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Fibre-infused gel scaffolds guide cardiomyocyte alignment in 3D-printed ventricles

Nature Materials volume 22, pages 1039–1046 (2023)Cite this article

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Abstract

Hydrogels are attractive materials for tissue engineering, but efforts to date have shown limited ability to produce the microstructural features necessary to promote cellular self-organization into hierarchical three-dimensional (3D) organ models. Here we develop a hydrogel ink containing prefabricated gelatin fibres to print 3D organ-level scaffolds that recapitulate the intra- and intercellular organization of the heart. The addition of prefabricated gelatin fibres to hydrogels enables the tailoring of the ink rheology, allowing for a controlled sol–gel transition to achieve precise printing of free-standing 3D structures without additional supporting materials. Shear-induced alignment of fibres during ink extrusion provides microscale geometric cues that promote the self-organization of cultured human cardiomyocytes into anisotropic muscular tissues in vitro. The resulting 3D-printed ventricle in vitro model exhibited biomimetic anisotropic electrophysiological and contractile properties.

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Fig. 1: Development of gelatin FIG inks for free-standing 3D-printed tissue scaffolds with cellular alignment cues.
Fig. 2: Anisotropic intra- and intercellular organization of cardiac tissues cultured on printed FIG scaffolds.
Fig. 3: Dynamics of electromechanical coupling of multidirectional anisotropic cardiac tissues.
Fig. 4: Structural, electrophysiological and contractile properties of human-stem-cell-based tissue-engineered 3D ventricle models.

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Data availability

Data generated or analysed during this study are included in the Article and its Supplementary Information files and publicly available at figshare at https://doi.org/10.6084/m9.figshare.22787714. Additional data may be obtained from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

G-code files used in this study are provided in the Supplementary Code. The contractility analysis of in vitro ventricle models was conducted using code from ref. 15 and is available at https://doi.org/10.5281/zenodo.6547775 (ref. 55).

References

  1. Frantz, C., Stewart, K. M. & Weaver, V. M. The extracellular matrix at a glance. J. Cell Sci. 123, 4195–4200 (2010).

    Article  CAS  Google Scholar 

  2. Blazeski, A. et al. Functional properties of engineered heart slices incorporating human induced pluripotent stem cell-derived cardiomyocytes. Stem Cell Rep. 12, 982–995 (2019).

    Article  CAS  Google Scholar 

  3. LeGrice, I., Pope, A. & Smaill, B. in Interstitial Fibrosis in Heart Failure (ed. Villarreal, F. J.) 3–21 (Springer New York, 2005).

  4. Ronaldson-Bouchard, K. et al. Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature 556, 239–243 (2018).

    Article  CAS  Google Scholar 

  5. Kleber, A. G. & Rudy, Y. Basic mechanisms of cardiac impulse propagation and associated arrhythmias. Physiol. Rev. 84, 431–488 (2004).

    Article  CAS  Google Scholar 

  6. Park, S. J. et al. Insights into the pathogenesis of catecholaminergic polymorphic ventricular tachycardia from engineered human heart tissue. Circulation 140, 390–404 (2019).

    Article  CAS  Google Scholar 

  7. Wang, G. et al. Modeling the mitochondrial cardiomyopathy of barth syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nat. Med. 20, 616–623 (2014).

    Article  CAS  Google Scholar 

  8. McCain, M. L., Agarwal, A., Nesmith, H. W., Nesmith, A. P. & Parker, K. K. Micromolded gelatin hydrogels for extended culture of engineered cardiac tissues. Biomaterials 35, 5462–5471 (2014).

    Article  CAS  Google Scholar 

  9. Feinberg, A. W. et al. Muscular thin films for building actuators and powering devices. Science 317, 1366–1370 (2007).

    Article  CAS  Google Scholar 

  10. Li, R. A. et al. Bioengineering an electro-mechanically functional miniature ventricular heart chamber from human pluripotent stem cells. Biomaterials 163, 116–127 (2018).

    Article  Google Scholar 

  11. Karp, J. M. et al. A photolithographic method to create cellular micropatterns. Biomaterials 27, 4755–4764 (2006).

    Article  CAS  Google Scholar 

  12. Wu, X. et al. Preparation of aligned porous gelatin scaffolds by unidirectional freeze-drying method. Acta Biomater. 6, 1167–1177 (2010).

