DEVELOPMENT OF A TISSUE ENGINEERED SKELETAL MUSCLE REPAIR CONSTRUCT FEATURING BIOMIMETIC PHYSICAL, CHEMICAL, AND MECHANICAL CUES

Abstract

Volumetric muscle loss (VML) injuries are particularly debilitating, and commonly result from trauma, infection, congenital defect, or surgical side effect. Unfortunately, current medical treatments for these injuries are lacking. The "gold standard" for treatment of critical-size defects remains autografting, which is restricted by donor site availability and morbidity, and further is often associated with incomplete repair of function or restoration of cosmesis. In the current work, we describe an in vitro tissue engineering approach incorporating biomimetic materials, cell supplementation, pharmacologic signaling, and mechanical strain that recapitulates some phenotypic aspects of motor end plates (MEPs) observed on innervated skeletal muscle in vivo. C2C12 cells cultured on a 2D fibrin biomaterial and treated with neural (Z+) agrin exhibited clusters of acetylcholine receptors (AChRs). When agrin was presented bound to the surface of a carrier microparticle, this response was spatially restricted to areas of cell – microparticle contact. Moreover, AChR clusters were observed from 16 – 72 hours after treatment when Z+ agrin was adsorbed to the surface of microparticles, but this time frame was extended to 120 hours and beyond when agrin was covalently linked to the microparticle surface. Similar AChR clustering was observed both when covalent agrin microparticles were allowed to settle from a culture medium suspension above a cell-seeded fibrin surface and when C2C12 cells were seeded on an agrin microparticle-containing fibrin surface. The latter microparticle delivery method enabled construction of 3D agrin-presenting fibrin scaffolds, which were seeded with expanded primary rodent muscle derived cells (MDCs). Incorporation of agrin-presenting microparticles in 3D fibrin constructs with surface-seeded rat MDCs resulted in little or no expression of AChR clusters. However, cyclic stretch of an agrin-presenting 3D fibrin scaffold seeded with MDCs resulted in dramatic enhancement of AChR clustering relative to samples lacking either agrin or stretch, providing evidence that mechanical strain and agrin supplementation may act synergistically in stimulating MEP-like structures in vitro. Taken together, these results describe a multidisciplinary approach leading to an in vitro phenotype that mimics some key structural features observed in innervated muscle in vivo. This represents an important first step toward preservation of native muscle phenotype following tissue engineered construct implantation in vivo.