Regulation of C2C12 Differentiation and Control of the Beating Dynamics of Contractile Cells for a Muscle-Driven Biosyncretic Crawler by Electrical Stimulation

Abstract

Biosyncretic robots have potential advantages associated with both living organisms and electromechanical systems. Skeletal muscle tissue is a candidate as bioactuators for biosyncretic robots because of its excellent contraction force and controllability. However, the low quality of myoblast (C2C12) differentiation into contractile myotubes and the lack of control research on biosyncretic robots are two of the main challenges in the development of biosyncretic robots. In this study, an approach with circularly distributed multiple electrodes (CEs) is proposed to improve C2C12 differentiation and to control the movement of a myotube-based biosyncretic crawling robot. To analyze the advantages of the proposed CEs, the electrical characteristics of CEs and a pair of traditional parallel stimulation electrodes (PEs) were simulated and compared with each other. Then, to determine the optimal electrical stimulation parameters and demonstrate the superiorities of the proposed CEs, electrical pulses with different parameters were used to stimulate two-dimensional and three-dimensional cells during culture with the proposed CEs and PEs. After this the control characteristics of the muscle tissue by the CEs were investigated from the relevance of pulse width–threshold voltage, voltage–contractility, frequency–contractility, and electric field direction–contractility by measuring the real-time responses of myotubes to different electrical stimulations. Moreover, to demonstrate the control of biosyncretic robots by the CEs, a biomimetic biosyncretic crawler actuated by myotubes was designed, fabricated, and controlled to move at different speeds by varying directions of electric field. This study not only provides a potential tool for the development and control of biosyncretic robots but is also informative for muscle tissue engineering and cardiomyocyte culture.

Introduction

Biosyncretic robots, which comprise living biological materials and artificial electromechanical components, have attracted considerable attention for their potential performance. Biosyncretic robots may have the potential to offer both the advantages of living organisms, such as a high degree of freedom, high energy conversion efficiency, high energy density, biocompatibility, and potential self-repair,^1–3 and qualities of electromechanical systems, including high accuracy,⁴ high strength, and favorable repeatability and controllability.⁵

As reported, traditional electromechanical systems operating on electrical energy perform with low energy transformation efficiency (<30%) during mechanical work and thus lead to a large heat loss. In comparison, biological actuators directly convert chemical energy into mechanical movement with a much higher efficiency (≥50%) that is impossible for conventional electromechanical actuators.⁶ Moreover, most biological actuators have the ability to produce and regulate their motion in response to the external environment, such as light stimuli,^7,8 electric fields,³ magnetic fields,⁹ temperature gradients,¹⁰ and chemical stimuli.¹¹

To construct a biosyncretic robot with the capability of generating force resulting in mechanical movement with the superior capabilities mentioned previously compared with conventional electromechanical robots, many researchers have explored the use of various types of living biological components of variable sizes, such as molecular motors,^6,12,13 microorganisms,^14–17 muscle cells,^{1,3,7,18–24} and insect dorsal vessel tissue (DVT).^25–27

Molecular motors, such as DNA, kinesin, myosin, and F1-ATPase, exert forces in the range of 1–45 pN⁶ and have been widely used as nanoscale actuators. Microorganisms, including flagellated bacteria, protozoa, and algae, autonomously swim in a certain direction at various speeds from 20 μm/s to 2 mm/s, with thrust forces between 0.3 and 200 pN,²⁸ depending on environmental factors such as illumination stimuli, chemical gradients, and temperatures. Bioactuators based on such microorganisms have demonstrated the capability of powering microscopic gears, moving microscale loads, and delivering drugs. Individual and clustered muscle cells, including cardiomyocytes and skeletal cells, produce even higher contractile forces between 80 nN and 3.5 μN,⁶ and have been used as bioactuators to actuate microstructures made of soft biocompatible materials. Insect DVTs, which are excised from inchworms, achieve a contraction force of 20 mN,²⁶ and have been used to power microdevices that move with environmental robustness and low maintenance.¹¹

Among live biological actuators, skeletal muscles seem to be highly appropriate and exhibit the potential to serve as actuators of biosyncretic robots because skeletal muscles are the main power generators of animals, including human beings. Skeletal muscles generate contraction forces (∼400 μN)^3,29 under neural stimulation or external stimulation.³⁰

Moreover, electrical stimulation from the central nervous system is one of the most important cues for functional muscle development.³¹ For example, denervated muscles in utero will lose the ability to develop into fully mature muscles.^31–33 Therefore, starting as far back as 1942, electrical stimulation has been engineered as a biomimetic tool to replace nerve stimulation in denervated skeletal muscles and preserve muscle tissue function.³⁴ Recent studies have shown that electrical stimulation not only enhances the proliferation, differentiation, and maturity of skeletal muscle cells^35–40 but also improves the alignment of differentiated myotubes.^35,41–43 Furthermore, electrical stimulation has widely been used to control and increase the contractility of muscle cells,^42,44,45 and, therefore, muscle cell-based biosyncretic robots.^1,3

As the optimization of electrical stimulation is important for inducing muscle development,⁴⁶ and unsuitable electrical stimulation may damage cells,^47–49 various electrical stimulation parameters,⁴³ including electrode materials,⁴⁷ amplitude,⁴³ duration,⁴³ and frequency,^47,49 and also different types of equipment, such as parallel electrodes^49,50 and substrate electrodes,^41,51–53 have been used to regulate the behaviors of two-dimensional (2D) and three-dimensional (3D) muscle tissues.

