Cardiomyocyte-Driven Structural Color Actuation in Anisotropic

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Cardiomyocyte-Driven Structural Color Actuation in Anisotropic Inverse Opals Yixuan Shang, Zhuoyue Chen, Fanfan Fu, Lingyu Sun, Changmin Shao, Wei Jin, Hong Liu, and Yuanjin Zhao ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b08230 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 20, 2018

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Cardiomyocyte-Driven Structural Color Actuation in Anisotropic Inverse Opals Yixuan Shang1,2, Zhuoyue Chen 2, Fanfan Fu 2, Lingyu Sun 2, Changmin Shao 2, Wei Jin*,1,2, Hong Liu*,2, Yuanjin Zhao*,1,2 1 Department

of Cardiology, Institute of Cardiovascular Diseases, Ruijin Hospital, Shanghai Jiao

Tong University School of Medicine, Shanghai 200025, China 2

State Key Laboratory of Bioelectronics, School of Biological Science and Medical

Engineering, Southeast University, Nanjing 210096, China. Email: [email protected] (W.J.); [email protected] (H.L.); [email protected] (Y.J.Z.)

ABSTRACT: Biohybrid actuators composed of living tissues and artificial materials have attracted increasing interest in recent years because of their extraordinary function of dynamically sensing and interacting with complex bioelectrical signals. Here, a compound biohybrid actuator with self-driven actuation and self-reported feedback is designed based on an anisotropic inverse opal substrate with periodical elliptical macropores and a hydrogel filling. The benefit of the anisotropic surface topography and high biocompatibility of the hydrogel is that the planted cardiomyocytes could be induced into a highly ordered alignment with recovering autonomic beating ability on the elastic substrate. Because of the cell elongation and contraction during cardiomyocyte beating, the anisotropic inverse opal substrates undergo a synchronous cycle of deformation actuations, which can be reported as corresponding shifts of their photonic band gaps and structural colors. These self-driven biohybrid actuators could be used as elements for the construction of soft-bodied structural color robot, such as a biomimetic guppy with a swinging tail.

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Besides, with the integration of a self-driven biohybrid actuator and microfluidics, the advanced heart-on-a-chip system with the feature of microphysiological visuality has been developed for integrated cell monitoring and drug testing. This anisotropic inverse opal-derived biohybrid actuator could be widely applied in biomedical engineering. KEYWORDS: structural color; actuator; heart on a chip; cardiomyocyte; inverse opal; microfluidics

Actuation, derived from traditional mechanical terminology, has been extended to be an interdisciplinary research area and has achieved extensive development in various fields.1,

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Inspired by natural examples from the animal world, a large number of actuators have been designed for complex applications that are capable of responding to stimulations such as light, electrical, magnetic, heat, pH, and moisture changes.3-5 In particular, by integrating synthetic structures that are appropriately engineered living biosystems, resultant biohybrid actuators with excellent responsiveness to complicated environmental signals could be created. These biohybrid actuators have been a source of inspiration in the exploration of surgical robots, smart skin, muscle tissue, sensing devices, etc.7-13 Most existing biohybrid actuators that depend on additional stimulation are intricate in their design and implementation and have a high response time. In addition, the current response forms of biohybrid actuators are mainly held up as a shape deformation, which gravely reduce intuitiveness. Therefore, there is a strong demand to exploit maneuverable systems with an efficient bioactuator and visualized feedback. In this paper, we present an integrated hybrid anisotropic inverse opal bioactuator with living tissue and with self-driven capability and self-reported structural color feedback (Figure 1). Inverse opals are a kind of structural material with three-dimensional (3D) and periodically


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arranged macropores that can be negatively replicated from colloidal crystal array templates.14-16 Because of their periodic macroporous structure, the inverse opal materials can strongly modulate electromagnetic waves and manipulate the propagation of photons in their photonic band gap (PBG), which gives rise to their vivid structural color properties.17,

