Structure-Property-Processing Analysis of Graphene Bioscaffolds for Viability and Differentiation of C2C12 Cells

We investigated the structure – property – processing correlation of graphene bioscaffolds produced using three different methods. Bioscaffolds were prepared by chemical vapor deposition (CVD), sublimation of Silicon Carbide (SiC), and printed solvent assisted exfoliated graphene ink. To gain insight into the roughness and topography of graphene, AFM was performed on each bioscaffold. Raman spectroscopy mapping demonstrated differences in the I2D/IG ratio for each scaffold. Young’s modulus was determined by nanoindentation and indicated that epitaxial graphene had the highest average stiffness, followed by CVD, with printed graphene demonstrating the lowest average stiffness. To investigate the biocompatibility of each scaffold, cellular morphology and gene expression patterns were investigated using the bipotential mouse C2C12 cell line. While it is well established that cell differentiation is influenced by the structure and mechanical properties of the substratum to which cells are attached, this study provides new information about differences in cellular response to graphene scaffolds prepared by specific production methods. Graphene production methods determine the structural and mechanical properties of the resulting bioscaffold, which in turn determine cell morphology, gene expression patterns and cell differentiation fate. Therefore, production methods for graphene bioscaffolds must be chosen carefully with the ultimate biomedical application in mind

Dystrophin-Glycoprotein Complex and Reactive Oxygen Species

Duchenne\u27s Muscular Dystrophy (DMD) is caused by a deficiency in dystrophin protein. DMD is distinguishable through muscle degeneration and weakness. Dystrophin protein is a necessary structural link between the sarcolemma and the cytoskeleton. Studies show Neuronal Nitric Oxide Synthase (nNOS), a critical enzyme in the sarcolemma, that catalyzes nitric oxide (NO), is a molecular component of the Dystrophin-Glycoprotein Complex (DGC). To rescue cells from the effects of a dystrophin deficiency, we will expose the muscle sarcolemma to NO by using gas plasma. Three methods will be tested: 1) treatment with air through a plasma device, as our control, 2) treatment with NO through the plasma device, and 3) treatment with NO via Cold Atmospheric Pressure Source (CAPS) to generate a NO plasma. Q-RT-PCR analysis and confocal microscopy will allow quantification of DGC stability at the plasma membrane. We propose to answer mechanistic questions such as: 1) does an increase in NO levels affect the expression of muscle specific genes in the presence and absence of dystrophin, 2) will increased levels of NO stabilize the DGC within the cell, and 3) are other types of muscle cells (skeletal, cardiac, and smooth) affected by increasing NO in cells. Thus, we predict NO treatment will rescue the deficiency in absence of dystrophin. Acknowledgment of Support: Research was supported by a Grant-in-Aid of Research administered by Sigma Xi, The Scientific Research Societ

Open-Source Automated Chemical Vapor Deposition System for the Production of Two-Dimensional Nanomaterials

The study of two- dimensional (2D) materials is a rapidly growing area within nanomaterials research. However, the high equipment costs, which include the processing systems necessary for creating these materials, can be a barrier to entry for some researchers interested in studying these novel materials. Such process systems include those used for chemical vapor deposition, a preferred method for making these materials. To address this challenge, this article presents the first open-source design for an automated chemical vapor deposition system that can be built for less than a third of the cost for a comparable commercial system. The materials and directions for the system are divided by subsystems, which allows the system to be easily built, customized and upgraded, depending upon the needs of the user. We include the details for the specific hardware that will be needed, instructions for completing the build, and the software needed to automate the system. With a chemical vapor deposition system built as described, a variety of 2D nanomaterials and their heterostructures can be grown. Specifically, the experimental results clearly demonstrate the capability of this open-source design in producing high quality, 2D nanomaterials such as graphene and tungsten disulfide, which are at the forefront of research in emerging semiconductor devices, sensors, and energy storage applications

\u3cem\u3eIn vitro\u3c/em\u3e Collagen Gel Model for Tissue Damage: Towards a Study of Dystrophin-Glycoprotein Complex and Reactive Oxygen Species

Duchenne\u27s Muscular Dystrophy is caused by a deficiency in the dystrophin protein, a component of the Dystrophin-Glycoprotein Complex (DGC), which serves as a structural link between the sarcolemma and the cytoskeleton. Neuronal Nitric Oxide Synthase is a critical enzyme in the sarcolemma responsible for catalyzing the formation of nitric oxide (NO). This enzyme is an important molecular component of the DGC. We studied the C2C12 pre-myogenic cell line by growing them in 3D collagen gels to form a model for muscle development. This muscle model maintains the cells in tension while they differentiate, and can be compared to cells grown in a stress-free environment as a control. We used the method q-RT-PCR to measure the expression of specific muscle markers in two distinct cellular environments. Histological images allowed us to assess cell morphology within each sample. This study provides preliminary data for future plans to test the effects of NO generated by plasma to answer mechanistic questions such as: 1) do increased levels of NO affect muscle-specific gene expression in the presence and absence of dystrophin, 2) will the increased NO level stabilize the DGC within the cell, and 3) are other types of muscle cells (skeletal, cardiac, and smooth) affected by increased NO in cells

\u3cem\u3eIn vitro\u3c/em\u3e Collagen Gel Model for Tissue Damage: Towards a Study of Dystrophin-Glycoprotein Complex and Reactive Oxygen Species