Recent studies with electrical stimulation have promoted the significant development of 2D/3D muscle tissues for regenerative therapy and biosyncretic robots. However, certain shortcomings remain, which may limit the application of electrical stimulation approaches for both inducing muscle maturation and controlling cell contraction. First, when a pair of parallel electrodes placed in the medium is used to stimulate muscle cells, the electric field will be diminished by the inherent resistance of the medium.⁴¹ Therefore, a high voltage is required to polarize and stimulate cells. However, the locally high current induced by a nonuniform electric field may damage the cells lying near the electrodes.^{43,47–49,54}

Moreover, the high voltage between the electrodes may exacerbate electrolysis of the medium and induce a local pH gradient and gas generation on the surface of the electrodes, which may damage the living cells in the medium.^44,47,51 Second, a parallel uniform electric field, which induces parallel aligned myotubes, is difficult to achieve using a pair of parallel electrodes in liquid medium unless the electrode length is more than twice that of the electrode spacing.⁵⁵ Third, although substrate electrodes may reduce damage to the medium and cells,^41,51 the direction of the electric field is immutable once the electrodes have been fabricated. In addition, because of the inflexible and variable direction of the electric field induced by the electrodes, few reports have closely examined the relationship between cellular contractility and the time-variable electric field direction.

In this study, a stimulation approach with circularly distributed multiple electrodes (CEs) was proposed to induce 2D and 3D myoblasts (C2C12) to differentiate into contractile myotubes. To analyze the advantages of the proposed CEs over a pair of traditional parallel stimulation electrodes (PEs), the electrical characteristics of CEs and PEs were simulated using ANSYS and compared with each other. Then, to determine the optimal electrical stimulation parameters and demonstrate the practicability of the proposed CEs, electrical pulses with different parameters were used with CEs and PEs, respectively, to induce 2D and 3D C2C12 differentiation. The real-time response of differentiated myotubes to various electrical stimulations was subsequently measured to investigate the control characteristics of muscle tissues under the CEs, including the relationships of pulse width–threshold voltage, pulse amplitude–contractility, stimulation frequency–contractility, and electric field direction–contractility.

Moreover, to demonstrate the control performance of the CEs for biosyncretic robots, a biomimetic biosyncretic crawler actuated by myotubes was designed, fabricated, and controlled to move by varying the direction of electric field through the CEs. The results of the theoretical simulation, the experiments of 2D and 3D cell differentiations, and the movement control of the biosyncretic crawlers have demonstrated the advantages and applicability of the proposed CEs. This study not only provides a potential tool for the development of biosyncretic robots but is also useful for muscle tissue engineering and cardiomyocyte culture.

Materials and Methods

Equipment and experimental setup

The proposed CEs contained 12 electrodes, which were uniformly distributed around a 60-mm Petri dish. The electrodes used in this study were constructed from gold modified conductive tapes by our laboratory. The size of each electrode was 10 mm in length, 5 mm in width, and 1 mm in thickness. When the CEs were used to stimulate the cells, the consecutive six electrodes were used as the positive electrodes and the remaining six electrodes were used as the negative electrodes. The step angle of the rotation magnetic field was generated by the CEs by changing the potentials of the electrodes. To compare the advantages of the proposed CEs, PEs were used, which consisted of a pair of glassy carbon electrodes with a spacing of 40 mm. The size of each glassy carbon electrode was 20 mm in length, 20 mm in width, and 2 mm in thickness. A modified bidirectional pulse stimulator (Master-9; AMPI, Israel) was used in this study. The Petri dishes with CEs and PEs were placed in an incubator for cell culture, and the wires were led out to connect the electrodes and the stimulator outside the incubator (Fig. 4a).

Simulation and analysis of electric fields

To theoretically investigate the underlying reason that the proposed CE is advantageous over PEs in stimulating myoblasts to differentiate into myotubes, the electric fields generated by CEs and PEs were simulated using the commercial software package ANSYS to study the characteristics of electric fields, including electrical potential, electric field intensity, and current density. The physical parameters of different materials used in the simulation, such as the conductive tapes, the glassy carbon electrodes, the medium, and the Petri dishes, were determined based on the real properties of the materials used in the experiment.

Cell preparation

In the experiments with 2D cells, mouse myoblasts (C2C12, less than six passages in age; American Type Culture Collection, Manassas, VA) were divided into three groups to investigate the effects of different electrical stimulations on C2C12 differentiation: a control group without electrical stimulation, an experimental group with CEs, and a comparison group with PEs. Myotubes differentiated from myoblasts were used to investigate the effects of different electrical stimulations on myotube contractility. The growth medium (GM) consisted of Dulbecco's modified Eagle medium (DMEM; HyClone), 100 U/mL penicillin, 100 mg/mL streptomycin (Gibco), and 10% fetal bovine serum (Gibco). The differentiation medium (DM) consisted of DMEM, 100 U/mL penicillin, 100 mg/mL streptomycin (Gibco), and 2% horse serum (Gibco).

