Nanofiber-Based Delivery of Bioactive Lipids Promotes Pro-regenerative Inflammation and Enhances Muscle Fiber Growth After Volumetric Muscle Loss

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.

The role of the immune microenvironment in bone, cartilage, and soft tissue regeneration: from mechanism to therapeutic opportunity

“…[ 290 ], Wiley online library. c Pseudotime analysis of the FTY720-induced increase in immune cell infiltration into a muscle defect area 3 days post-VML injury [ 291 ]. Copyright 2018, Elsevier.…”

“…The ECM scaffolds induce IL-4-dependent M2 macrophage polarization through activation of the mTOR/Rictor-dependent T helper 2 pathway, thus guiding a pro-regenerative response that facilitates muscle regeneration. The effects of local delivery of RK35, a myostatin inhibitor, by HA/muscle-derived ECM scaffolds on muscle regeneration have been investigated [ 291 ] (Fig. 5 c).…”

“…The scaffolds show a prolonged release of RK35, thereby promoting pro-regenerative M2 macrophages and FoxP3 + Tregs, and increasing anti-inflammatory cytokine expression. In a recent study, PLGA/PCL electrospun nanofiber scaffolds loaded with FTY720, an agonist of the sphingosine-1-phosphate signal, have been demonstrated to promote pro-regenerative local injury milieu formation, as shown by increased numbers of M2 macrophages and muscle stem cells, thus improving muscle regeneration after volumetric loss [ 291 ].…”

Identifying dysregulated immune cell subsets following critical volumetric muscle loss with pseudo-time trajectories

“…UMAPs generated as previously reported (27, 45, 46). Briefly, UMAP was used to embed high-dimensional flow cytometry data into a space of two dimensions, cells are visualized in a scatter plot where similarity is demonstrated via proximity to other points.…”

“…SPADE trees generated as previously reported (27, 45, 46). Briefly, SPADE was performed through MATLAB and the source code is available at http://pengqiu.gatech.edu/software/SPADE/.…”