3D Nano-Biohybrid Carbon Nanotube Forest for Cardiac Tissue Engineering

Biohybrid cell-material structures have applications in tissue engineering, disease modeling, biorobotics, etc. Conductive materials, in particular, are useful in applications such as bioelectronics and pacing. The scaffolds that are used for making biohybrid structures have so far been limited to those with low electrical conductivities. Carbon nanotube forests have high electrical conductivity as well as mechanical compliance and stability, which are not offered by other commonly used cell scaffolds. Here, we developed a new 3D biohybrid structure using CNT forests and cardiomyocytes. The CNT Forest was fabricated on a silicon wafer by a chemical vapor deposition technique under atmospheric pressure. The sterilization procedure of CNT forests started with soaking the samples in 70% ethanol, followed by UV light exposure overnight. After sterilization, samples were coated with 1 wt.% gelatin. Physical characterization of the scaffolds was performed by scanning electron microscopy (SEM), four-point probe conductivity measurement, and electrochemical impedance measurement. The cardiomyocytes were isolated from 1-3 day old neonatal rat hearts and cultured on the CNT forest - gelatin scaffold for 19 days. All animal protocols were approved by the Michigan Technological University Institutional Animal Care and Use Committee. The cytocompatibility of the CNT forest - gelatin scaffold was assessed through the live/dead assay on day 2, and PrestoBlue assay on days 2, 8, and 11 from the start of culturing. The performance of cardiomyocytes on the scaffold was investigated using immunofluorescence cardiomyocyte labeling (Alpha actinin and Troponin T) and SEM imaging. The morphology and microstructure of the CNT forest - gelatin scaffolds were studied with SEM imaging. The CNT Forest had wavy and entangled CNTs with an average height of ~ 200 microns. The SEM images of CNTs before and after coating demonstrated that the CNTs were bent and formed micron-scale islands due to electrocapillary forces produced by gelatin. The electrical characterization of the scaffold showed that the coated CNT scaffold is conductive in liquid, and the impedance decreases due to the presence of gelatin. The live-dead assay indicated 89% viability on day 3 for the CNT forest-gelatin scaffold, which was in the range with the control. The PrestoBlue assay, which quantifies absorbance by dead cells, showed no toxicity for the scaffolds. The SEM images of cardiomyocytes on the scaffold showed the cells were spread and well-adhered to the structure on top and along with the height of CNT forests forming a 3D network. The cells expressed mature cardiomyocyte markers for alpha-actinin (actin-binding proteins cardiac marker) and troponin T (cytoskeletal organization cardiac marker) in fluorescence imaging. The cardiomyocyte cells were well-adhered to the CNT forest-gelatin scaffolds with no sign of toxicity for 11 days of culturing. They formed a 3D network and showed markers of mature cardiomyocytes. Our results indicate that the unique multi-scale structure of the CNT forest scaffolds makes them ideal for a highly conductive scaffold for biohybrid material-cardiomyocyte structures.