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In particular, when the

inverse opals are composed of soft polymers, their volume or shape could change under different stimuli, leading to shifts in their structural colors or PBG spectra. This property gives the inverse opal materials significant technological value in self-reported molecular detection and physical sensing.19-26 However, most of the recently self-reported inverse opals are based on statically isotropic structures, which restrict their potentials in biological and biomimetic applications referring to tissue arrangements. Herein, we developed a designed anisotropic inverse opal with stretch-derived periodical elliptical macropores for tissue assembling and for constructing a biohybrid actuator. It was found that the cultured cardiomyocytes could be induced into a highly ordered alignment with recoverable autonomous beating on the hydrogel-filled anisotropic inverse opal substrates. The cardiomyocyte beating processes were accompanied by cell elongation and contraction; the anisotropic inverse opal substrates underwent a synchronous cycle of self-driven deformation actuations, which could be described as corresponding shifts in their PBGs and structural colors. Based on this self-driven biohybrid actuator, an elaborate soft structural color robot, such as a biomimetic guppy with a swinging tail, could be created. In addition, by simply integrating this biohybrid actuator into microfluidics, a combined visualizable microphysiological heart-on-a-chip system was constructed for integrated cell monitoring and drug testing. These features of the anisotropic inverse opal-derived biohybrid actuators indicated their wide perspective for biomedical applications.


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RESULTS AND DISCUSSION In a typical experiment, the inverse opal substrates were derived from colloidal silica crystal templates (Figure S1, Supporting Information). In this process, such templates were first obtained by a vertical deposition method to gain highly ordered arrays and then sintered at 500 °C to increase the compactness of the silica nanoparticles. Because of its excellent flexibility and stability, a poly(vinylidene fluoride) (PVDF) material dissolved in dimethylformamide (DMF) solution was then chosen and dropwise filled into the interpenetrating pores of the colloidal silica crystal templates, which was further solidified with DMF evaporation. Next, PVDF inverse opal substrates with 3d-ordered macroporous arrays were obtained by etching the silica nanoparticles. To obtain a functionalized anisotropic substrate, the PVDF substrates underwent a final uniaxial stretch to gain periodical elliptical macropores. Consistent with the isotropic inverse opals, the anisotropic substrate with stretch-derived periodical elliptical macropores possessed the capability of modulating photons in PBGs. Thus, certain light wavelengths in the PBG were blocked and reflected by the anisotropic substrate. In particular, the main characteristic reflection peak of the substrate with specific structural color can be calculated by the Bragg–Snell equation: λ = 1.633D(n2average - cos2θ)

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(1)

where D is the distance to the diffracting plane, naverage refers to the average refractive index of the substrate, and θ is the Bragg glancing angle of incidence of the light falling on the nanostructures. Equation (1) shows that there are several approaches to regulating the structural colors of the substrate, such as changing the diffracting plane spacing D or the Bragg glancing angle θ. According to Equation (1), when the stretching degree increases, D decreases along with it in the stretching direction, and the structural color of the stretched inverse opal substrate exhibits blue


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shifts at a fixed observation angle (Figure S2, Supporting Information). Once the stretching degree is determined, together with the diffracting plane spacing D, only the glancing angle refers to the change in structural color of these substrates. To further confirm the relationship between θ and structural color, the stretched substrate was fixed on one side, and its reflection peak was measured on the other side. When the incident angle increased from 0° to 60°, there was a blue shift in the structural color of the substrate from red to blue, and also the wavelength diminished from 660 to 450 nm (Figure S3, Supporting Information). These optical properties laid the foundation for an efficient visualized feedback of the designed biohybrid actuator. Firstly, inverse opals with different stretching degrees were constructed and characterized to quantify their optical properties and surface morphologies (Figure 2). Generally, as the stretching degree increased, the round holes of the inverse opal substrate were gradually squashed into ovals, causing a descending diffracting plane spacing D (Figure 2a). In addition, a methacrylated gelatin hydrogel was used to cover the substrate and infused into the macropores of the substrate for better biocompatibility and for cardiac facilitation (Figure S4, Supporting Information). We found that the structural color of the stretched inverse opal substrate presented blue shift as expected, but a red shift of the reflection peak occurred after hydrogel infusion (Figure 2b and c). However, the hydrogel-covered substrates maintained excellent optical properties as photonic crystals. To investigate the potential of the elliptical macropores affecting cells and tissues, inverse opals with different stretching degrees were used as substrates for a rat cardiomyocyte culture (Figure 3). It could be observed that the cardiomyocytes, which were stained by 4',6-diamidino-2phenylindole (DAPI) for the nucleus and by phalloidine for F-actin, showed a narrow angular distribution on stretched substrates and spread widely along the long axis of the elliptical topography, especially on the substrate that was stretched six times (Figure 3a and b). This effect


