Three-dimensionally printed biological machines powered by skeletal muscle

Abstract

Combining biological components, such as cells and tissues, with soft robotics can enable the fabrication of biological machines with the ability to sense, process signals, and produce force. An intuitive demonstration of a biological machine is one that can produce motion in response to controllable external signaling. Whereas cardiac cell-driven biological actuators have been demonstrated, the requirements of these machines to respond to stimuli and exhibit controlled movement merit the use of skeletal muscle, the primary generator of actuation in animals, as a contractile power source. Here, we report the development of 3D printed hydrogel "bio-bots" with an asymmetric physical design and powered by the actuation of an engineered mammalian skeletal muscle strip to result in net locomotion of the bio-bot. Geometric design and material properties of the hydrogel bio-bots were optimized using stereolithographic 3D printing, and the effect of collagen I and fibrin extracellular matrix proteins and insulin-like growth factor 1 on the force production of engineered skeletal muscle was characterized. Electrical stimulation triggered contraction of cells in the muscle strip and net locomotion of the bio-bot with a maximum velocity of ∼ 156 μm s(-1), which is over 1.5 body lengths per min. Modeling and simulation were used to understand both the effect of different design parameters on the bio-bot and the mechanism of motion. This demonstration advances the goal of realizing forward-engineered integrated cellular machines and systems, which can have a myriad array of applications in drug screening, programmable tissue engineering, drug delivery, and biomimetic machine design.

Keywords: bioactuator; stereolithography.

Fig. 1.

Fabrication of hydrogel structures and formation of 3D muscle strips. (A) Computer-aided design software was used to design bio-bots with desired dimensions. Measurements are in millimeters. (B) An SLA was used to polymerize hydrogel structures in an additive process. (C) The cell–matrix solution consisted of C2C12 skeletal muscle myoblasts, matrix proteins (fibrin or collagen I), and Matrigel. (D) Fabricated bio-bot (i, side view) and holder (ii, top view). Cell–matrix solution was pipetted into a polymerized holder containing the bio-bot structure (iii and iv, side view). Cells and matrix compacted around the pillars to form a solid muscle strip (v and vi, top view with immunostaining for MF-20, green, and DAPI, blue) and the device was released from the holder (vii, side view). All scale bars, 1 mm.

Fig. 2.

Biological characterization of muscle strips. (A) Longitudinal fibrin muscle strip slice (day 9). Scale bar, 500 μm. (Inset) Scale bar, 200 μm. (B) Myotubes aligned along the fibrin muscle strip perimeter. Scale bar, 200 μm. (C) Longitudinal collagen muscle strip slice (day 14). Scale bar, 500 μm. (D) Multinucleated myotubes in the collagen muscle strip. Scale bar, 50 μm. (E–H) H&E staining of fibrin muscle strip sections (day 9). All scale bars, 200 μm. (E) Longitudinal and (F) transverse muscle strip sections. (G) Quantitative analysis of transverse sections demonstrated 75% of cells within 200 μm from the edge (n = 8). (H) Perimeter of transverse section of the muscle strip.

Fig. 3.

Analysis of bending and passive tension. (A) Over time, bio-bot beams polymerized with varying energy doses exhibited different bending profiles. Scale bar, 1 mm. (B) Elastic modulus of the beam as a function of energy dose (R² > 0.99, n = 5). Increasing polymerization energy resulted in a higher modulus and less bending. (C) Finite-element analysis simulation demonstrating global deflection of the bio-bot beam and pillars on day 8. (D) Passive tension in the muscle strip as a function of time, modulus, and addition of IGF-1 (n = 3, 489.3 kPa; n = 4, others). Increasing beam stiffness resulted in a higher muscle strip tension. (Inset) Addition of IGF-1 significantly increased the average tension by 70.7% (for 319.4-kPa stiffness) over time period shown. Statistics represent one-way ANOVA and Tukey’s test, with ** = P < 0.001. All data are presented as mean ± SD (shaded region in D).

Fig. 4.

Electrical control and pacing. (A) During stimulation, bio-bots were placed standing up on the surface of the dish, with the muscle strip’s longitudinal axis parallel to the electrodes and perpendicular to the applied field. (B) IGF-1-supplemented bio-bots were reliably paced at stimulation frequencies of 1, 2, or 4 Hz. Bio-bots not supplemented with IGF-1 did not respond to stimulation at any frequency in the observed time period. (C) A Kelvin–Voigt viscoelasticity model was used to extract active tension generated by the muscle strip in response to electrical stimulation. (D) Muscle strip time-varying active tension was calculated for a bio-bot undergoing varying electrical stimulation (0, 1, 2, 3, 4, 0 Hz) during one experiment, and followed a positive force–frequency relationship.

Fig. 5.

Simulation and movement profiles of symmetric and asymmetric bio-bots supplemented with IGF-1. (A) von Mises stress of a symmetric and asymmetric bio-bot. The range of one contraction is shown over a period of 10 s (10 “steps” at 1-Hz stimulation). (B and E). Top-view time-lapse images of the symmetric (B) and asymmetric (E) bio-bot’s movement. All scale bars, 1 mm. (C and F) Relative ratios of pillar movement over time during 1-Hz stimulation. The bio-bots moved in the direction of greater pillar movement during contraction. (D) Displacement (μm) of a symmetric bio-bot at 1-Hz stimulation during a 25-s interval when contraction produced maximum displacement along the surface. (G) Active tension force of the asymmetric bio-bot during locomotion. (H) The increased number of contractions with increasing stimulation frequency within a given time period resulted in an increased average velocity of the asymmetric bio-bot. (Inset, Left) Velocity (gray) and total displacement. (Inset, Right) Zoom of boxed region.