased myofiber diameter and alignment across the defect space followed by enhanced revascularization and reinnervation of the injured muscle. These findings indicate that localized modulation of S1P receptor signaling via nanofiber scaffolds, which resemble the native extracellular matrix ablated upon injury, provides great potential as an immunotherapy for bolstering endogenous mechanisms of regeneration following VML injury.
Highlights
Though skeletal muscle possesses robust potential for healing after injury, large volumetric wounds that occur during combat, accidents or surgical resection often do not heal completely, resulting in fibrotic scarring and limited range of motion (Garg et al, 2015)
We have previously demonstrated that nonclassical anti-inflammatory Ly6Clo monocytes express relatively high levels of sphingosine-1-phosphate receptor 3 (S1PR3), which can be leveraged to modulate their response in vivo to promote the onset of the regenerative phase of muscle healing (Awojoodu et al, 2013)
scanning electron microscopy (SEM) and atomic force microscopy (AFM) were utilized to assess the morphology and mechanical properties between unloaded, blank nanofibers and those loaded with FTY720 to ensure that any differences in healing outcomes after injury were not attributed to variations in biomaterial characterization (Figures 1B,C)
Summary
Though skeletal muscle possesses robust potential for healing after injury, large volumetric wounds that occur during combat, accidents or surgical resection often do not heal completely, resulting in fibrotic scarring and limited range of motion (Garg et al, 2015). Current standard of care involves the autologous transfer of tissue but this treatment produces limited functional restoration and results in complications at both the donor and injury site (Beth and Pollot, 2016). Extensive soft tissue injury and concomitant damage to collateral blood vessels results in inadequate vascularization to the regenerating or grafted tissue. This unsuccessful muscle regeneration outcome following a traumatic injury has been linked to a dysfunctional immune response. The immune response is overwhelmed by pro-inflammatory myeloid cells that exacerbate the pro-inflammatory phase of inflammation to a point where regeneration is not able to take place. The prolonged pro-inflammatory phase induces fibrosis and longterm loss of function (Aguilar et al, 2018; Larouche et al, 2018)
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