Duchenne\u27s Muscular Dystrophy is caused by a deficiency in the dystrophin protein, a component of the Dystrophin-Glycoprotein Complex (DGC), which serves as a structural link between the sarcolemma and the cytoskeleton. Neuronal Nitric Oxide Synthase is a critical enzyme in the sarcolemma responsible for catalyzing the formation of nitric oxide (NO). This enzyme is an important molecular component of the DGC. We studied the C2C12 pre-myogenic cell line by growing them in 3D collagen gels to form a model for muscle development. This muscle model maintains the cells in tension while they differentiate, and can be compared to cells grown in a stress-free environment as a control. We used the method q-RT-PCR to measure the expression of specific muscle markers in two distinct cellular environments. Histological images allowed us to assess cell morphology within each sample. This study provides preliminary data for future plans to test the effects of NO generated by plasma to answer mechanistic questions such as: 1) do increased levels of NO affect muscle-specific gene expression in the presence and absence of dystrophin, 2) will the increased NO level stabilize the DGC within the cell, and 3) are other types of muscle cells (skeletal, cardiac, and smooth) affected by increased NO in cells

Synthesis of Laser-Induced Graphene via Laser Irradiation

Laser-Induced Graphene (LIG) is a graphitic material with properties comparable to more pure forms of graphene, but with a simpler and cheaper synthesis process. By irradiating a carbon-rich precursor with a laser, LIG samples can be formed in a matter of seconds instead of hours. In this work, we aim to show that the synthesis of LIG using consumer-grade equipment is an effective method. The graphene content of the LIG was confirmed by Raman analysis, and Hall measurements showed a low sheet resistance. Our findings indicate that LIG could be better suited to certain applications, such as biomedical research, than other methods of graphene synthesis

Measurement of Signal‐to‐Noise Ratio In Graphene‐Based Passive Microelectrode Arrays

This work aims to investigate the influence of various electrode materials on the signal‐to‐noise ratio (SNR) of passive microelectrode arrays (MEAs) intended for use in neural interfaces. Noise reduction substantially improves the performance of systems which electrically interface with extracellular solutions. The MEAs are fabricated using gold, indium tin oxide (ITO), inkjet printed (IJP) graphene, and chemical vapor deposited (CVD) graphene. 3D‐printed Nylon reservoirs are adhered to glass substrates with identical MEA patterns and filled with neuronal cell culture media. To precisely control the electrode area and minimize the parasitic coupling of metal interconnects and solution, SU‐8 photoresist is patterned to expose only the area of the electrode to solution and cap the remainder of the sample. Voltage signals with varying amplitude and frequencies are applied to the solution using glass micropipettes, and the response is measured on an oscilloscope from a microprobe placed on the contact pad external to the reservoir. The time domain response signal is transformed into a frequency spectrum, and SNR is calculated. As the magnitude or the frequency of the input signal gets larger, a significantly increased signal‐to‐noise ratio was observed in CVD graphene MEAs compared to others. This result indicates that 2‐dimensional nanomaterials such as graphene can provide better signal integrity and potentially lead to improved performance in hybrid neural interface systems

The Effects of 2D and 3D Graphene Bioscaffolds on Osteogenic Differentiation of Mesenchymal Stem Cells

Stem cells differentiation occurs naturally in a 3D environment with cell response linked to structure. It is not clearly defined how stem cells respond to 3D environments. Graphene is a novel and promising material with conductive and thermal capabilities that make it a potential viable bioscaffold for 2D and 3D structures. The osteogenic differentiation capabilities of murine mesenchymal stem cells (mMSC) were tested with and without mechanical stimulation in a variety of environments: glass, graphene film, collagen gel, and graphene foam. Graphene foam has shown to be a biocompatible micro environment that promotes cell proliferation with pore sizes of approximately 300-500 um. This study examines osteogenesis of mMSC’s in osteogenic media in known 2D and 3D environments (glass and collagen gel) against their graphene analogs (film and foam) while comparing the low-intensity vibration (LIV) against a non-vibrated group. Completion of this study will facilitate future studies in testing the efficacy of electrical compared to mechanical stimulus

CVD Growth and Electrochemical Transfer of Graphene Films

Graphene— a hexagonal lattice encompassing a single layer of carbon atoms—has made great advancements in electronic devices, flexible electronics, and more recently as an electrically conductive bioscaffold for stem cell growth and differentiation. For this study, chemical vapor deposition(CVD) is used for graphene growth with copper as the metal catalyst. Graphene films, transferred from the metal catalyst to glass slides via chemical or electrochemical techniques, are used as bioscaffolds for the growth and differentiation of C2C12 stem cells. The ability to transfer graphene from the metal catalyst to a substrate of choice is a highly desirable and beneficial property. Traditional transfer techniques demonstrate potential limitations for tissue engineering applications as they require the use of a harmful, highly corrosive copper etchant, iron(III) chloride (Fecl3). Additional concerns are associated with the presence of metal catalyst even after long processing times. An alternative method, based on water electrolysis, electrochemical delamination, provides a transfer method with high efficiency, low cost recyclability, and minimal use of etching chemicals, and has been successfully performed. This project aims to compare the two transfer techniques to determine the optimal transfer method for graphene transfers to establish an ideal environment for C2C12 cell growth and differentiation