Electrospun nanofibers scaffold of PMMA coated collagen or laminin composite as a patch for treating muscular disease

Abstract

Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate) (PMMA) nanofiber (PM) scaffolds were fabricated using electrospinning techniques and coated with extracellular matrix (ECM) proteins, such as collagen (PM-C) or laminin (PM-L), in the presence or absence of genipin (G). Subsequently, PM, PM-C, and PM-L scaffolds were coated with polypyrrole (Ppy) to mimic the electroconductive property of muscle tissue. Muscle cells containing myoblasts and fibroblast were isolated from skeletal muscle tissues, and cultured on coated and non-coated nanofiber mesh. PMMA can produce smooth fibers with an average diameter of 360±50 nm. Adsorption of collagen (1040±51µg/mg nanofiber) and laminin (900±65 µg/mg nanofiber) on PM scaffolds is significantly enhanced in the presence of G, which introduces roughness to the nanofiber surface without affecting fiber diameter and mechanical properties. It was also demonstrated that laminin-coated PM scaffolds significantly enhance myoblast proliferation (0.0081±0.0007 h−1) and migration (0.26±0.04 µm/min), while collagen-coated PM scaffolds favors fibroblasts proliferation (0.0097±0.0009 h−1) and migration (0.23±0.03 µm/min). Consequently, coating with Ppy introduces the electroconductivity on PM, PM-C-G, and PM-L-G. Cells attachment and spreading were also facilitated by Ppy coating. Similar to PM-L-G, PM-L-G with Ppy demonstrated significantly higher myoblast population (63.86±7.06%) after 7 days of culture. The angiogenic potential was also evaluated via supplementing conditioned medium (CM) that contains secretory factors of muscle cells cultured on different conditions to human umbilical vein endothelial cells (HUVEC) on Matrigel™. Supplementation of CM from PM-C-G and PM-L-G resulted in the increases of mean meshes, but decreases in number of meshes and branching points compared to other conditions, indicating the simulation of paracrine effects of angiogenic factors by myoblast and fibroblast on 3D nanofiber scaffolds. Consequently, the myoblast population was enriched on laminin-coated PM scaffolds throughout the culture process. Therefore, laminin and Ppy coating of nanofiber scaffolds could be a potential scaffold for the development of a tissue-engineered muscle substitute.