Patterned Silicon Oxynitride (SiONx) Scaffolds Enhance Alignment and Myogenic Differentiation of C2C12 Muscle Cells

Abstract

Recently, engineered biomaterial scaffolds became a crucial branch in the tissue regeneration field due to its promising results. Engineered scaffolds could play an important role by providing structural support for cell attachment and development into a functional tissue. Functionality of myoblast and their highly aligned myotubes have greatly benefited efforts in tissue repair and muscle regeneration. Furthermore, organized myoblasts to form aligned myotubes is a critical step in myogenesis (myotubes formation, alignment, and fusion) that mimics the natural muscle architecture. Different scaffolds can promote the myoblasts alignment, fusion, and yield aligned myotubes with very different results. Here, we offer an alternative approach that propose the use of patterned silicon oxynitride (SiONx) films to enhance myogenesis in C2C12 muscle cells. Micropatterned scaffolds were designed and developed on Si-wafer using photolithography techniques “using UV light to transfer a geometric pattern from a photomask to the substrate”, then 100 nm layer of SiONx was deposited by Plasma Enhanced Chemical Vapor Deposition technique (PECVD). C2C12 mouse myoblast cell line was used for the in-vitro studies, and MTS assay and fluorescence microscopy were used to study the cell viability, proliferation, and differentiation. X-ray diffraction and Energy Dispersive X-ray results confirmed the chemical structure of a uniform amorphous SiONx films on the pattern surfaces. Atomic force microscopy and Scanning Electron Microscopy (SEM) elucidated the surface morphology with a uniform 2μm grating. Contact angle measurements revealed that SiONx has a hydrophilic surface with high surface energy that enhances cells attachment and leads to rapid myotubes formation. Fluorescence images of DAPI-stained nuclei and myosin heavy chain antibody-stained the formed myotubes confirmed higher number of cells attached and differentiated into myotubes onto the SiONx films compared to the tissue culture plate (TCP) as a positive control. In addition, the Fusion Index was significantly higher for SiONx compared to TCP. Furthermore, SEM and fluorescence images exhibited highly aligned myotubes in a parallel direction to the micropattern SiONx scaffolds. We concluded that SiONx films enhance the C2C12 muscle cells adhesion, growth, and yield aligned myotubes on the pattern surfaces. Thus, these SiONx engineered scaffolds could be a promising material for accelerating functional muscles tissue, which may facilitate their future use in translational and clinical applications for muscle regeneration applications in the near future. Fluorescence images of DAPI-stained nuclei (blue) and myosin heavy chain antibody (MHC, green)-stained myotubes of C2C12 myoblasts shows the aligned myotubes on SiON scaffolds, and the Fusion Index graph. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.