    Article  CAS  Google Scholar 

  13. MacQueen, L. A. et al. A tissue-engineered scale model of the heart ventricle. Nat. Biomed. Eng. 2, 930–941 (2018).

    Article  CAS  Google Scholar 

  14. Capulli, A. K. et al. Jetvalve: rapid manufacturing of biohybrid scaffolds for biomimetic heart valve replacement. Biomaterials 133, 229–241 (2017).

    Article  CAS  Google Scholar 

  15. Chang, H. et al. Recreating the heart’s helical structure-function relationship with focused rotary jet spinning. Science 377, 180–185 (2022).

    Article  CAS  Google Scholar 

  16. Wang, Z., Lee, S. J., Cheng, H. J., Yoo, J. J. & Atala, A. 3D bioprinted functional and contractile cardiac tissue constructs. Acta Biomater. 70, 48–56 (2018).

    Article  CAS  Google Scholar 

  17. Yue, K. et al. Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. Biomaterials 73, 254–271 (2015).

    Article  CAS  Google Scholar 

  18. Lind, J. U. et al. Instrumented cardiac microphysiological devices via multimaterial three-dimensional printing. Nat. Mater. 16, 303–308 (2017).

    Article  CAS  Google Scholar 

  19. Zhao, Y. et al. A platform for generation of chamber-specific cardiac tissues and disease modeling. Cell 176, 913–927 (2019).

    Article  CAS  Google Scholar 

  20. Capulli, A. K., MacQueen, L. A., Sheehy, S. P. & Parker, K. K. Fibrous scaffolds for building hearts and heart parts. Adv. Drug Deliv. Rev. 96, 83–102 (2016).

    Article  CAS  Google Scholar 

  21. Yi, H. G. et al. A bioprinted human-glioblastoma-on-a-chip for the identification of patient-specific responses to chemoradiotherapy. Nat. Biomed. Eng. 3, 509–519 (2019).

    Article  CAS  Google Scholar 

  22. Daly, A. C., Davidson, M. D. & Burdick, J. A. 3D bioprinting of high cell-density heterogeneous tissue models through spheroid fusion within self-healing hydrogels. Nat. Commun. 12, 753 (2021).

    Article  CAS  Google Scholar 

  23. Brassard, J. A., Nikolaev, M., Hubscher, T., Hofer, M. & Lutolf, M. P. Recapitulating macro-scale tissue self-organization through organoid bioprinting. Nat. Mater. 20, 22–29 (2021).

    Article  CAS  Google Scholar 

  24. Jia, W. et al. Direct 3D bioprinting of perfusable vascular constructs using a blend bioink. Biomaterials 106, 58–68 (2016).

    Article  CAS  Google Scholar 

  25. Ashammakhi, N. et al. Bioinks and bioprinting technologies to make heterogeneous and biomimetic tissue constructs. Mater. Today Bio 1, 100008 (2019).

    Article  CAS  Google Scholar 

  26. Ouyang, L. et al. Expanding and optimizing 3D bioprinting capabilities using complementary network bioinks. Sci. Adv. 6, abc5529 (2020).

    Article  Google Scholar 

  27. Choi, J. R., Yong, K. W., Choi, J. Y. & Cowie, A. C. Recent advances in photo-crosslinkable hydrogels for biomedical applications. BioTechniques 66, 40–53 (2019).

    Article  CAS  Google Scholar 

  28. Markstedt, K. et al. 3D bioprinting human chondrocytes with nanocellulose-alginate bioink for cartilage tissue engineering applications. Biomacromolecules 16, 1489–1496 (2015).

    Article  CAS  Google Scholar 

  29. Nadernezhad, A. et al. Nanocomposite bioinks based on agarose and 2D nanosilicates with tunable flow properties and bioactivity for 3D bioprinting. ACS Appl. Bio Mater. 2, 796–806 (2019).

    Article  CAS  Google Scholar 

  30. Schwab, A. et al. Printability and shape fidelity of bioinks in 3D bioprinting. Chem. Rev. 120, 11028–11055 (2020).

    Article  CAS  Google Scholar 

  31. Noor, N. et al. 3D printing of personalized thick and perfusable cardiac patches and hearts. Adv. Sci. 6, 1900344 (2019).

