Hiroki Kondo1 Tomonori Ichikawa1 Kenji Ishikawa1 Hiromasa Tanaka1 Takayoshi Tsutsumi1 Keigo Takeda2 Makoto Sekine1 Masaru Hori1 Mineo Hiramatsu2

1, Nagoya Univ, Nagoya, , Japan
2, Meijo University, Nagoya, , Japan

In recent years, nanomaterials, such as carbon nanotubes, graphene nanoflakes, and so forth, have attracted much attention as a cell culturing scaffold. It has been reported that an introduction of nanometer level fine structures affects cell differentiation induction. Very recently, it has also been reported that an electric stimulation through conductive scaffold of resin containing carbon nanotubes can affect proliferation and differentiation of cultured cells. Based on these backgrounds, we have focused on carbon nanowalls (CNWs), in which multiple layers of graphene sheets are vertically grown on a substrate and form randomly intricate wall structures like maze. Due to their unique morphology, the CNWs have very high aspect ratio over 100 and high specific surface area. In addition, they also have high conductivity and robustness against chemical treatments. Therefore, the CNWs are promising as electrically conductive cell culturing scaffold with nanometer level fine structures. We have reported that the wall density and the chemical termination of CNWs influence the proliferation rate and morphology of HeLa cell. Furthermore, electric stimulation through the CNWs scaffold increased proliferation rate of human osteoblast-like cells (Saos-2) and suppress their ossification only if frequency of electrical stimulation was 10 Hz. In this study, the effects and mechanisms of such the electrical stimulation through the CNWs scaffold with the different wall densities were investigated. Proliferation rates and intra- and extra-cellarer calcium amounts were measured for the Saos-2 cells cultured on the CNWs scaffold with the electric stimulation.
The CNWs were grown on Ti substrates by a radical injection type plasma excited chemical vapor deposition (RI-PECVD) system. In order to change the wall densities, the total pressures during the growth were controlled from 1 to 3 Pa, which realizes density controls of hydrogen and methyl radicals. According to scanning electron microscopy (SEM) images, distances between neighboring walls were 208 and 341 nm, when the total pressures were 1 and 3 Pa, respectively. Saos-2 cells were cultured on these CNWs scaffolds in an environment at 37°C and with CO2 concentration of 5%. Electric stimulation with a frequency of 10 Hz, square wave shape and a peak-to-bottom voltage of 226 mV was supplied for 24 hours after seeding the cells. Then, after culturing for a total of 100 hours, the number of cells and morphology were observed. A 58% increase in proliferation rate was observed on the CNWs with a wall density of 341 nm, while that on the CNWs with a wall density of 208 nm hardly changed. At the same time, it was confirmed that aggregates of cells were formed only on the on the CNWs with a wall density of 341 nm. This suggested that an intercellular adhesion can be controlled by the electrical stimulation on the CNWs scaffold. These results could open the way of novel cell control system.