Functionalization of hyaluronic acid hydrogels with ECM-derived peptides to control myoblast behavior

Volumetric muscle loss (VML) occurs when skeletal muscle injury is too large for the body to fully self-repair. Typically, fibrotic tissue fills the void, which reduces muscle functionality and limb movement. Although a wide variety of natural and synthetic scaffolds have been studied with the purpose of providing the appropriate structural support, to date no scaffold has significantly restored muscle functionality after VML. Satellite cells, adult stem cells within the muscle capable of restoring smaller injuries, are sensitive to the stiffness and composition of the surrounding environment. Scaffolds that only address structural support are not sufficient to restore functionality and instead need to be designed to both promote satellite cell activation and prevent excessive fibroblast recruitment. The objective of this study was to design a scaffold that mimicked the regenerative environment and determine how the biomechanical properties differentially influence myogenic precursor and connective tissue cells. One of the main extracellular matrix (ECM) molecules upregulated during regeneration is hyaluronic acid (HA). Therefore, thiol-modified HA and poly(ethylene glycol) diacrylate hydrogels were generated and functionalized with peptides based on ECM known to influence regeneration, including fibronectin, laminin and tenascin-C. Scaffolds with different stiffness were created by varying HA content. The influence of HA stiffness and peptide functionalization on myogenic precursor and connective tissue cell proliferation, migration and gene expression was quantified. Our results indicated that HA hydrogels functionalized with the laminin peptide, IKVAV, show potential due to the enhanced promotion of myogenic cell behaviors including migration, proliferation and an increase in relevant transcription factors. STATEMENT OF SIGNIFICANCE: The goal of this study was to identify hyaluronic acid (HA) hydrogels with peptide and stiffness combinations that will direct muscle-derived cells towards regenerating phenotypes. While the interaction of skeletal muscle with RGD-functionalized HA hydrogels has been investigated, none of the other peptides described in this study had been used in the context of HA-based scaffolds and skeletal muscle-derived cells. Notably, the response of cells to variations in mechanics was dependent on ECM coating and lineage. The 3% HA functionalized with the laminin peptide, IKVAV, showed the most promise for future in vivo studies, as these hydrogels best promoted myoblast cell proliferation, attachment and spreading, enhanced migration over connective tissue cells and upregulated transcription factors associated with activated satellite cells.