    Article  Google Scholar 

  32. Kupfer, M. E. et al. In situ expansion, differentiation, and electromechanical coupling of human cardiac muscle in a 3D bioprinted, chambered organoid. Circ. Res. 127, 207–224 (2020).

    Article  CAS  Google Scholar 

  33. Lee, A. et al. 3D bioprinting of collagen to rebuild components of the human heart. Science 365, 482–487 (2019).

    Article  CAS  Google Scholar 

  34. Campostrini, G., Windt, L. M., van Meer, B. J., Bellin, M. & Mummery, C. L. Cardiac tissues from stem cells: new routes to maturation and cardiac regeneration. Circ. Res. 128, 775–801 (2021).

    Article  CAS  Google Scholar 

  35. Ahrens, J. H. et al. Programming cellular alignment in engineered cardiac tissue via bioprinting anisotropic organ building blocks. Adv. Mater. 34, e2200217 (2022).

    Article  Google Scholar 

  36. Gladman, A. S., Matsumoto, E. A., Nuzzo, R. G., Mahadevan, L. & Lewis, J. A. Biomimetic 4D printing. Nat. Mater. 15, 413–418 (2016).

    Article  Google Scholar 

  37. Siqueira, G. et al. Cellulose nanocrystal inks for 3D printing of textured cellular architectures. Adv. Funct. Mater. 27, 1604619 (2017).

    Article  Google Scholar 

  38. Jinkins, K. R., Chan, J., Jacobberger, R. M., Berson, A. & Arnold, M. S. Substrate‐wide confined shear alignment of carbon nanotubes for thin film transistors. Adv. Electron. Mater. 5, 1800593 (2018).

    Article  Google Scholar 

  39. Caliari, S. R. & Burdick, J. A. A practical guide to hydrogels for cell culture. Nat. Methods 13, 405–414 (2016).

    Article  CAS  Google Scholar 

  40. Hausmann, M. K. et al. Dynamics of cellulose nanocrystal alignment during 3D printing. ACS Nano 12, 6926–6937 (2018).

    Article  CAS  Google Scholar 

  41. Pasqualini, F. S., Sheehy, S. P., Agarwal, A., Aratyn-Schaus, Y. & Parker, K. K. Structural phenotyping of stem cell-derived cardiomyocytes. Stem Cell Rep. 4, 340–347 (2015).

    Article  CAS  Google Scholar 

  42. Bray, M. A., Sheehy, S. P. & Parker, K. K. Sarcomere alignment is regulated by myocyte shape. Cell Motil. Cytoskeleton 65, 641–651 (2008).

    Article  Google Scholar 

  43. Kuo, P. L. et al. Myocyte shape regulates lateral registry of sarcomeres and contractility. Am. J. Pathol. 181, 2030–2037 (2012).

    Article  CAS  Google Scholar 

  44. Geisse, N. A., Sheehy, S. P. & Parker, K. K. Control of myocyte remodeling in vitro with engineered substrates. Vitr. Cell. Dev. Biol. Anim. 45, 343–350 (2009).

    Article  Google Scholar 

  45. Grosberg, A. et al. Self-organization of muscle cell structure and function. PLoS Comput. Biol. 7, e1001088 (2011).

    Article  CAS  Google Scholar 

  46. Bray, M. A. et al. Nuclear morphology and deformation in engineered cardiac myocytes and tissues. Biomaterials 31, 5143–5150 (2010).

    Article  CAS  Google Scholar 

  47. Lee, H. et al. Cytoskeletal prestress regulates nuclear shape and stiffness in cardiac myocytes. Exp. Biol. Med. 240, 1543–1554 (2015).

    Article  CAS  Google Scholar 

  48. Carmeliet, E. Conduction in cardiac tissue. Historical reflections. Physiol. Rep. 7, e13860 (2019).

    Article  Google Scholar 

  49. Kleber, A. G. & Jin, Q. Coupling between cardiac cells—an important determinant of electrical impulse propagation and arrhythmogenesis. Biophys. Rev. 2, 031301 (2021).

