Electrically Driven Microengineered Bioinspired Soft Robots

Abstract

To create life-like movements, living muscle actuator technologies have borrowed inspiration from biomimetic concepts in developing bioinspired robots. Here, the development of a bioinspired soft robotics system, with integrated self-actuating cardiac muscles on a hierarchically structured scaffold with flexible gold microelectrodes is reported. Inspired by the movement of living organisms, a batoid-fish-shaped substrate is designed and reported, which is composed of two micropatterned hydrogel layers. The first layer is a poly(ethylene glycol) hydrogel substrate, which provides a mechanically stable structure for the robot, followed by a layer of gelatin methacryloyl embedded with carbon nanotubes, which serves as a cell culture substrate, to create the actuation component for the soft body robot. In addition, flexible Au microelectrodes are embedded into the biomimetic scaffold, which not only enhance the mechanical integrity of the device, but also increase its electrical conductivity. After culturing and maturation of cardiomyocytes on the biomimetic scaffold, they show excellent myofiber organization and provide self-actuating motions aligned with the direction of the contractile force of the cells. The Au microelectrodes placed below the cell layer further provide localized electrical stimulation and control of the beating behavior of the bioinspired soft robot.

Keywords: bioactuators; bioinspiration; cardiac tissue engineering; flexible microelectrodes; hydrogels.

Figure 1. Schematic of the device

(a) Schematic of the cartilage joints and muscle patterns of a string ray. (b) Schematic illustration of the layer-by-layer structure of the construct. The bottom layer was composed of a PEG hydrogel with vertical line patterns for alignment. The upper layer in contact with the cells was made of patterned CNT-GelMA hydrogel with a pattern, which was perpendicular to the PEG hydrogel patterns. The microelectrodes were embedded in between the two layers. (c, d) Schematic design of the sting ray movement in the macro and micro scale: relaxed cardiomyocytes (c) and contracted cardiomyocytes (d).

Figure 2. The optimization of PEG- and CNT-GelMA hydrogel patterns

(a) Image of the bio-inspired actuator without the Au microelectrode. (b) SEM image of the pattern of the dense CNT-GelMA hydrogel lines aligned perpendicular to the sparse PEG hydrogel lines. (c) SEM image of the fractal-like surface of the CNT-GelMA hydrogel pattern. (d) Schematic illustration of the CNTs embedded into the GelMA hydrogel. (e) Fluorescent images of cardiomyocytes on the CNT-GelMA hydrogel pattern with a 50-, 75-, and 150-μm spacing. (f) Spontaneous beating rates of cardiac tissues seeded on the multilayer bio-inspired actuator with different spacing between the CNT-GelMA hydrogel patterns on day 5. (*p< 0.05 and **p < 0.005) (h) Alamar blue assay of cardiomyocyte cultured on the bio-construct after 7 days of incubation revealed high cell viability. (*p < 0.05) (i) The rolling morphologies of the bio-inspired constructs with the PEG hydrogel pattern with a 200-, 300-, and 500-μm spacing. (j) Table showing the index of the PEG hydrogel pattern and the CNT-GelMA hydrogel pattern optimization process: different spacings between the lines of PEG and CNT-GelMA hydrogel patterns have been inspected. For each device made with a different combination of patterns, the cell spreading, alignment, and beating behavior was analyzed. (Beating level indicated: 0, Very weak or no beating; 1, Weak beating; 2, Strong beating; 3, Very Strong beating. Rolling score indicated: -1, Rolling; 0, No Rolling).

Figure 3. The characterization of the cardiomyocytes on the bio-inspired scaffold

(a) Schematic illustration of the contraction behavior of the cultured cardiac muscle tissue on the bio-inspired scaffold. The cultured cardiac muscle tissue showed a pseudo-3D structure, which could be separated into four layers, the (i-1 and -2) upper, (ii) middle, and (iii) bottom, based on aligned and random cardiomyocyte morphology. (b) Confocal fluorescent images showed different morphology in the (i-1 and -2) upper, (ii) middle, and (iii) bottom of cardiomyocytes cultured on the bio-inspired scaffold for day 5. (c) Spontaneous beating rates of the cardiomyocytes on the bio-inspired scaffold from day 3 to day 9. (d) (i) Photograph of a free-standing bio-inspired soft robot cultured for 5 days at 0, 0.18, and 0.3 sec. The blue line represents the longitudinal axis displacement while the green line represents the transverse axis displacement. (ii) Particle Image Velocimetry measurement of the bio-inspired soft robot spontaneously moved within 0.3 sec. All arrows indicated direction and magnitude of the beating motion. (e) Displacement of the two major axes during stimulated contractions (2.0 Hz, 1 V/cm). The blue line represents the longitudinal axis displacement (corresponding to the blue line in Figure 3d) while the green line (corresponding to the green line in figure 3d) represents the transverse axis displacement. The frame taken in correspondence to the lines marked with 1 and 2 are shown in Figure 3d. (f) Young’s modulus of the PEG hydrogel pattern, CNT-GelMA hydrogel pattern, and the CNT-GelMA hydrogel pattern fabricated on the Au microelectrode. (*p < 0.05) (g) Schematic of the mechanism of tail longitudinal displacement which induces the soft robot displacement along the vertical direction, mainly on the tail part, when the cells contract. (h) Beating response of the bio-inspired soft robot when stimulated with an AC external electrical field at 1V/cm and with various frequencies from 0.5 to 2.0 Hz.

Figure 4. Characterization of the Au microelectrodes and their incorporation into the bio-inspired soft robot

(a) E-beam-evaporated Au microelectrodes with a serpentine pattern. (b) AFM image of the Au microelectrodes. (c) and (d) SEM image of the Au microelectrode successfully transferred onto the PEG hydrogel. (e) Optical microscope image of the Au microelectrodes successfully embedded in the CNT-GelMA hydrogel pattern. (f) Obtained bio-inspired soft robot with embedded Au microelectrodes. Copper wires were connected to the structure using silver paste to make an electrical contact for local electrical stimulation. (g) Variations in the Au microelectrodes resistance embedded in the bio-inspired scaffold during 5 days of incubation in cell culture media at 37 °C. (h) Measured impedance modulus of Au microelectrodes transferred on the PEG hydrogel pattern (green), embedded in between PEG and CNT-GelMA hydrogel patterns (blue), and PEG and CNT-GelMA hydrogel patterns with cardiomyocytes layer (red). (i) Confocal fluorescence image of the cardiomyocytes, randomly spread among the Au microelectrodes (red signal) on the unpatterned central body. (j) The cardiomyocytes exhibited a random network organization on the unpatterned central body. (k) Well-elongated and aligned cardiomyocytes were showed on the CNT-GelMA hydrogel pattern which is indicated by the white dots. (l) Partial uniaxial sarcomere alignment and interconnected sarcomeric structure was observed on the patterned areas. (m) Top views of the numerically calculated electric potential contour plot volume distribution when a square wave signal (Peak Amplitude: 1 V, DC offset value: 0V, Frequency: 2.0 Hz, Pulse width: 50 ms, Duty Cycle: 10%) was applied to the embedded microelectrodes. (n) Excitation threshold voltage required at different frequencies (0,5, 1.0 and 2.0 Hz) when electrical stimulation was applied via the embedded Au microelectrodes. (o) Spontaneous beating behavior of the bio-inspired soft robot with embedded Au microelectrodes after electrical stimulation.