The Effect of Electrical Stimulation on the Cellular Response of Human Mesenchymal Stem Cells on SiC-coated CNW Scaffolds

<b>Introduction</b><br/>In recent years, regenerative medicine has been actively researching ways to heal damaged tissues in the body by applying electrical stimulation (ES) to cells on implanted scaffolds, which controls cell differentiation and growth. Scaffold materials are important factor in this process. Currently, polymer materials are primarily used as scaffold materials for electrically stimulated scaffolds in vivo. However, polymer materials have issues with mechanical properties and electrical conductivity, so they are not ideal.<br/>Carbon nanowalls (CNWs) have excellent conductivity and mechanical properties, and graphene sheets oriented perpendicularly to the substrate, giving them a unique surface morphology. This material has been reported to promote osteoblast differentiation due to its unique surface structure and to enhance cell proliferation when combined with ES in vitro. However, using CNWs as scaffold electrodes in vivo has safety concerns due to the reported genotoxicity of graphene, which consists of CNWs. To improve this issue, I thought that coating CNWs with biocompatible silicon carbide (SiC) could solve the problems related to using CNWs as scaffold electrodes in the body. SiC is chemically stable and has excellent mechanical properties, so it is used as a coating for stents and implants. Additionally, its excellent mechanical properties have been reported to induce bone formation in osteoblasts, which suggests that SiC is an attractive material for cell culture scaffolds. This suggests that SiC is suitable as a coating material for CNWs.<br/>However, the typical chemical vapor deposition (CVD) method for depositing SiC has a high deposition temperature of over 1200°C, which can cause CNWs to etch due to residual oxygen, making coating difficult. Our previous research successfully deposited SiC at a growth temperature of 700°C using thermal CVD with vinylsilane, a using novel precursor. We believe that using this precursor can achieve SiC coating on CNWs. Additionally, there are no reports of applying ES to cells on SiC-coated CNWs, so the effects of SiC on cells under ES remain unclear. Thus, it is necessary to investigate the effects of ES on cells with SiC-coated scaffolds.<br/>In this study, we aim to investigate the cellular response to ES on mesenchymal stem cells on SiC-coated CNW scaffolds, with the goal of applying carbon materials as scaffold electrodes in vivo.<br/><br/><b>Method</b><br/>We deposited SiC on CNWs using thermal CVD method with 5 sccm of vinylsilane, 500 sccm of Ar dilution gas, a growth pressure of 1 Torr, a surface temperature of 700°C, and a growth time of 5 to 30 minutes. Human mesenchymal stem cells (hMSCs) were used for cell culture. We used CNWs only, SiC-coated CNWs with varying deposition times, and a 48-well plate as a control for these samples as cell culture scaffolds. ES of 226 mVpp at a frequency of 10 Hz was applied one day after cell seeding for 3 days. The surface morphology of SiC-coated CNWs was evaluated using Scanning electron Microscope (SEM). Cell proliferation on each scaffold was assessed using the MTS assay.<br/><br/><b>Result and Discussion</b><br/>SiC-coated CNWs retained their unique high-aspect-ratio structure, with the thickness of the CNWs walls increasing with longer growth times. Without ES, cell proliferation increased as the wall thickness of CNWs coated with SiC increased. In the samples with 15 minutes of SiC coating, the cell proliferation rate was lower than the control without ES, but it was comparable to the control when ES was applied. For samples with 30 minutes of SiC coating, there was no significant difference in cell proliferation rates with or without ES, and the rates were comparable to the control. This indicates a synergistic effect between SiC coating and ES, as the effect of ES was more pronounced on SiC-coated CNWs than on CNWs only.