Surface-Patterned Silicon Oxynitride for Aligned Myotubes and Neurite Outgrowth In Vitro

Abstract

Traumatic injuries lead to volumetric muscle loss (VML) and nerve damage that cause chronic functional deficits. Due to the inability of mammalian skeletal muscle to regenerate after VML damage, engineered scaffolds have been explored to address this challenge, but with limited success in functional restoration. We introduce novel bioactive amorphous silicon oxynitride (SiONx) biomaterials with surface properties and Si ion release to accelerate muscle and nerve cell differentiation for functional tissue regeneration. Micropatterned scaffolds were designed and developed on Si-wafer to test the effect of SiONx on myogenesis and neurogenesis. The scaffolds were created using UV photolithography to first pattern their surface, followed by the deposition of SiONx through plasma enhanced chemical vapor deposition (PECVD). X-ray diffraction (XRD) and energy dispersive spectroscopy (EDS) confirmed the uniform chemical structure of an amorphous SiONx film on the patterned surfaces. Atomic force microscopy and scanning electron microscopy (SEM) elucidated the surface morphology with a uniform 2 μm grating microstructure. The 2 µm pattern size is within the range of cellular dimensions, allowing for effective cell-surface interactions. Further, 2 µm features provide sufficient contact points for cell adhesion without overwhelming the cell’s ability to interact with the surface. Two separate studies were conducted with SiONx biomaterials and Si ions alone. This was done to understand how Si ions impact cell response separate from the surfaces. C2C12 mouse myoblasts and NG108 neuronal cells were cultured on SiONx biomaterials. In separate studies, we tested the effect of Si ion treatments with these cells (cultured on tissue culture plastic). Cell culture studies demonstrated enhanced C2C12 myoblast attachment and proliferation on SiONx surfaces. High-resolution SEM and fluorescence images revealed highly aligned myotubes (from C2C12 cells) and axons (from NG108 cells) in a parallel direction to the micropatterned SiONx scaffolds. GAP43 expression, neurite outgrowth, and alignment were significantly increased with the Si-ions and SiONx biomaterials. These findings suggest that SiONx scaffolds enhance muscle and nerve cell adhesion and growth and promote the formation of aligned myotubes and axons on the pattern surfaces.

Impact Statement

Our findings demonstrate that silicon oxynitride biomaterials, with tailored surface chemistry and topography, significantly enhance cell adhesion and alignment without extracellular matrix (ECM) surface coating. Scientifically, we discovered a set of surface chemistries and topography that induce chemical and physical cues needed for neuronal and muscle cell alignment.