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demonstrated an aligned orientation of cardiomyocytes on the anisotropic inverse opals, contrasting with the disordered distribution on the isotropous one (Figure S5, Supporting Information). As expected, cardiomyocytes cultured on common inverse opal substrates presented as out-of-order with spherical shapes and random orientations, whereas the cardiomyocytes displayed a conspicuous alignment along the stretching direction on the substrates that were stretched six times; additionally, cell pseudopods extended to the longest and generated compact cell-cell connections. To obtain further the response of cardiomyocytes to different stretched substrates accurately, the angle between the cardiomyocyte growth direction and the stretching direction of the inverse opals was measured and analyzed (Figure 3c). The cell orientation angles, which ranged from 0° to 90°, were quantified by the Image J software package. The 0° orientation angle indicated that the long axis of the cardiomyocytes was consistent with the stretching direction of the substrates, whereas the angle of 90° represented the orthogonal direction between them. By counting the number of cardiomyocytes with an incremental orientation angle interval of 10°, it was demonstrated that there was no significant distribution of cell orientation on the isotropous substrate. However, nearly 70% of the cardiomyocytes showed a striking orientation within 30° to the substrate that was stretched three times, and this value progressively increased to almost 100% for the anisotropic substrates that were stretched six times. These values demonstrated that the inverse opals with stretch-derived elliptical topography could effectively induce cardiomyocytes into a stretching orientation. Because of the different morphology of cardiomyocytes on different inverse opals, the autonomous beating frequencies of the cultured cell were also analyzed for fundamental cardiac evaluation. The cardiomyocytes cultured on the inverse opals usually restored autonomic beating


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after two days and displayed the most intense consistent beating behavior on day 4 (Figure 3d). In this culture period from day 4 to day 9, the beat frequencies of the cardiomyocytes on the stretched substrates decreased by around 40%, compared with a 45% decrease of the unstretched substrates during the same period. In addition, the wavelength shifts of the structural color of several substrates actuated by cultured cardiomyocytes were counted (Figure S6, Supporting Information), and they ranged from 30 (the isotropous substrate) to 100 nm (the anisotropic substrate stretched six times). Thus, compared to former hierarchy films, the substrate which needed complex replication, the present stretched substrate demonstrated a prominent orientation induction of cardiomyocytes, and its deformation with a structural color change by cardiac autonomous behavior provided a visualized approach for self-sensing biohybrid actuators. To demonstrate the self-driven deformation property, a soft-bodied structural color robot was laid out to imitate a swimming guppy (Figure 4a and b). An elliptical macroporous substrate was shaped as a guppy with the self-actuating property at the tail. With a joint connection, the guppyshaped substrate was composed of a fixed body with an invariable structural color and a flexible tail with self-assembled highly oriented cardiac tissue, which displayed corresponding synchronous contractions and relaxations, and could self-wave at a certain frequency in the medium (Movie S1, Supporting Information). When the cardiomyocytes shrank, the blue shift that appeared on the guppy tail from the edge spread gradually inward following the incremental bending angle (Figure 4c and d). It was also found that each position of the bionic guppy corresponding to each degree had a distinctive structural color fingerprint according to Equation (1). This indicated that the self-driven bionic robots could gain self-feedback and had enormous potential with multiple types of cells integrated in biomaterials, sensing, and other fields.