First, the cells in the three groups were cultured in 60-mm Petri dishes at 37°C under a 5% carbon dioxide (CO₂) atmosphere in the GM for 3 days until they proliferated to 90% confluence. Then, the cells were washed three times with phosphate-buffered saline (PBS; HyClone) after the GM was removed. Next, the cells were cultured with 5 mL of DM in a cell culture incubator, and the medium was exchanged for fresh DM every day. After culturing for 24 h in DM, the cells in each experimental group and comparison group were stimulated in four subgroups based on stimulation with an alternating pulse voltage of 0.5, 1, 1.5, or 2 V/cm to optimize the electrical stimulation parameters for inducing myoblast cells to differentiate. During the stimulation, the same pulse width (10 ms) and frequency (1 Hz) were used according to previous reports.^41,47 After stimulating with the corresponding electrodes and electrical pulse parameters for 24 h (1-h rest every other 1-h stimulation), the cells in the experimental and comparison groups were cultured in DM. After culturing in DM for 8 days (in total), the cells in the three groups were immunostained, stimulated to beat, and measured to study their morphology and contractility.

Immunofluorescence staining and measurement of myotubes

To analyze the C2C12 differentiation in the three different groups under different stimulation conditions, the relative proteins were stained with corresponding biological stains. The myosin heavy chain (MHC) protein, nuclei, and cytoskeleton were labeled with the reagents of Anti-Myosin Heavy Chain Alexa Fluor 488 (eBioscience), 4′,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich, St. Louis, MO), and Tubulin-Tracker Red (Beyotime, China), respectively.

The following staining process was applied: (1) the culture medium was removed from the dish and the cells were washed three times with PBS, each for 5 min, (2) the cells were fixed with fixative (Beyotime) for 10 min, (3) the cells were then washed three times with cell-scrubbing solution (Beyotime), each for 5 min, (4) the cells were covered with Anti-Myosin Heavy Chain Alexa Fluor 488 solution at 1/100 prepared in secondary antibody dilution buffer (Beyotime), with a volume one-fifth that of the primary complete GM, (5) the cells were incubated in staining solution while avoiding light for 30 min, (6) the staining solution was removed, and the cells were washed three times with cell-scrubbing solution, each for 5 min (staining of 3D muscle tissue cytoskeleton was subsequently carried out by repeating steps 4–6 by replacing the Anti-Myosin Heavy Chain Alexa Fluor 488 with Tubulin-Tracker Red), (7) the cells were covered with DAPI staining solution for 10 min while avoiding light, (8) the cells were washed three times with cell-scrubbing solution, each for 5 min, and (9) the cells were covered with PBS and imaged using a commercial fluorescence microscope (Ti-e; Nikon, Tokyo, Japan).

Measurement of cellular contractility with atomic force microscopy

Differentiated myotubes contract and beat under external electrical stimulation, and the beating behavior of single myotubes is significantly affected by the parameters of electrical stimulation. The beating frequency and amplitude of myotubes determine the movement characteristics of muscle cell-based microrobots. Therefore, it is of great significance to understand how electrical stimulation affects the beating behaviors of myotubes.

In this study, to characterize the electroresponsive beating behaviors of single myotubes under electrical stimulation, external electrical pulses generated by the CEs were applied with four different parameters of electrical stimulation, specifically frequency, amplitude, width, and direction, to stimulate the myotubes to beat (Fig. 1). According to a previous report,⁵⁶ the vertical beating of a cell is positively correlated with cellular contractility; therefore, a BioScope Catalyst atomic force microscopy (AFM; Bruker, Santa Barbara, CA) that was set on an inverted microscope (Ti; Nikon) was used to measure the vertical beating amplitude of the myotubes (Fig. 1). To noninvasively measure the beating of the myotubes, AFM contact scanning mode with zero scanning size was used. In this mode, theoretically, the deflection of the cantilever is maintained at a small preset set point by the scanner with feedback from the position sensitive detector signal. Owing to the retardance of the scanner actuation, there is an oscillating deflection of the cantilever with a small amplitude around the set point, and the oscillation of the cantilever deflection causes errors in the beating measurements. To reduce the effects of the oscillating deflection of the cantilever on the beating measurements, the beating signal of the myotubes is corrected by means of a linear combination of the deflection data of the cantilever and the displacement data of the scanning piezoelectric ceramic transducer tube (scanner) of the AFM system, which were obtained by the AFM controller and recorded with a digital oscilloscope (Tektronix DPO7054C). Subsequently, the beating signals of the myotubes under electrical stimulation with different parameters and directions were filtered and measured with MATLAB.

FIG. 1.

Schematic diagram of the equipment for the stimulation and measurement of muscle cells. (a) The electrical stimulation of a myotube by the proposed CEs and the measurement of cellular beating patterns by the AFM. (b) The electrical stimulation patterns (voltage of 1 V/cm, pulse width of 10 ms, and frequency of 1 Hz) on the myotubes and the cellular beating response obtained by the AFM. AFM, atomic force microscopy; CEs, circularly distributed multiple electrodes; PSD, position sensitive detector. Color images available online at www.liebertpub.com/soro

Manufacture of a 3D biosyncretic crawler

A 3D biosyncretic crawler was designed and fabricated to verify the validity of the proposed CEs for 3D muscle tissue formation and to demonstrate the utility of the proposed CEs in the control of biosyncretic robots. Established procedures were used for rapid fabrication of the mold for the formation of the 3D muscle tissue used to fabricate the biosyncretic crawler,⁸ as shown in Figure 2a and b. In brief, based on the bionic characteristics of a crawler, the geometric structure of the mold was designed using computer-aided design software (Fig. 2a). According to the design, a polymethyl methacrylate (PMMA) negative mold (for the fabrication of the polydimethylsiloxane [PDMS] mold for the formation of 3D muscle tissue) was manufactured by a mini-type miller (Roland EGX-400; Japan). Then, the uncured PDMS (10:1) was poured into the PMMA negative mold and cured at 75°C for 4 h.

FIG. 2.

