Etching anisotropic surface topography onto fibrin microthread scaffolds for guiding myoblast alignment

To regenerate functional muscle tissue, engineered scaffolds should impart topographical features to induce myoblast alignment by a phenomenon known as contact guidance. Myoblast alignment is an essential step towards myotube formation, which is guided in vivo by extracellular matrix structure and micron-scale grooves between adjacent muscle fibers. Fibrin microthread scaffolds mimic the morphological architecture of native muscle tissue and have demonstrated promise as an implantable scaffold for treating skeletal muscle injuries. While these scaffolds promote modest myoblast alignment, it is not sufficient to generate highly functional muscle tissue. The goal of this study is to develop and characterize a new method of etching the surface of fibrin microthreads to incorporate aligned, sub-micron grooves that promote myoblast alignment. To generate these topographic features, we placed fibrin microthreads into 2-(N-morpholino)ethane-sulfonic acid (MES) acidic buffer and evaluated the effect of buffer pH on the generation of these features. Surface characterization with atomic force microscopy and scanning electron microscopy indicated the generation of aligned, sub-micron sized grooves on microthreads in MES buffer with pH 5.0. Microthreads etched with surface features had tensile mechanical properties comparable to controls, indicating that the surface treatment does not inhibit scaffold bulk properties. Our data demonstrate that etching threads in MES buffer with pH 5.0 enhanced alignment and filamentous actin stress fiber organization of myoblasts on the surface of scaffolds. The ability to tune topographic features on the surfaces of scaffolds independent of mechanical properties provides a valuable tool for designing microthread-based scaffolds to enhance regeneration of functional muscle tissue.