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To implement further these bio-actuator applications, a heart-on-a-chip system integrated with biohybrid actuators and microfluidics was constructed (Figure 5). As an artificial organ, an organon-a-chip system is a multichannel 3D microfluidic cell culture chip, which could mimic the activities, mechanics, and physiological response of entire organs and organ systems. This biomimetic microfluidic system could revolutionize many fields, including the development of human in vitro models for healthy or diseased organs, toxicity screening, and target discovery, and could potentially replace animal testing.26-32 In this heart-on-a-chip system, the microfluidics contained bifurcated channels for numerous tributaries to provide an engineered anisotropic ventricular myocardium with a homogeneous medium for culture and drug evaluation (Figure 5a and b). As shown above, the elliptical macropores of the substrate contributed to the concentration of cardiac contractility, so that the substrate would bend along the anisotropic organization in turns (Figure 5c). This process was self-reported by a blue shift in the structural color and reflection peak, as a result of the Bragg glancing angle decreasing in the anisotropic inverse opal. With a specific reflection peak fingerprint, the integrative system could be used to investigate the cellular behavior of cardiomyocytes with different stimulations. For demonstration, different concentrations of isoproterenol were injected into microfluidics, providing different conditions for cardiomyocytes. It was observed that the structural color of the annotated position on the substrates changed from red to orange under normal conditions, whereas the color changed to green when 1μM isoproterenol was infused (Movie S2, Supporting Information). With incremental isoproterenol concentration, such a degree of blue shift manifested a synchronous increase in Figure 5d-e. The beat frequency of the cardiomyocytes presented a positive, time-varying response for isoproterenol concentrations, consistent with the actual reaction of isoproterenol in vivo (Figure 5d and f). The visualizable microphysiological


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chip also possessed an additional piezoelectric property because of the PVDF substrate, which presented broad prospects for optoelectronic monitoring platforms, biological research, and drug screening.

CONCLUSION In summary, we have developed a structural color bioactuator driven by assembling engineered cardiomyocytes on elliptical topological PVDF inverse opals. Because of the high biocompatibility and the outstanding orientation-regulating capacity of the composite substrates, the cardiomyocytes could rapidly restore their autonomous orderly beating and drive the synchronous deformations of the substrate. To simulate basic behaviors of living organisms, we constructed a substrate with a composite morphology that resembled a guppy waving its tail at a certain frequency in the medium, like a swimming fish. It was demonstrated that inverse opals driven by cardiomyocytes could provide certain possibilities for biohybrid actuators with self-reporting ability. In addition, we designed a visualizable microphysiological organ-on-a-chip system with integration of biohybrid actuators and microfluidics, for cardiomyocyte monitoring and drug testing. Compared with other substrates in bioactuators, the elliptical architecture of the inverse opal acted as a critical design component for ordered beating and output magnifying because of its extremely effective cardiomyocyte-induced orientation. We demonstrated that such subtle cellular forces could be characterized by vivid structural color changes or reflection spectra shifts of the designed heart-on-a-chip. It is foreseeable that with the integration of optogenetic control strategies and visual intelligent bioactuators, real-time detection and a self-feedback system could be constructed eventually. Therefore, such anisotropic inverse opal-derived biohybrid actuators present significant potential for a wide range of biomedical applications.