Schematic diagram for the fabrication and control of biomimetic biosyncretic crawlers actuated by 3D muscle tissue. Dimensions (a) and 3D image (b) of the mold for 3D muscle tissue formation. (c) Schematic diagram of the fabrication of a biosyncretic crawler from 3D muscle tissue cut with a surgical knife. (d) Components of the mixed biological material for 3D muscle tissue formation. 3D, three-dimensional. Color images available online at www.liebertpub.com/soro

Based on previous reports,²⁹ living cells, Matrigel (Solarbio, Beijing, China), fibrinogen (Sigma Aldrich), and thrombin (Sigma Aldrich) were used for the 3D muscle tissue. To maintain the structure and retain a certain recurrent force of the muscle tissue for effective movement of the biosyncretic crawlers when the 3D muscle ring was extracted from the mold and cut off to form the biomimetic biosyncretic crawler (Fig. 2c), the parameters of the components in the mixed biological materials were optimized as follows: the final concentration of the myoblasts was 5 × 10⁶ cells/mL in GM, the Matrigel volume was 30%, the fibrinogen concentration was 8 mg/mL, and the thrombin concentration was 4 U/mL (Fig. 2d).

Three groups were tested to investigate the effects of differential electrical stimulation on the formation of 3D muscle tissue, including control group without electrical stimulation, experimental group with CEs, and comparison group with PEs. First, 100 μL of ice-cold mixed biological material was poured into each PDMS mold (Fig. 2b) in each group, and then the material was cultured at 37°C under a 5% CO₂ atmosphere in an incubator for 1 h. After preliminary solidification of the mixed biological material, 5 mL of GM was added into each Petri dish, and the cells were cultured and induced to differentiate following the procedures used for 2D cell culture. In brief, the mixed material was cultured in each mold for 3 days. Then, the GM was replaced by DM, and the medium was exchanged daily with fresh DM. After culturing for 24 h, the cells in the experimental and comparison groups were stimulated with the corresponding electrodes. For optimization of the electrical stimulation parameters and to improve the formation of 3D muscle tissues, cells in different molds for the experimental and comparison groups were stimulated with different electrical amplitudes (0.5, 1, 1.5, and 2 V/cm) and the same stimulation frequency (1 Hz) and pulse width (10 ms) as those used in the 2D cell culture experiments. After stimulating for 24 h, the 3D muscle tissues were cultured in DM for 6 days (8 days in DM in total). Then, the 3D muscle tissues in each group were observed and extracted from the molds for fabrication and experiments with the biosyncretic crawlers (Fig. 2c).

Data acquisition and processing

The data obtained by the oscilloscope in this study were analyzed using MATLAB and smoothed by the available median filtering function of MATLAB with a “window” modulus of 100 according to data size. The fluorescence intensity (FI) of the MHC and the nuclei and the size and arrangement of the differentiated myotubes were measured by ImageJ software. Hypothesis testing (two-sample t-test) was used to correlate the data from different groups. A significant difference was assessed at p < 0.05.

Results and Discussion

Simulation and analysis of CEs and PEs

To study the electrical characteristics of the proposed CEs and PEs in the simulation process, a direct current voltage was applied to the corresponding electrodes for the purpose of creating a static electric field intensity of 2.0 V/cm with the proposed CEs and PEs. The electrical resistance parameters of the materials, specifically the conductive tapes, the glassy carbon electrodes, the medium, and the Petri dishes used in the simulation, were set at 2.4 × 10⁻⁸, 5.0 × 10⁻⁵, 6.67 × 10⁻², and 10.21 Ω·m, respectively, which means the resistance of the CEs and PEs are so little compared with the medium that the effect of the difference in the electrical resistance parameters of CEs and PEs on the electric field in the medium could be ignored.

The simulation results showed that the electrical potential generated by the CEs had a larger gradient with apparent directivity (Fig. 3f) than that generated by the PEs (Fig. 3b), which may improve the directivity of the myotubes; the region of the locally high electric field intensity generated by the PEs (Fig. 3c), which may harm the cells, was much larger than that of the electric field generated by the CEs. Although the CEs generated a uniform electric field in the main area of the Petri dish (Fig. 3g), the PEs generated a locally high-density current around the electrodes (Fig. 3d, h), which would electroporate the cells. These results indicated that the CEs may provide more effective and suitable electrical stimulation for C2C12 culture than the PEs.

FIG. 3.

Simulation results of the electrical characterization of the PEs and the proposed CEs. (a, e) The 3D models of the PEs and CEs, respectively. (b, f) The simulation results of the electrical potential distribution generated by the PEs and CEs, respectively. (c, g) The simulation results of the electric field intensity distribution generated by the PEs and CEs, respectively. (d, h) The simulation results of the total current density distribution generated by the PEs and CEs, respectively. The color bar at the right of the figure represents increasing values of the simulation results from bottom to top. PE, parallel stimulation electrodes. Color images available online at www.liebertpub.com/soro

Differentiation results for C2C12 stimulated by CEs and PEs

To study the effects of the PEs and CEs on the differentiation and maturation of the myoblast cells (Fig. 4a–c), the differentiated myotubes and nuclei of the cells in each group were stained with Anti-Myosin Heavy Chain Alexa Fluor 488 and DAPI (Fig. 4d–f) and were measured by ImageJ software.

FIG. 4.

