Bio-inspired Hybrid Carbon Nanotube Muscles

Abstract

There has been continuous progress in the development for biomedical engineering systems of hybrid muscle generated by combining skeletal muscle and artificial structure. The main factor affecting the actuation performance of hybrid muscle relies on the compatibility between living cells and their muscle scaffolds during cell culture. Here, we developed a hybrid muscle powered by C2C12 skeletal muscle cells based on the functionalized multi-walled carbon nanotubes (MWCNT) sheets coated with poly(3,4-ethylenedioxythiophene) (PEDOT) to achieve biomimetic actuation. This hydrophilic hybrid muscle is physically durable in solution and responds to electric field stimulation with flexible movement. Furthermore, the biomimetic actuation when controlled by electric field stimulation results in movement similar to that of the hornworm by patterned cell culture method. The contraction and relaxation behavior of the PEDOT/MWCNT-based hybrid muscle is similar to that of the single myotube movement, but has faster relaxation kinetics because of the shape-maintenance properties of the freestanding PEDOT/MWCNT sheets in solution. Our development provides the potential possibility for substantial innovation in the next generation of cell-based biohybrid microsystems.

Figure 1. PEDOT/MWCNT-based cell culture platform and analysis of myotube alignment.

(a) Schematic illustration of the cell culture protocol. (Left) C2C12 myoblasts seeded on a PEDOT/MWCNT sheet. (Middle) 2 days of cell culture. (Right) Aligned myotubes after 7 days of differentiation. (b) Scanning ion-conductance microscope (SICM) 3D image of the myotubes on the PEDOT/MWCNT sheet. The scan area is 40 × 40 μm. (c) Myotube width and height profiles were obtained from the cross-sectional line profile shown in (b). Each letter on the line profile corresponds to the same letter on the cross-section of (b). (d) The average myotube length and width were obtained from confocal microscope images of MyHC⁺ myotubes. (e–g) Confocal microscope images of MyHC⁺ myotubes grown on a PEDOT/MWCNT sheet. Scale bar = 50 μm. (h) Myotube alignment ratio analyzed by constructing an angular spread distribution histogram (n = 200). 93% of the myotubes were positioned in the range of −10° to +10°.

Figure 2. Single myotube contraction.

(a) Schematic diagram of the device with an edge-detection system and electric field stimulation (EFS) for tracing contraction and relaxation. (b) Snapshot images from the monitoring system when a myotube relaxes and contracts. Red and green lines the indicate line scan profile of the left and right sides, respectively, of a single myotube. (c) Contraction distance changes of a single myotube at 1 Hz EFS. Red and green lines correspond to the movement of (1) and (2) indicated in (b). The average distance is 1.3 μm at 1 Hz EFS. (d) The frequency-contraction distance relationship in response to varying EFS frequencies. The inset shows the enlarged signal recorded at 1 Hz EFS. (e) Contraction distances were measured at each EFS frequency (1.4 ± 0.2 μm at 0.5 Hz; 1.2 ± 0.3 μm at 1 Hz; 0.6 ± 0.3 μm at 2 Hz; 0.3 ± 0.1 μm at 4 Hz; n = 500). (f) Baseline movement is increased as EFS frequency increases.

Figure 3. Fabrication of hornworm-like hybrid muscle.

(a) Schematic illustration of the procedures for fabrication of the hybrid muscle using a PTFE mold. A window frame patterned PTFE mold was mounted on a PEDOT/MWCNT sheet and C2C12 cells were seeded in the open window regions of the mold. After 7 days of differentiation, the mold was removed and then the hornworm-like hybrid muscle was actuated by EFS. (b) A transmission electron microscope image shows myotube-free (left) and myotube regions (right). Confocal microscope images of the cells located in the dashed squares in (b) are presented in (c–e) (scale bar: 200 μm). (c) The PTFE mold separates the two regions and poorly-aligned myotubes are abundant at around the boundary region (yellow dashed line). (d,e) No myotube are observed in (d), but the well-aligned myotubes are plentiful in region (e). Transmission microscope images are inserted on the left of the confocal microscope images (Scale bar: 50 μm). Confocal image scale bars: 100 μm in (c); 25 μm in (d,e).

Figure 4. Contraction behaviors of hornworm-like hybrid muscle.

(a) Photographs of the EFS-stimulated hybrid muscle (left, relaxation state; right, contraction state; scale bars, 10 mm). (b) Frequency-contraction distance relationship of the hybrid muscle in response to varying EFS frequencies (1–8 Hz). (c) Baseline movement in response to EFS frequency changes was compared between the hybrid muscle and the single myotube. Baseline movement is normalized to the amplitude at 1 Hz. (d) Normalized contraction behaviors of a single myotube and hybrid muscle system at 1 Hz. (e) The half-contraction and half-relaxation times (t_½) were compared between the single myotube and the hybrid muscle system.