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EXPERIMENTAL SECTION Materials. Monodisperse silicon dioxide spheres with diameters of 660 nm and 410nm were purchased from Nanjing Nanorainbow Biotechnology Co., Ltd. PBS solution (pH 7.4) was freshly prepared. Polyvinylidene Fluoride (PVDF) was purchased from Sigma Aldrich (St. Louis, MO). N, N-dimethylformamide (DMF) was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Hydrofluoric acid (HF) and ethanol were purchased from Aladdin, Shanghai. Gelatin hydrogel was self-prepared. Cellulose dialysis membranes [molecular weight cutoff (MWCO), 8000-14,000] for hydrogel synthesis were acquired from Shanghai Yuanye Biotechnology Corporation (Shanghai, China). Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12), Hanks' Balanced Salt Solution (HBSS 1X) and fetal bovine serum (FBS) were purchased from Life technologies (USA). Penicillin-streptomycin and isoproterenol were obtained from Gibco (USA). 5-bromo-2-deoxyuridine (BrdU) was obtained from Sigma Aldrich (St. Louis, MO). Collagenase type 2 was purchased from Worthington. Alexa Fluor 488 Phalloidin and 4', 6-diamidino-2-phenylindole (DAPI) were obtained from Life Technologies (USA). Deionized water with a resistivity of 18 MΩ·cm-1 was acquired from a Millipore Milli-Q system. Preparation of PVDF inverse opal films. These Opal films composed of monodisperse silica spheres were deposited on a glass by using vertical deposition method. Then, the glass with the silica nanostructures was calcined at 500°C for 4 hours to improve their mechanical strength, and the silica colloidal crystal templates were thus obtained. After that, 0.04 g/ml PVDF/DMF solution was then infiltrated the opal template. Last, the inverse opal PVDF films were obtained by etching (2 wt % hydrofluoric acid) the silica nanoparticles. Afterwards, the film was uniaxially stretched


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by Vernier caliper (Masterproof, Germany) to get different degrees elongation (three and six times, respectively) in an 80 °C water bath. The gelatin pregel solution with a concentration of 0.05 g/ml was covered on the stretched PVDF film and infused into the nanopores of the film. Finally, the pregel solution was exposed into UV light and polymerized in the nanopores of films. The biomimic swimming guppy and the heart-on-a-chip system were both based on the six times stretched PVDF substrates. Extraction of rat cardiomyocytes. The 2-3 days neonatal Sprague Dawley rat pups were supplied by the department of comparative medicine of Jinling Hospital (Nanjing, China). The rat pups were fixed with tweezers and their ventral faces were cut open along the chest to remove the heart. The removed heart was placed in a Petri dish containing D-Hanks solution, washed three times, and removed the tissue at the edge of the heart and possible clots in the heart. Then the tissue mass of 0.5-1mm³ size was produced by separation and shearing, and transferred to an HBSS (0.02% trypsin, 0.02% pancreatin, and 0.05% collagenase) for enzymes digesting method. The mixture solution with cardiomyocytes and cardiac fibroblasts was added into a DMEM/F-12 medium (20% FBS) solution. Then, the digested cells were collected by centrifugation (5 min) at 1250 rpm. The supernatant was discarded and about 15 mL of DMEM/F-12 medium (10% FBS and 0.1mM BrdU) was added and homogenized to prepare a cell suspension. Subsequently, the cell suspension was transferred to a 75 cm² cell culture flask and cultured for 1.5 hours (37 °C; CO2, 5%). Finally, the resulting cell suspension inoculated into the desired plate, cell culture flask or material surface, according to the normal operation to continue the culture of cardiomyocytes. Cells cultured and image. Cardiomyocytes were cultured and passaged in the DMEM/F-12 medium containing 10% FBS and 1% penicillin-streptomycin (37 °C; CO2, 5%). The PVDF inverse opal films were first sterilized with one-night UV light exposure and washed with sterile