(a) Equipment to culture and induce cellular differentiation. (b) A Petri dish with a pair of parallel glassy carbon electrodes. (c) A Petri dish with a set of CEs. (d) Immunofluorescence image of the cells in the control group. (e) Immunofluorescence image of the cells in the experimental group induced by PEs with a stimulation voltage of 1.5 V/cm. (f) Immunofluorescence image of the cells in the experimental group induced by CEs with a stimulation voltage of 1.5 V/cm. The inset in (f) is an enlarged view of the secondary myotube. The green and blue colors in (d–f) illustrate the MHC and nuclei of the cells, respectively. The scale bars in (d–f) are 200 μm. MHC, myosin heavy chain. Color images available online at www.liebertpub.com/soro

MHC of cells stimulated under different conditions

MHC is one of the sarcomere contractile proteins, and it is a marker of skeletal muscle tissue differentiation.⁵⁷ Therefore, the MHC FI of the myotubes in the control, comparison, and experimental groups was measured to observe and assess the differentiation level and quality of the cells in each group. As shown in Figure 5a, the MHC FI of the cells in the groups with CEs and PEs under a stimulation voltage of 0.5 V/cm was similar to those in the control group. When the stimulation voltage was >1.0 V/cm, the MHC FI of the cells in the control group was clearly lower than those in the experimental groups. Moreover, the increase in the MHC FI from the proposed CEs was greater than those from the PEs. Whereas when the stimulation voltage increased from 1.5 to 2.0 V/cm, the MHC FI of the cells increased indistinctively.

FIG. 5.

Statistical graphs of cell morphology in the control and experimental groups. (a) Statistical graph of the MHC protein FI of the cells in each group. (b) Statistical graph of the lengths of the myotubes in each group. (c) Statistical graph of the widths of the myotubes in each group. (d) Statistical graph of the alignment of the myotubes in each group. The green lines are the mean values in the control group; the green dashed lines and the yellow arrows indicate the SDs. *Significant difference between the different groups stimulated with CEs and PEs, p < 0.05. FI, fluorescence intensity; SDs, standard deviations. Color images available online at www.liebertpub.com/soro

Length of the myotubes stimulated under different conditions

Myotube size is related to muscle maturation; therefore, the length and width of the myotubes in each group were measured and analyzed. As shown in Figure 5b, there was no significant difference between the length of the myotubes in the control, experimental, and comparison groups when the stimulation pulse amplitude was 0.5 V/cm. When the stimulation voltage was 1.0 V/cm, the lengths of the myotubes in the experimental and comparison groups were larger than those of the myotubes in the control group. However, there was no significant difference between the lengths of the myotubes in the experimental and comparison groups. When the stimulation voltage was set at 1.5 V/cm, the lengths of the myotubes in the experimental and comparison groups were clearly increased. Moreover, the increased length with the proposed CEs was higher than that with the PEs. In contrast, when the stimulation voltage was increased to 2.0 V/cm, the lengths of the myotubes in the experimental and comparison groups decreased. In addition, the lengths of the myotubes in the comparison groups with PEs were critically affected.

Width of myotubes stimulated under different conditions

There was no significant difference between the widths of the myotubes in all groups when the stimulation voltage was 0.5 V/cm (Fig. 5c). When the stimulation voltages were 1.0 and 1.5 V/cm, the widths of the myotubes in the experimental and comparison groups increased, and the effects of the proposed CEs on the width of the myotubes were more significant than that of the PEs. However, when the stimulation pulse amplitude was increased to 2.0 V/cm, the widths of the myotubes in the experimental and comparison groups were smaller than the width of the myotubes stimulated with a voltage of 1.5 V/cm. Moreover, the width of the myotubes induced by the PEs was markedly reduced. Of note, the width of the myotubes induced by the proposed CEs with a pulse voltage of 1.5 V/cm was significantly larger than that of the other groups. This is attributable to the fact that the differentiated myotubes in the control group were at the level of primary myotubes (Fig. 4d),⁵⁸ and proper electrical stimulation transitions primary myotubes to secondary myotubes.^49,59 Therefore, the proposed CEs may be more effective to induce C2C12 differentiation and secondary myotube formation (Fig. 4f) than the PEs (Fig. 4e).

Alignment of myotubes stimulated under different conditions

Myotube alignment is a basic requirement for the formation of engineered muscle tissue⁶⁰ and is important for the generation of maximal muscle contractility^61,62 to actuate biosyncretic robots. Therefore, the alignment of the myotubes in the control, experimental, and comparison groups was measured and assessed. As shown in Figure 5d, the alignments of the myotubes in all the groups were similar when the stimulation voltage was 0.5 V/cm. When the stimulation voltages were 1.0 and 1.5 V/cm, the alignment of the myotubes in the experimental and comparison groups obviously increased with stimulation voltage. However, when the stimulation voltage was 2.0 V/cm, the alignments of the myotubes in all groups were not as good as those by a voltage of 1.5 V/cm. Moreover, the alignment of the myotubes in the experimental group was higher than that of the control and comparison groups. A comparison of the directional distributions of the myotubes in the control, experimental, and comparison groups with the stimulation voltage of 1.5 V/cm is shown in Figure 6.

FIG. 6.

Statistical graphs of the alignment of the myotubes in (a) the control group, (b) the comparative group induced by PEs, and (c) the experimental group induced by CEs with a stimulation voltage of 1.5 V/cm. Color images available online at www.liebertpub.com/soro

The results (Fig. 5a–d) are consistent with previous reports showing that electrical stimulation improves the maturation,⁶³ size,⁴² and alignment⁴³ of differentiated myotubes and demonstrating the advantages of the proposed CEs in terms of C2C12 differentiation level and quality. However, the present results show that higher stimulation voltages (such as 2.0 V/cm) do not obviously improve C2C12 maturation, size, and alignment, and, furthermore, this voltage may exert negative effects on cellular differentiation. These results are attributable to the fact that cells may be electroporated by a high stimulation voltage, and the pH value of the medium may be affected, which would damage the health of the cells (Fig. 5a–d), among other possible explanations.

