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Phototactic guidance of a tissue-engineered soft-robotic ray

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Science  08 Jul 2016:
Vol. 353, Issue 6295, pp. 158-162
DOI: 10.1126/science.aaf4292
  • Fig. 1 Bioinspired concept design of the tissue-engineered ray.

    (A) A live Little skate, Leucoraja erinacea, swimming and (B) its musculoskeletal structure. (C to E) Tissue-engineered ray with (C) four layers of body architecture, (D) concept, and (E) phototactic control. Upon optical stimulation, the tissue-engineered ray induces sequential muscle activation via serpentine-patterned muscle tissues, generates undulatory locomotion, and sustains steady forward swimming. It changes direction by generating asymmetric undulating motion between left and right fins, modulated by light pulse frequency.

  • Fig. 2 Engineering solutions.

    (A) System-level design for skate (top) and tissue-engineered ray (bottom left) comparable with one penny and a two Euros coin (bottom middle and right). (B and C) Musculoskeletal (B) meso- and (C) micro-architecture of a skate, L. erinacea (top), is replicated in a tissue-engineered ray (bottom). Horizontal sections of the skate were stained with hematoxylin and eosin (top), and the engineered tissue was immunostained with a light-sensitive membrane protein, ChR2 (red, bottom left), sarcomeric α-actinin [red, (B), bottom right, and (C)], and nuclei (blue, (B), bottom right). (C) Orientation of the Z-lines (skate, black arrow and black triangles, top; and tissue-engineered ray, black arrow with gray distribution and white triangles, bottom). In both cases, Z-lines are perpendicular to the skeleton rays (pink arrows). (D) Muscle circuits with preprogrammed activation pattern. A point light stimulus directed at the front of the fins with 1.5 Hz frequency triggers the calcium wave that propagates along the predefined serpentine patterns. (E) Operating range of the muscle circuits. The circuit with intermediate serpentine pattern density represents the best tradeoff between contraction time reproducibility (SEM) and overlap with batoids’ operating range [Taeniura lymma (20) and Potamotrygon orbignyi (22), black symbols]. Black, red, and blue indicates muscle circuit without serpentine patterns and with intermediate and dense serpentine patterns, respectively. Each colored band indicates SEM of number of traveling waves.

  • Fig. 3 Kinematics and hydrodynamics.

    (A to D) PIV flow measurements highlight the production of alternated positive and negative vortices by the tissue-engineered ray. The viscosity associated with the relatively small Re is responsible for the dissipation of the vortex street in the wake. (E) Correlation between calcium activity and undulatory locomotion. (F) The moving distance during four strokes. (G) Comparison of swimming speed between the tissue-engineered rays stimulated by point and field stimulations. Undulatory locomotion produced by sequential muscle activation (point, 1.85 mm/s) improved swimming speed significantly compared with pulsatile propulsion generated by global muscle activation (field, 1.03 mm/s; matched pairs test, P = 0.014, n = 3 rays). Gray and red lines indicate the speed of individual rays and their average, respectively. (H and I) Out-of-plane fin deflection in both (H) a live stingray, P. orbignyi, and (I) a tissue-engineered ray (maximum amplitude, 2.54 ± 0.02 mm). (J) Comparison of swimming performance between tissue-engineered rays (n = 7 rays) and aquatic swimmers [batoid fish (20) and larval zebrafish (32)] following the scaling analysis (23). [Figure adapted from (23).] (K to N) PIV analysis of live Little skate, L. erinacea.

  • Fig. 4 Phototactic steering of the tissue-engineered ray through an obstacle course.

    (A) The ray completed a course that required complex coordination and maneuvering. Motion began with a forward protocol (at 0 s) to gain acceleration. A following left turn protocol allowed the ray to overcome forward momentum, making a left turn (at ~50 s). Next, another forward protocol was used to dissipate counterclockwise angular momentum and regain directionality (at ~100 s). While the ray made its way back to the other side of the obstacles, a final right turn protocol was given to make a right turn, winding the last obstacle. Grids, 1 cm. (B) Corresponding kinematic analysis relative to light frequency modulation protocols used for guidance.