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HBSS for three times. Then the obtained cardiomyocytes were seeded on the inverse opal films in the DMEM/F-12 solution (10% FBS, 1% penicillin-streptomycin and 0.1mM BrdU) and cultured for three days. Then, the cardiomyocytes were continued cultured with the normal medium. To demonstrate the biocompatibility, different substrates including the multi-well plate, the PVDF substrates after plasma treatment and the PVDF substrates covered with the methacrylated gelatin hydrogel were measured by the MTT cell activity assay. After the cardiomyocytes adhered on these substrates, the MTT solution (0.5 g MTT in 100 mL PBS) was added in the culture medium at the ratio of 1:10 every 48 h and incubated at 37 °C for 4h. Then, the formazan crystal in the cardiomyocytes were dissolved by DMSO for OD value measurement. The films with cardiomyocytes were immunostained after 6 days. At first, films were fixed for 30 min in 4% (v/v) paraformaldehyde-phosphatebuffered saline (PBS) solution and then permeabilized with 0.3% (v/v) Triton X-100-PBS solution for 30 min. After that, samples were counterstained with Alexa Fluor 488 phalloidin (1:400 dilution) for F-actin staining. Subsequently, 4', 6-diamidino-2phenylindole (DAPI) diluted in PBS solution (1:1000 dilution) was used for the nuclei. In each step, the samples were washed with PBS solution at least three times. Confocal microscopy images were acquired using a Zeiss LSM700 laser scanning microscope (Zeiss, Heidenheim, Germany). To characterize the morphology of cardiomyocytes, we washed the samples repeatedly and dehydrated them with gradient ethanol (20, 40, 60, 80, and 100%) before SEM imaging. Characterization of the PVDF substrates and cardiomyocytes. Reflection spectra were measured at a fixed glancing angle, using an optical microscope equipped with a fiber-optic spectrometer (Ocean Optics; USB2000-FLG) and an angular resolution spectrometer (Idea Optics; R1). SEM images of samples were taken with a scanning electron microscope (Hitachi S-3000N). Confocal microscopy images were obtained using a Zeiss LSM700 laser-scanning microscope


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(Zeiss, Heidenheim, Germany). Microscopy images of the samples were obtained with an optical microscope (Olympus BX51) equipped with a CCD camera (Media Cybernetics Evolution MP5.0) and a digital camera (Canon5D Mark II).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], *E-mail: [email protected], *E-mail: [email protected].

ACKNOWLEDGMENT Funding Sources: This work was supported by the National Natural Science Foundation of China (grant nos. 51522302, 81670266, and 21473029), the National Science Foundation of Jiangsu (grant no. BE2018707), and the Science and Technology Commission of Shanghai Municipality (17140902500).

ASSOCIATED CONTENT Supporting Information Available Schematic diagram and SEM images of the generation process of the inverse opal substrate.; The optical properties of inverse opal substrates; Dynamic reflectance wavelengths of the stretched inverse opal substrate when incident angle increased from 0 until 60; MTT assays of cardiomyocytes cultured on multi-well plates, PVDF substrates after plasma treatment and PVDF substrates covered with the methacrylated gelatin hydrogel for 2 days, 4 days, and 6 days,


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respectively; Shift of the reflection spectra of the inverse opals with different stretching degrees; Optical images of a guppy shaped soft-bodied robot with a self-wave tail; The varing structural color of biohybrid actuator in a heart-on-a-chip system under different conditions. This material is available free of charge via the Internet at http://pubs.acs.org. Conflict of Interest: The authors declare no competing financial interests.

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13. Xu, B. Z.; Lin, X. D.; Li, W.; Wang, Z. X.; Zhang, W. C.; Shi, P. Cell Generator: a SelfSustaining Biohybrid System Based on Energy Harvesting from Engineered Cardiac Microtissues. Adv. Funct. Mater. 2017, 27, 1606169. 14. Lu, J.; Zou, X.; Zhao, Z.; Mu, Z. D.; Zhao, Y. J.; Gu, Z. Z. Cell Orientation Gradients on an Inverse Opal Substrate. ACS Appl. Mater. Interfaces 2015, 7, 10091-10095. 15. Ding, W. C.; Li, Y. J.; Xia, H. B.; Wang, D. Y.; Tao, X. T. Synthesis of Janus Particles via Strain-Driven Microphase Separation and Their Assembly into Nanoscale Vesicles. ACS Nano 2014, 8, 11206-11213. 16. Zhang, Y. S.; Zhu, C. L.; Xia, Y. N. Inverse Opal Scaffolds and Their Biomedical Applications. Adv. Mater. 2017, 29, 1701115. 17. Kuang, M. X.; Wang, J. X.; Jiang, L. Bio-Inspired Photonic Crystals with Superwettability. Chem. Soc. Rev. 2016, 45, 6833-6854. 18. Fu, F. F.; Chen, Z. Y.; Zhao, Z.; Wang, H.; Shang, L. R.; Gu, Z. Z.; Zhao, Y. J. Bio-Inspired Self-Healing Structural Color Hydrogel. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 5900-5905. 19. He, L.; Janner, M.; Lu, Q. P.; Wang, M. S.; Ma, H.; Yin, Y. D. Magnetochromatic Thin-Film Microplates. Adv. Mater. 2015, 27, 86-92. 20. Qin, M.; Huang, Y.; Li, Y. N.; Su, M.; Chen, B. D.; Sun, H.; Yong, P. Y.; Ye, C. Q.; Li, F. Y.; Song, Y. L. A Rainbow Structural-Color Chip for Multisaccharide Recognition. Angew. Chem. Int. Ed. 2016, 55, 6911-6914. 21. Fu, F. F.; Shang, L. R.; Zheng, F. Y.; Chen, Z. Y.; Wang, H.; Wang, J.; Gu, Z. Z.; Zhao, Y. J. Cells Cultured on Core–Shell Photonic Crystal Barcodes for Drug Screening. ACS Appl. Mater. Interfaces 2016, 8, 13840-13848.


