Achieving Acetylcholine Receptor Clustering in Tissue-Engineered Skeletal Muscle Constructs In vitro through a Materials-Directed Agrin Delivery Approach.

John B Scott,Benjamin S Harrison,Catherine L Ward,Justin M Saul,Benjamin T Corona,George J Christ,Michael R Deschenes

doi:10.3389/fphar.2016.00508

Abstract

Volumetric muscle loss (VML) can result from trauma, infection, congenital anomalies, or surgery, and produce permanent functional and cosmetic deficits. There are no effective treatment options for VML injuries, and recent advances toward development of muscle constructs lack the ability to achieve innervation necessary for long-term function. We sought to develop a proof-of-concept biomaterial construct that could achieve acetylcholine receptor (AChR) clustering on muscle-derived cells (MDCs) in vitro. The approach consisted of the presentation of neural (Z+) agrin from the surface of microspheres embedded with a fibrin hydrogel to muscle cells (C2C12 cell line or primary rat MDCs). AChR clustering was spatially restricted to areas of cell (C2C12)-microsphere contact when the microspheres were delivered in suspension or when they were incorporated into a thin (2D) fibrin hydrogel. AChR clusters were observed from 16 to 72 h after treatment when Z+ agrin was adsorbed to the microspheres, and for greater than 120 h when agrin was covalently coupled to the microspheres. Little to no AChR clustering was observed when agrin-coated microspheres were delivered from specially designed 3D fibrin constructs. However, cyclic stretch in combination with agrin-presenting microspheres led to dramatic enhancement of AChR clustering in cells cultured on these 3D fibrin constructs, suggesting a synergistic effect between mechanical strain and agrin stimulation of AChR clustering in vitro. These studies highlight a strategy for maintaining a physiological phenotype characterized by motor endplates of muscle cells used in tissue engineering strategies for muscle regeneration. As such, these observations may provide an important first step toward improving function of tissue-engineered constructs for treatment of VML injuries.

Highlights

The ability to voluntarily control skeletal muscle is critically important to key daily functions such as locomotion, breathing, and postural support
We investigated the enhancement of acetylcholine receptor (AChR) clusters on either a myoblast cell line (C2C12 cells) or on primary rat muscle-derived cell (MDC) in order to assess the efficacy of the strategy for tissue-engineered muscle repair (TEMR) applications
In samples treated with agrin dissolved within the culture medium, detectable AChR clusters were observed on numerous cells within 16 h after treatment, though again without spatial organization or predictability (Figure 3B)

Summary

Introduction

The ability to voluntarily control skeletal muscle is critically important to key daily functions such as locomotion, breathing, and postural support. The repair process consists of a series of phases that start with inflammation followed by satellite cell mobilization and, to maturation of myofibers and remodeling of muscle architecture (Charge and Rudnicki, 2004; Jarvinen et al, 2005; Ambrosio et al, 2009; Ciciliot and Schiaffino, 2010). This repair process is insufficient in more extensive injuries, resulting in extensive scar tissue formation (Jarvinen et al, 2005). Traumatic VML injuries in the civilian population result from causes such as automobile accidents (and gun shot wounds) (Mazurek and Ficke, 2006) and in military personnel from high impact forces (Mazurek and Ficke, 2006; Owens et al, 2008; Lew et al, 2010)

Methods

Results

Conclusion