Supplementary Materials

  • Phototactic guidance of a tissue-engineered soft-robotic ray

    Sung-Jin Park, Mattia Gazzola, Kyung Soo Park, Shirley Park, Valentina Di Santo, Erin L. Blevins, Johan U. Lind, Patrick H. Campbell, Stephanie Dauth, Andrew K. Capulli, Francesco S. Pasqualini, Seungkuk Ahn, Alexander Cho, Hongyan Yuan, Ben M. Maoz, Ragu Vijaykumar, Jeong-Woo Choi, Karl Deisseroth, George V. Lauder, L. Mahadevan, Kevin Kit Parker

    Phototactic guidance of a tissue-engineered soft-robotic ray

    Sung-Jin Park, Mattia Gazzola, Kyung Soo Park, Shirley Park, Valentina Di Santo, Erin L. Blevins, Johan U. Lind, Patrick H. Campbell, Stephanie Dauth, Andrew K. Capulli, Francesco S. Pasqualini, Seungkuk Ahn, Alexander Cho, Hongyan Yuan, Ben M. Maoz, Ragu Vijaykumar, Jeong-Woo Choi, Karl Deisseroth, George V. Lauder, L. Mahadevan, Kevin Kit Parker

    Phototactic guidance of a tissue-engineered soft-robotic ray

    Sung-Jin Park, Mattia Gazzola, Kyung Soo Park, Shirley Park, Valentina Di Santo, Erin L. Blevins, Johan U. Lind, Patrick H. Campbell, Stephanie Dauth, Andrew K. Capulli, Francesco S. Pasqualini, Seungkuk Ahn, Alexander Cho, Hongyan Yuan, Ben M. Maoz, Ragu Vijaykumar, Jeong-Woo Choi, Karl Deisseroth, George V. Lauder, L. Mahadevan, Kevin Kit Parker

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Table S1
    • Full Reference List