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22. Lee, S. Y.; Choi, J.; Jeong, J. R.; Shin, J. H.; Kim, S. H. Magnetoresponsive Photonic Microspheres with Structural Color Gradient. Adv. Mater. 2017, 29, 1605450. 23. Liu, C. H.; Ding, H. B.; Wu, Z. Q.; Gao, B. B.; Fu, F. F.; Shang, L. R.; Gu, Z. Z.; Zhao, Y. J. Tunable Structural Color Surfaces with Visually Self-Reporting Wettability. Adv. Funct. Mater. 2016, 26, 7937-7942. 24. Kim, S. H.; Park, J. G.; Choi, T. M.; Manoharan, V. N.; Weitz, D. A. Osmotic-PressureControlled Concentration of Colloidal Particles in Thin-Shelled Capsules. Nat. Commun. 2014, 5, 3068. 25. Wang, M. S.; Yin, Y. D. Magnetically Responsive Nanostructures with Tunable Optical Properties. J. Am. Chem. Soc. 2016, 138, 6315-6323. 26. Fu, F. F.; Shang, L. R.; Chen, Z. Y.; Yu, Y. R.; Zhao, Y. J. Bioinspired Living Structural Color Hydrogels. Sci. Robot. 2018, 3, eaar8580. 27. Hou, X.; Zhang, Y. S.; Santiago, G. T.; Alvarez, M. M.; Ribas, J.; Jonas, S. J.; Weiss, P. S.; Andrews, A. M.; Aizenberg, J.; Khademhosseini, A. Interplay Between Materials and Microfluidics. Nat. Rev. Mater. 2017, 2, 17016. 28. Lind, J. U.; Yadid, M.; Perkins, I.; O'Connor, B. B.; Eweje, F.; Chantre, C. O.; Hemphill, M. A.; Yuan, H. Y.; Campbell, P. H.; Vlassak, J. J.; Parker, K. K. Cardiac Microphysiological Devices with Flexible Thin-Film Sensors for Higher-Throughput Drug Screening. Lab Chip 2017, 17, 3692-3703. 29. Wang, J.; Zou, M. H.; Sun, L. Y.; Cheng, Y.; Shang, L. R.; Fu, F. F.; Zhao, Y. J. Microfluidic Generation of Buddha Beads-Like Microcarriers for Cell Culture. Sci. China Mater. 2017, 60, 857-865.


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30. Grosberg, A.; Alford, P. W.; McCain, M. L.; Parker, K. K. Ensembles of Engineered Cardiac Tissues for Physiological and Pharmacological Study: Heart on a Chip. Lab Chip 2011, 11, 4165-4173. 31. Shang, L. R.; Cheng, Y.; Zhao, Y. J. Emerging Droplet Microfluidics. Chem. Rev. 2017, 117, 7964-8040. 32. Lind, J. U.; Busbee, T. A.; Valentine, A. D.; Pasqualini, F. S.; Yuan, H. Y.; Yadid, M.; Park, S. J.; Kotikian, A.; Nesmith, A. P.; Campbell, P. H.; Vlassak, J. J.; Lewis, J. A.; Parker, K. K. Instrumented Cardiac Microphysiological Devices via Multimaterial Three-Dimensional Printing. Nat. Mater. 2017, 16, 303-308.


