Aligned carbon nanotube-based flexible gel substrates for engineering bio-hybrid tissue actuators

Abstract

Muscle-based biohybrid actuators have generated significant interest as the future of biorobotics but so far they move without having much control over their actuation behavior. Integration of microelectrodes into the backbone of these systems may enable guidance during their motion and allow precise control over these actuators with specific activation patterns. Here, we addressed this challenge by developing aligned CNT forest microelectrode arrays and incorporated them into scaffolds for stimulating the cells. Aligned CNTs were successfully embedded into flexible and biocompatible hydrogel exhibiting excellent anisotropic electrical conductivity. Bioactuators were then engineered by culturing cardiomyocytes on the CNT microelectrode-integrated hydrogel constructs. The resulting cardiac tissue showed homogeneous cell organization with improved cell-to-cell coupling and maturation, which was directly related to the contractile force of muscle tissue. This centimeter-scale bioactuator has excellent mechanical integrity, embedded microelectrodes and is capable of spontaneous actuation behavior. Furthermore, we demonstrated that a biohybrid machine can be controlled by an external electrical field provided by the integrated CNT microelectrode arrays. In addition, due to the anisotropic electrical conductivity of the electrodes provided from aligned CNTs, significantly different excitation thresholds were observed in different configurations such as the ones in parallel vs. perpendicular direction to the CNT alignment.

Keywords: Bioactuators; Carbon Nanotubes; Cardiac tissue engineering; Hybrid hydrogels; Microelectrode arrays.

Figure 1

A schematic illustrating the fabrication steps to produce 3D bio-hybrid actuators composed of cardiac tissue on top of a multilayer hydrogel sheet impregnated with aligned CNT microelectrodes.

Figure 2

SEM images of (a) vertically aligned CNT forests (width: 460 μm and height: 300 μm), (b–c) magnified parts of the CNT forest surface. (d & e) HRTEM images of multiple and individual MWNTs isolated from the CNT forest. (f) Diameter distribution of the MWNTs in the CNT forests.

Figure 3

Two-point probe I-V curves from the CNT microelectrodes in parallel and perpendicular configurations relative to the nanotube alignment direction of CNT microelectrodes were measured. Au contacts were deposited at the edges of the CNT microelectrodes.

Figure 4

SEM images show that (a) arrays and (b) alignment of CNTs were preserved after they were transferred to the PEG substrate. (c) Cardiac fibroblast cell viability using LIVE/DEAD staining after 24h of seeding on CNT forest microelectrodes. (d) SEM images show porous surfaces of a 1 mg/ml CNT-GelMA layer. This image shows nanofibrous networks of CNTs across and inside a porous gelatin framework.

Figure 5

Cardiac tissue organization on composite hydrogel layers incorporating CNT forest electrodes. (a) Phenotype of cardiac cells. Immunostaining of sarcomeric α-actinin (green), nuclei (blue), and Cx-43 (red) revealed that cardiac tissues (5-day culture) were created on top of a multilayer hydrogel sheet impregnated with aligned CNT microelectrodes. Magnified image showing the well interconnected sarcomeric structures of cardiac tissue which is located (b) above locations between the electrodes (red dot arrow) and (c) above the electrodes (yellow dot arrow). (d) Photograph of a free-standing 3D bio-hybrid actuator cultured for 8 days. (e) Schematic illustration of a multilayer hydrogel sheet impregnated with aligned CNT microelectrodes (side view). Phase contrast image of the boundary between the CNT forest electrodes and the hydrogel layer shows that the cardiac tissues remained attached to the top hydrogel layer snd stayed intact. (f) The displacement of the CNT forest electrode in multilayer hydrogel sheets (yellow circled tip in b) over time under electrical stimulation (Square wave form, 1.2 V/cm, Frequency: 0.5 Hz – 3 Hz, 50ms pulse width).

Figure 6

(a) and (b) Phase contrast images that show the direction of the applied field which is along the parallel and the perpendicular direction to the alginment of the CNT forests. (c) The excitation threshold of cardiac tissue in the parallel configuration was three times lower than that in the perpendicular configuration. (d) Numerically calculated maximum voltage applied on the cardiac cell layer using the two different electrode configurations (Frequency range: 0.5 Hz – 3 Hz, Wave: Pulse signal, Pulse width: 50ms). Top views of the numerically calculated electric field applied to the muscle cells in perpendicular (e) and parallel (f) electrode configurations with top and cross sectional views.