Micro‐patterned Bioactive Amorphous Silicon Oxynitride Enhances Adhesion, Growth, and Myotubes and Axon Alignment in Muscle and Nerve Cells
Authors: AWAD KAMAL R. , Ahuja Neelam , Fiedler Matthew , Huang Jian , Brotto Leticia , Aswath Pranesh , Brotto Marco , Varanasi Venu 
Published: April 2020

Introduction

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 the damaged tissue in VML injuries, muscle flap procedures have been used. Functional muscle transfer, including vasculature and innervation, has been shown to improve strength to an injured muscle. However, most muscle flap procedures are not intended to restore muscle function except in limited circumstances. Thus, these procedures have not adequately addressed this issue and novel engineered materials that regenerate the damaged tissue and restore the functionality is crucially needed. Here, we introduce novel bioactive amorphous silica‐base biomaterials that accelerate muscle, nerve, and vascular healing for fully functional tissue regeneration.

Materials and Methods

Micro‐patterned scaffolds were designed and developed on Si‐wafer using photolithography, then 200 nm layer of SiONx was deposited by Plasma Enhanced Chemical Vapor Deposition technique. C2C12 mouse myoblast and NG108‐15 neuroblastoma x rat glioma hybrid cell lines were used for in‐vitro studies. In‐vitro studies using different ionic silicon concentration (0.1, 0.5, and 1.0 mM) were performed to determine the optimal concentration for muscle and nerve cells. MTS assay and fluorescence microscopy were used to study the cell viability, growth, and differentiation. Also, qRT‐PCR was used to investigate the targeted gene expression.

Results

In‐vitro studies revealed that 0.1 mM of Si4+ significantly enhance the C2C12 cell growth, fusion index (1.5 fold), and the area covered by myotubes (*** p<0.001) compared to the control (Fig.
1). Also, 0.1 mM of Si4+ significantly increase the NG108‐15 neural cell viability after 12 hours in growth media. qRT‐PCR data revealed that 0.1 mM of Si4+ increases MyoD gene expression 3‐folds compared to the control by day 1, and MyoG expression 2‐folds by day 5. Also, 0.5 mM of Si4+ significantly increases the GAP43 gene expression 3‐folds by day 1 of neural cell culture. Intriguingly, 0.5 mM of Si4+ significantly increase Neurturin (Myokine) protein concentration at day 3 of C2C12 cell culture. Most intriguingly, the formed myotubes and axons were perfectly aligned (Fig.
2) on the surface of micro‐patterned SiONx scaffolds (Without laminin coating).

Conclusion

This study confirms that 0.1 mM of Si4+ is the optimal concentration for muscle and nerve cells that enhance the cell differentiation and increase the myogenic/neurogenic gene expression as well as myogenic/neurogenic markers. Surface pattern successfully support axons and myotubes alignment on the SiONx surface.

Support or Funding Information

National Institutes of Health (Grant No. 1R56DE027964‐01A1‐01)University of Texas STARS award Program

Si‐ions (0.1 mM) enhance Fusion index, total number of cells, and area covered by myotubes in C2C12 cells after 4 days of differentiation.

Figure 1

aligned axons and myotubes on the patterned SiONx. A) DAPI and MHC Stained myotubes, b) Refractive microscopy image of the aligned myotubes, C) Neural network formation on the smooth surface of SiON, and D) axon alignment on the patterned SiON.

Figure 2