Beating response of single myotubes to electrical stimulation

To characterize the electroresponsiveness of single myotubes, cellular beating patterns under different electrical stimulation parameters using the CEs, including different frequencies, voltage amplitudes, pulse widths, and electric field directions (a research area that has received little attention), were measured by using an AFM.

Relationship between threshold voltage and pulse width with different electrodes

To demonstrate the advantages of the proposed CEs and to determine the optimal pulse width needed to stimulate a myotube to beat for the production of biosyncretic robot control with an electrical pulse, different pulse widths generated by different electrodes were used to stimulate the differentiated myotubes to beat, and the corresponding threshold voltages were recorded. The threshold voltage was defined as the voltage that leads a myotube to beat with the average amplitude of the minimum and maximum beating amplitudes of the myotube stimulated with low and high voltages, respectively.

As shown in Figure 7, the threshold voltage amplitude decreased with an increasing pulse width of the electrical stimulation generated by CEs and PEs. This result is consistent with a previous report,⁴³ which accounted for this phenomenon by showing that the available pulse energy is related to cell contractility. Moreover, the threshold voltage of CEs was lower than that of PEs with the same pulse width (Fig. 7). It should be considered that a high stimulation pulse amplitude may electrochemically damage living cells,⁴³ and a long pulse width may generate sufficient joule heating to damage living cells.⁶⁴ According to the results shown in Figure 7 and previous studies,^41,43 the proposed CEs were used to determine a suitable stimulation for the myotubes, and the artificial electrical stimulation was set at 10 ms in this study.

FIG. 7.

Relationship between the threshold voltage of the myotubes and the electrical stimulation pulse width generated by different electrodes with the same stimulation frequency of 1 Hz. *Significant difference between the different groups stimulated with CEs and PEs, p < 0.05. Color images available online at www.liebertpub.com/soro

Relationship between cellular beating amplitude and pulse amplitude

The relationship between the cellular beating amplitude and the pulse amplitude has been studied for the production of biosyncretic robot control with different stimulation pulse amplitudes. In this study, the voltage amplitude was varied from 0.5 to 1.0 V/cm, with an external stimulation of 1 Hz and a 10-ms pulse width.

As shown in Figure 8, the beating amplitude of the cells changed with different stimulation electrical pulse amplitudes. When the pulse amplitude was <0.7 V/cm, the beating amplitude of the myotubes increased slowly with stimulation voltage, and the cellular beating frequency was less than the stimulation frequency (illustration (1) in Fig. 8). When the voltage amplitude was increased to 0.75 V/cm from 0.7 V/cm, the cellular beating amplitude suddenly changed from 0.81 to 1.10 μm. This may be attributed to the fact that the threshold voltage of the trigger ion channel of the myotubes stimulated by the proposed CEs was ∼0.75 V/cm when the other electrical parameters had a frequency of 1 Hz and a duration of 10 ms. In addition, when the stimulation voltage was >0.8 V/cm and <0.9 V/cm, the cellular beating amplitude increased with voltage amplitude. Furthermore, when the voltage amplitude was >0.75 V/cm, the cellular beating frequency was equal to that of the stimulation pulse (illustration (2) in Fig. 8). However, when the voltage was >0.9 V/cm, the beating amplitude of the myotubes would decrease with increasing voltage amplitude. This may be attributable to the fact that a high voltage may impact living cells. This result is consistent with results obtained by immunofluorescence (Fig. 5).

FIG. 8.

Relationship between the beating amplitude and the electrical stimulation voltage amplitude. The pink dotted line indicates the saltation of cellular beating amplitude. The blue arrow labels the optimal pulse amplitude. Illustrations (1) and (2) are the beating patterns of myotubes stimulated by electrical pulses of 0.6 and 0.9 V/cm, respectively, with a frequency of 1 Hz and a duration of 10 ms. The red arrowed lines in the illustrations represent the measured beating amplitudes of myotubes. Color images available online at www.liebertpub.com/soro

Relationship between beating amplitude and stimulation frequency

The beating amplitude of the myotubes is related to the actuation force and step size of biosyncretic robots. Therefore, we studied the relationship between the beating amplitude of differentiated myotubes and the frequency of the stimulation electrical pulse for the production of biosyncretic robot control with different stimulation frequencies. In this study, the frequency of the external electrical pulse with a voltage of 0.9 V/cm and a 10-ms pulse width was varied from 1 to 11 Hz with 1-Hz increments.

As shown in Figure 9, the beating amplitude of the contractile myotubes changed with increasing stimulation frequency. When the stimulation frequency was <3 Hz, the myotubes displayed normal beating behaviors with an amplitude of ∼1.2 μm (illustration (1) in Fig. 9). When the stimulation frequency was >3 Hz, the beating amplitude decreased with increasing electrical stimulation frequency (illustration (2) in Fig. 9). Specifically, the beating amplitude clearly decreased from a stimulation frequency of 5 to 6 Hz. This effect may be attributed to the fact that these frequencies are similar to the cellular threshold frequency associated with the mechanical properties and action potentials of the myotubes. When the stimulation frequency was >6 Hz, the myotubes displayed tetanic contraction (illustration (2) in Fig. 9).