    Images, Video, and Other Media

    Movie S1
    Undulatory locomotion of a Little skate Lateral view movie of a Little skate, L. erinacea, swimming freely in a recirculating flow tank at a speed of 2.0 body lengths per second (~ 15 cm/s). High-speed video at 250 Hz revealed the motion of the wing and lateral view videos like this one are synchronized with dorsal view videos to allow three-dimensional reconstruction of wing motion. Scale bar, 2 cm.
    Movie S2
    Micro-CT scan of three dimensional structure of the tissue-engineered ray The micro-CT scan data was reconstructed along horizontal (middle), sagittal (right) and coronal (top) planes. The tissue-engineered ray has a three-dimensional structure of the dorsoventral disk, showing decreasing thicknesses from proximal to distal and from anterior to posterior. Red and blue lines indicate the locations of displayed sagittal and coronal planes, respectively, among the horizontal plane. Scale bars, 2 mm (horizontal plane) and 0.5 mm (sagittal and coronal plane).
    Movie S3
    Calcium propagation in muscle circuit with the serpentine pattern of choice The muscle circuit was fabricated by culturing rat cardiac cells on the fibronection (FN)-patterned elastomer substrate. The calcium propagation in the muscle circuit was monitored with a calcium indicator, X-Rhod-1. The synchronous optical pacing was applied at the anterior part of muscle circuits in both fins using a light source of 470nm wavelength with frequencies of 1, 1.5, 2, 2.5, and 3 Hz. The muscle circuit with the serpentine pattern of choice was designed to have 10 parallel sections of serpentine patterns on each fin. Parallel sections were separated by gaps at least 100 μm wide in order to prevent cardiac cells from spanning over the adjacent parallel sections. Calcium waves initiated by local optical stimulation at the anterior part propagated through the serpentine pattern along anterior-posterior axis. The movie is playing 5Ã- slower. Scale bar, 2 mm.
    Movie S4
    Calcium propagation of the muscle circuits without serpentine pattern and with a dense serpentine pattern Muscle circuits with different serpentine patterns were designed to compare calcium propagation with the serpentine pattern of choice (10 parallel sections, movie S3); one without the serpentine pattern (top) and the other with a dense serpentine pattern having 20 parallel sections (bottom). The calcium propagation of both muscle circuits was monitored with a calcium indicator, X-Rhod-1, and the synchronous optical pacing was applied at the anterior part of both fins with 470nm wavelength light and 1.5, 2, and 3 Hz frequencies. The calcium imaging shows that the number of waves present in the fins is controllable by varying pacing frequency or serpentine pattern density (number of parallel section serpentine patterns per given fin area). The movie is playing 5Ã- slower. Scale bar, 2 mm.
    Movie S5
    Asynchronous optical pacing for muscle circuit The muscle circuit with the serpentine pattern of choice was calcium-imaged with a calcium indicator, X-Rhod-1, while the anterior part of the muscle circuits on both sides were paced asynchronously with paired frequencies of 1/1.5 Hz (top) and 3/1.5 (bottom). The individual muscle circuits can be controlled independently by asynchronous pacing. The movie is playing 5Ã- slower. Scale bar, 2 mm.
    Movie S6
    Undulatory locomotion of the tissue-engineered ray paced at 1.5 Hz The undulatory locomotion of the tissue-engineered ray in Tyrode’s solution was recorded at 100 frames per second. The synchronous optical pacing at 1.5 Hz frequency was applied at the anterior part of both fins. The tissue-engineered ray swims with undulating motion of both fins in a rhythmic fashion following the optical pacing. The muscle contraction was initiated at the anterior of both fins and the muscular wave propagated from anterior to posterior. The tissue-engineered ray showed slower forward locomotion when the wave was propagating, after which it moved at its maximum speed when the wave reached the posterior part of the fin. In addition, the tissue-engineered ray displayed an asymmetric deflection pattern as the batoid fish show: deflection amplitude of fins increased until mid-disc and then remained constant through anterior-posterior axis, while amplitude increases across mediolateral fin axes. The movie is playing 5Ã- slower. Scale bar, 2 mm.
    Movie S7
    Comparison between undulation by point pacing and pulsatile propulsion by field pacing The locomotion of the same tissue-engineered ray was compared when two different stimulation methods were applied: the optical point stimulation was applied at the anterior part of both fins with 400-μm optical fibers, while the electrical field stimulation was applied to the entire muscle tissue of both fins by placing the ray between two 8 cm separated platinum electrodes. Both optical stimulation frequencies are 1.5 Hz. The optical point stimulation induced sequential muscle activation, generating undulation, while the electrical field stimulation induced global contraction of both fins, generating pulsatile propulsion. The optical point stimulation improved the swimming speed compared with the electrical field stimulation. The movie is playing in real-time. Grid, 1 cm.
    Movie S8
    Locomotion of the tissue-engineered ray with symmetrical body To evaluate the effect of an asymmetrical body shape on swimming efficiency, the tissue-engineered ray with the symmetrical body was designed as a control. The fin deflection of the tissue-engineered ray with the symmetrical body was significantly greater than that of the ray with the asymmetrical body (movie S6). As a consequence, the symmetrical tissue-engineered ray’s speed was reduced, compared to the asymmetrical tissue-engineered ray (movie S6). The optical stimulation frequency was 1.5 Hz. The movie is playing in real-time. Scale bar, 1 cm.
    Movie S9
    Locomotion of the tissue-engineered ray with various structural designs To evaluate the effect of gold skeleton and fin structures on swimming efficiency, the tissue-engineered rays without a gold skeleton (top left, 1Hz pacing), with a denser gold skeleton (top right, 2Hz pacing), and with thicker (bottom left, 2Hz pacing) and thinner (bottom right, 1Hz pacing) fins were designed as controls. The swimming speed of these tissue-engineered rays decreased dramatically. The thinner fins (9.18 μm) deflected with large amplitude because of the decreased stiffness of the substrate. When the deflection amplitude exceeds a critical value, the benefit of the displacing larger masses of fluid is counterbalanced and dominated by the increased drag associated with a larger effective frontal area, thus impairing forward speed. The movie is playing in real-time. Scale bar and grid, 1 cm.