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Figure 1 Schematic diagram of the biohybrid actuator with the features of self-driven actuation and self-reported structural color feedback. (a) Fabrication of the biohybrid actuator: the stretchderived anisotropic inverse opal substrates were filled with gelatin hydrogel and planted with cardiomyocytes; (b) The corresponding shifts of substrates’ structural colors actuated by cardiomyocytes.


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Figure 2 Topological and optical property of actuation substrate. (a, b) SEM and optical images of the inverse opal substrate with different stretching degrees: (i) unstretched substrate, (ii) three times stretched substrate, (iii) six times stretched substrate, (iv) six times stretched substrate covered with gelatin hydrogel; (c) Reflection peaks of inverse opal substrates assembled by using 660 nm silica nanoparticles: the red line is correspond to (i), the orange line is correspond to (ii), the blue line is corresponds to (iii), the green line is correspond to (iv). Scale bars are 1μm in (a) and 2 mm in (b).


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Figure 3 The effect of anisotropic surface topography on cardiomyocytes. (a) Schematic diagram of cardiomyocytes cultured on inverse opal substrates with different stretching degrees: (i) 0 times, (ii) 3 times, (iii) 6 times; (b) Confocal laser scanning microscopy (CLSM) images of the cardiomyocytes cultured on inverse opal substrates with different stretching degrees: (i) 0 times, (ii) 3 times, (iii) 6 times, (iv) 6 times; (c) Orientation angle frequency distribution of cardiomyocytes cultured on different stretched inverse opal substrates; (d) Beating characterization of the cardiomyocytes on different stretched inverse opal substrate. These dates were the average values of each day (10 min each time and five times every day). Error bars represent SD. Scale bars are 30 μm.


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Figure 4 The construction of soft-bodied structural color robot. (a) The multicolor of a swimming guppy; (b) Schematic image of the guppy-shaped substrate with self-actuate property at the tail; (c) Relationship between the bending angles and the characteristic reflection peak values in different positions of the guppy tail; (d) Optical microscope images of varying structural color of the guppy tail during one myocardial cycle. Scale bar is 1 mm.


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Figure 5 The heart-on-a-chip system with the integration of biohybrid actuators and microfluidics. (a, b) Schematic and image of the integrated heart-on-chip; (c) Optical microscope images of varying structural color of biohybrid actuator in a heart-on-a-chip system during one myocardial cycle; (d) Relationship between the bend-up process and the reflection peak shift values at the position noted with dotted line in (c) of the biohybrid actuator treated with different concentrations of isoproterenol; (e, f) Relationships of the average peak shift values and the frequency of the bending cycle of the biohybrid actuator treated with different concentrations of isoproterenol. Error bars represent SD. Scale bars are 1cm in (b) are 0.5mm in (c).


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TOC graphic Cardiomyocyte-Driven Structural Color Actuation in Anisotropic Inverse Opals Yixuan Shang1,2, Zhuoyue Chen 2, Fanfan Fu 2, Lingyu Sun 2, Changmin Shao 2, Wei Jin*,1,2, Hong Liu*,2, Yuanjin Zhao*,1,2

A cardiomyocyte-driven structural color actuator: Cardiomyocytes on the anisotropic inverse opal substrate could be induced into a highly ordered alignment with recovering autonomic beating ability. During their beating, the substrates could undergo synchronous deformations, which can be reported as corresponding shifts of their structural colors. Thus, soft structural color robot and heart-on-a-chip system could be developed for cell monitoring and drug testing.


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Cardiomyocyte-Driven Structural Color Actuation in Anisotropic

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