FIG. 9.

Relationship between beating amplitude and electrical stimulation frequency. The arrowed dotted lines indicate the ranges of the unattenuated beating and tetanic beating. Illustrations (1) and (2) are the beating patterns of myotubes stimulated by electrical pulses of 1 and 6 Hz, with a voltage amplitude of 0.9 V/cm and a duration of 10 ms. The red arrowed lines in the illustrations represent the measured beating amplitudes of myotubes. Color images available online at www.liebertpub.com/soro

Relationship between cellular beating amplitude and electric field direction

To investigate the control method for biosyncretic robots by electric field direction, the beating amplitude responses of differentiated myotubes to different directional electrical pulse stimulations generated by CEs with the same electrical parameters (stimulation frequency of 1 Hz, pulse amplitude of 0.9 V/cm, and pulse width of 10 ms) were measured.

As shown in Figure 10, the cellular beating amplitude varied with the angles between the myotube axis and the electric field (as shown by the illustrations (1), (2), and (3) in Fig. 10). The results showed that an electrical pulse at a smaller angle to the myotube axis led to a greater myotube beating amplitude. In short, myotubes achieve maximal contraction when the external stimulation is parallel to the myotubes and minimal contraction when the external stimulation is perpendicular to the myotubes. In addition, there was no significant difference between the cellular beating amplitudes stimulated by electric fields with symmetrical directions (such as electrical directions of 30°, 150°, 210°, and 330°, which all have the same angle of 30° to the myotube axis) by stimulation pulses with the same electrical parameters. The change in beating amplitude was attributable to the fact that the axial direction of the myotubes may be more suitable for conduction of the electrical pulse and for triggering the appropriate cellular ion channels for sarcomere contraction. This relationship between amplitude and electric field direction may be used to control the movement of biosyncretic robots.

FIG. 10.

Relationship between the beating amplitude and the angle between the electric field and the myotube axis. Illustrations (1), (2), and (3) are schematic diagrams of cases with angles of 0°, 90° and 150°. The yellow arrows represent the electric fields of different directions. #No significant difference between the cellular beating amplitudes with symmetrical directions, p > 0.05. Color images available online at www.liebertpub.com/soro

Manufacture and control of a 3D biosyncretic crawler

To compare the effects of the proposed CEs and PEs on the formation of 3D myotubes, myoblasts were cultured in 3D molds and electrically stimulated to differentiate into contractile 3D muscle tissues, which were used to fabricate 3D biomimetic biosyncretic crawlers. Myoblasts were stimulated by different electrical pulse amplitudes (0.5, 1.0, 1.5, and 2.0 V/cm) and the same pulse frequency (1 Hz) and pulse width (10 ms) generated by the proposed CEs and PEs as those used in the 2D cell culture experiments.

The results showed that the quantities of differentiated myotubes in 3D muscle tissues induced by the CEs and PEs with pulse amplitudes of 0.5 and 1.0 V/cm were lower than that induced with a pulse amplitude of 1.5 V/cm. Moreover, the geometry of the 3D muscle tissue induced by the PEs and CEs with a pulse amplitude of 2.0 V/cm was irregular compared with that induced by a pulse amplitude of 1.5 V/cm. These phenomena are attributable to the fact that a low pulse amplitude was unable to induce sufficient myoblasts to differentiate into myotubes. A high pulse amplitude may impair some living cells, such that nonuniform differentiation of the myoblasts leads to uneven 3D muscle tissue. These results were consistent with the results obtained in the experiments investigating 2D cell differentiation induced by the proposed CEs and PEs.

To compare the effects of the different electrodes on the formation of 3D muscle tissues, the muscle rings were released from the PDMS molds (Fig. 11a) and stained to label the MHC, microtubules, and nuclei of the cells in the muscle tissues (Fig. 11b, c). The results showed that the differentiation quality of the cells in the 3D muscle tissues induced by the proposed CEs with a pulse amplitude of 1.5 V/cm (Fig. 11b) was greater than that induced by the PEs (Fig. 11c). These results were consistent with the results of the related 2D cell culture experiment.

FIG. 11.

Manufacture and control of the biosyncretic crawlers with the proposed CEs. (a) Picture of the 3D muscle tissue in the mold and the biosyncretic crawler obtained by cutting off the muscle ring. (b, c) Confocal fluorescence images of the 3D muscle tissues induced by the proposed CEs and PEs with a pulse amplitude of 1.5 V/cm, respectively. The green, red, and blue regions in (b, c) mark the MHC, microtubules, and nuclei of the cells in the muscle tissues, respectively. (d–i) Pictures of a biosyncretic crawler stimulated by an electrical pulse with the following parameters: 1 Hz frequency, 0.9 V/cm pulse amplitude, and 10 ms pulse width, from 0 to 60 s. The gray dotted lines and green arrows in (d–i) represent the original position and displacement of the crawler, respectively. The scale bars in (a), (d–i) are 5 mm, and the scale bars in (b, c) are 100 μm. Color images available online at www.liebertpub.com/soro

To control the biosyncretic robots induced by the CEs, the 3D muscle tissues were cut off to form the biosyncretic crawlers (Fig. 11a) and were stimulated to move by the CEs with different directional electric fields using the optimized electrical parameters determined in the 2D cell stimulation study: 1 Hz frequency, 0.9 V/cm pulse amplitude, and 10 ms pulse width (Supplementary Movie S1; Supplementary Data are available online at www.liebertpub.com/soro).