    Movie S10
    Fluid motion of the tissue-engineered ray generated by undulatory locomotion The fluid motion generated by undulatory locomotion of the tissue-engineered ray was recorded with a particle image velocimetry (PIV) method at 100 frames per second. The synchronous optical pacing at 1.5 Hz frequency is applied at the anterior part of both fins. Silver coated hollow glass microspheres with 5-15 μm diameters and neutral buoyancy (1.08 g/mm density) were used to enhance the intensity of reflected light, but affected the viability of cardiac cells showing weak contraction. The PIV data shows that the undulating motion of the fins produced alternated positive and negative vortices. The movie is playing 5Ã- slower. Scale bar, 2 mm.
    Movie S11
    Fluid motion of the Little skate generated by undulatory locomotion Lateral view movie of a Little skate, L. erinacea, swimming freely in a laser light sheet within a recirculating flow tank at a speed of 2.0 body lengths per second (~ 15 cm/s). A continuous wave laser was used to illuminate particles in the flow and reveal body and wake flow patterns during locomotion. Scale bar, 2 cm.
    Movie S12
    Acceleration and deceleration of the tissue-engineered ray induced by optical stimulation The change in thrust was monitored when initiating (right) and seizing (left) the synchronous pacing with 1.5 Hz frequency. As soon as we optically stimulated the tissue-engineered rays that were initially moving in uncoordinated spontaneous motions, they reached a stable swimming speed with coordinated motion within 5 strokes. As soon as the stimulation was seized, the tissue-engineered rays stopped or decreased their speed with uncoordinated spontaneous motions. The movie is playing in real-time. Grid, 1 cm.
    Movie S13
    Sustainable directional locomotion with synchronous pacing The directional locomotion of the tissue-engineered ray was monitored while synchronous optical pacing was applied at the anterior part of both fins. The synchronous pacing induced a coordinated undulating motion of the fins and generated unidirectional locomotion (99.5 mm moving distance) with stable cruising speed. The movie is playing in real-time. Grid, 1 cm.
    Movie S14
    Undulation with various pacing frequency (slow motion video) The tissue-engineered ray was stimulated with synchronous optical pacing with varying pacing frequencies (1, 1.5, 2, 2.5 and 3 Hz). The locomotion of the tissue-engineered ray was recorded at 100 frames per second. The swimming speed of the tissue-engineered ray reached the maximum and minimum at the frequencies of 2 Hz and 1 Hz, respectively. The red line indicates the initial position of the tissue-engineered ray. The movie is playing 5Ã- slower. Scale bar, 2 mm.
    Movie S15
    Undulation with various pacing frequencies (video from a 45 degree angle view toward the dorsal of the fins) The tissue-engineered ray was stimulated with synchronous optical pacing with varying pacing frequencies (1.5, 2 and 3 Hz). The locomotion of the ray was recorded from a 45-degree angle view toward the dorsal of the fins, to monitor the propagation of wave best. The swimming speed of this tissue-engineered ray reached the minimum and maximum speed at frequencies of 3 Hz and 2 Hz, respectively. The movie is playing in real-time. Grid, 1 cm.
    Movie S16
    Undulation with various pacing frequencies (video from dorsal view) The tissue-engineered ray was stimulated with synchronous optical pacing with varying pacing frequencies (1, 1.5, 2 and 3 Hz). The locomotion of the ray was recorded from the dorsal view. This tissue-engineered ray reached the minimum and maximum speed at frequencies of 1 Hz and 1.5 Hz, respectively. The maximum speed is 3.2 mm/s. The movie is playing in real-time. Grid, 1 cm.
    Movie S17
    Counterclockwise and clockwise turns with asynchronous pacing (1.5/1 Hz) The counterclockwise and clockwise turns of the tissue-engineered ray were monitored while it was asynchronously paced with pairing frequencies (1.5 Hz and 1 Hz). The 1.5 Hz pacing generated faster locomotion than the 1Hz pacing, so the ray turned to 1Hz pacing direction. The movie is playing 2Ã- faster. Grid, 1 cm.
    Movie S18
    Counterclockwise turn with asynchronous pacing (1.5/3 Hz). The counterclockwise turn of the tissue-engineered ray was monitored while it was asynchronously paced with pairing frequencies (1.5 Hz and 3 Hz). The 1.5 Hz pacing applied on the left fin generated faster locomotion than the 3Hz pacing applied on the right fin, so the ray turned counterclockwise. The movie is playing 2Ã- faster. Grid, 1 cm.
    Movie S19
    Phototactic guidance of tissue-engineered ray. The obstacle course was designed to challenge maneuverability of the tissue-engineered ray. The three obstacles were placed with 7.5 cm distance which was longer than the average turning radius of the tissue-engineered ray, 4.5 cm. The direction of the tissue-engineered ray was controlled by combinational pacing protocols (synchronous: 1.5 Hz on both fins and asynchronous pacing: paired 1.5 and 3 Hz), which were rapidly manipulated by digital trigger signals to an LED light controller through the custom LabVIEW program (fig. S14C). The tissue-engineered ray completed the obstacle course by generating counterclockwise and clockwise turns as well as directional locomotion. The movie is playing 5Ã- faster. Grid, 1 cm.
    Movie S20
    Durability measurements of a tissue-engineered ray The locomotion of the tissue-engineered ray was monitored for 8 days. After 5 days in culture, the ray was released (top left). The swimming speed was found to increase during two days (a total of 7 days in culture, top right), to then stabilize within 80% of initial speed for up to six days (a total of 11 days in culture, bottom left). After this time, substantial performance degradation was observed (a total of 13 days in culture, bottom right). The locomotion of the tissue-engineered ray was recorded at 60 frames per second while the ray was stimulated with synchronous optical pacing at 1.5 Hz frequency. The movie plays in real-time. Grid, 1 cm.

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