The results showed that the biosyncretic crawler moved with different speeds (ranging from 4.10 to 40.09 μm/s) under electric fields of different directions, and electric fields at smaller angles with the crawler axis led to higher speeds and it was shown that there was significant difference between the movement speeds of the crawler stimulated by the electric fields of adjacent directions, such as 0° and 30° and 30° and 60° (Fig. 12). In addition, there was no significant difference between the crawler speeds stimulated by electrical pulses in symmetrical directions, such as 30° and 150° and 60°, and 120°. These results were consistent with the results obtained with 2D cells stimulated with the proposed CEs.

FIG. 12.

Relationship between the speed of the biosyncretic crawler and the angle between the direction of the electric field and the crawler axis. The yellow arrows represent the electric fields of different directions. *Significant difference between the movement speeds of the crawler under the electric fields of different direction, p < 0.05. Color images available online at www.liebertpub.com/soro

However, it was noticed that the movement speeds of the 3D biosyncretic crawler to the electrical pulses in the directions of 60° and 90° were similar (Fig. 12), but the beating amplitudes of myotubes were significantly different under the electrical pulses in the directions of 60° and 90° (Fig. 10). This phenomenon could be attributable to the fact that the crawler was curving, and the angle between the electric field and crawler was inconclusive.

Although this study only examined the speed control of a biosyncretic crawler, our results demonstrate the advantages of the proposed CEs for C2C12 differentiation and the feasibility of the control method for biosyncretic robots. Control of the speed and direction of biosyncretic robots with the CEs will be studied in depth in the future by designing smart robotic structures actuated by 3D muscle tissues, whose contractility is controlled by different electrical pulse parameters, including stimulation frequency, electrical direction, pulse amplitude, and width, generated by the CEs. In addition, few recent studies have focused on the control of biosyncretic robots through the application of various electric field directions, which is useful for the control of biosyncretic robots in special cases, such as environments where photic stimulation is unavailable.

Conclusions

Biosyncretic robots, which comprise living biological tissues and artificial electromechanical components, may have the potential to perform the advantages of both living organisms and electromechanical systems. To explore the feasibility of combining living biological materials and nonviable materials, different types of living biological actuators have been used in hybrid devices. Skeletal muscle tissues, which are the main power generators of animals, including human beings, appear appropriate and have the potential to be actuators of biosyncretic robots. However, differentiation of myoblasts into contractile myotubes and the control of biosyncretic robots based on muscle cells are two of the main challenges facing the development of biosyncretic robots.

In this article, CEs were proposed to improve C2C12 differentiation and the flexible control of biosyncretic robots. To optimize the electrical stimulation for C2C12 differentiation, electrical pulses with different pulse parameters were used to stimulate 2D/3D myoblasts to differentiate into myotubes with the proposed CEs compared with PE stimulation. The results showed that cells stimulated by CEs with the electrical parameters of a 1.5-V/cm pulse amplitude, 10-ms pulse width, and 1-Hz stimulation frequency have the preferred characteristics, including differentiation, myotube length, myotube width, and myotube alignment. The results that the cells stimulated by the proposed CEs have better differentiation characters than those by PEs were consistent with the ANSYS simulation results that the characteristics of the electric field generated by CEs were more effective and suitable for cell cultures than those by PEs.

Next, the beating responses of differentiated single myotubes to different electrical pulse stimulations, including different pulse amplitudes and widths, stimulation frequencies, and electric field directions, generated by the proposed CEs, were studied. The results showed that the beating amplitude of single myotubes is affected by different electrical pulse amplitudes, widths, stimulation frequencies, and electrical directions. The myotubes may exhibit the greatest beating amplitude when they were stimulated by an electrical pulse with the following parameters: 10-ms pulse width, 1–3-Hz stimulation frequency, 0.9-V/cm pulse amplitude, and a 0° angle between the electric field and the cellular axes.

Moreover, to demonstrate control of biosyncretic robots by the CEs, a biomimetic biosyncretic crawler actuated by myotubes, which were induced by the proposed CEs, was designed, fabricated, and controlled to move by different directional electrical pulses with the same electrical parameters optimized in the study investigating the response of single myotubes to differential electrical stimulation. The results showed that the biosyncretic crawler stimulated by an electric field direction of 0° angle demonstrated a higher speed (40.09 μm/s) than those stimulated with other directional electrical pulses with the same parameters (1-Hz pulse width, 0.9-V/cm pulse amplitude, and 10-ms pulse width). This result was consistent with the results obtained in the study examining single myotube responses to differential electrical stimulation.

The main object of this study was to demonstrate the advantages of the proposed CEs for C2C12 differentiation and the responses of single myotubes and also the feasibility of biosyncretic robot movement control with the CEs. Movement characteristics, such as the speed and direction of the biosyncretic robots, based on muscle tissues controlled by different electrical stimulation parameters, including stimulation frequency, electric field direction, and pulse amplitude and width, will be studied in depth in future studies based on this work. This study not only provides a potential tool for the development of biosyncretic robots but is also useful for muscle tissue engineering, regenerative medicine, and cardiomyocyte culture and stimulation.

Footnotes

Acknowledgments

This study was supported by the National Natural Science Foundation of China (Grant Nos. 61673372, 91748212, 61522312, and 61433017), the Key Research Program of Frontier Sciences, CAS (Grant No. QYZDB-SSW-JSC008), and the CAS/SAFEA International Partnership Program for Creative Research Teams.

Author Disclosure Statement

No competing financial interests exist.

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