    Article  CAS  Google Scholar 

  50. Buckberg, G., Hoffman, J. I., Mahajan, A., Saleh, S. & Coghlan, C. Cardiac mechanics revisited: the relationship of cardiac architecture to ventricular function. Circulation 118, 2571–2587 (2008).

    Article  Google Scholar 

  51. Rezakhaniha, R. et al. Experimental investigation of collagen waviness and orientation in the arterial adventitia using confocal laser scanning microscopy. Biomech. Model. Mechanobiol. 11, 461–473 (2012).

    Article  CAS  Google Scholar 

  52. Lian, X. et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nat. Protoc. 8, 162–175 (2013).

    Article  CAS  Google Scholar 

  53. Al Tanoury, Z. et al. Prednisolone rescues Duchenne muscular dystrophy phenotypes in human pluripotent stem cell–derived skeletal muscle in vitro. Proc. Natl Acad. Sci. USA 118, e2022960118 (2021).

    Article  CAS  Google Scholar 

  54. Liberzon, A. et al. OpenPIV/openpiv-python: fixed windows conda-forge failure with encoding. Zenodo https://doi.org/10.5281/zenodo.3566451 (2019).

  55. SeasDBG. SeasDBG/FRJS: recreating the heart’s helical structure-function relationship with focused rotary jet spinning-data analysis (analysis). Zenodo https://doi.org/10.5281/zenodo.6547775 (2022).

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Acknowledgements

We thank M. Rosnach for photography and illustrations and A. G. Kleber for discussions regarding cardiac physiology. This work was sponsored by the John A. Paulson School of Engineering and Applied Sciences at Harvard University, the National Science Foundation through the Harvard University Materials Research Science and Engineering Center (DMR-1420570, DMR-2011754 to K.K.P.) and the National Institutes of Health and National Center for Advancing Translational Sciences (UH3HL141798 to W.T.P. and K.K.P.; UG3TR003279 to W.T.P. and K.K.P.) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00248477, 2022R1A2C2012738 to K.Y.L.). This work was also performed in part at the Harvard University Center for Nanoscale Systems, a member of the National Nanotechnology Coordinated Infrastructure Network, which is supported by the National Science Foundation under award no. ECCS-2025158. Microcomputed tomography imaging reported in this publication was supported by Harvard University, Center for Nanoscale Systems under National Institutes of Health award no. S10OD023519. We gratefully acknowledge financial support by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 810104-PoInt to A.R.B.) and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, Project ID No. 201269156—SFB 1032 to A.R.B.). H.A.M.A. thanks the American Chemical Society for support through the Irving S. Sigal Postdoctoral Fellowship. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors and Affiliations

  1. Disease Biophysics Group, John A. Paulson School of Engineering and Applied Sciences, Harvard University, Boston, MA, USA

    Suji Choi, Keel Yong Lee, Sean L. Kim, Luke A. MacQueen, Huibin Chang, John F. Zimmerman, Qianru Jin, Michael M. Peters, Herdeline Ann M. Ardoña, Rudy Gabardi, Collin Richardson & Kevin Kit Parker

  2. Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Republic of Korea

    Keel Yong Lee

  3. Department of Chemical and Biomolecular Engineering, Samueli School of Engineering, University of California, Irvine, CA, USA

    Herdeline Ann M. Ardoña

  4. Department of Cardiology, Boston Children’s Hospital, Boston, MA, USA

    Xujie Liu & William T. Pu

  5. Fuwai Hospital Chinese Academy of Medical Sciences, Shenzhen, China

    Xujie Liu

  6. Department of Bioscience, TUM School of Natural Sciences, Technische Universität München, Garching, Germany

    Ann-Caroline Heiler & Andreas R. Bausch

  7. Center for Functional Protein Assemblies, Technische Universität München, Garching, Germany

    Ann-Caroline Heiler & Andreas R. Bausch

  8. Center for Organoid Systems (COS), Technische Universität München, Garching, Germany

    Ann-Caroline Heiler & Andreas R. Bausch

  9. Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA

    William T. Pu & Kevin Kit Parker

  10. Max Planck School Matter to Life, Max Planck Schools, Heidelberg, Germany

    Andreas R. Bausch

  11. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA

    Kevin Kit Parker

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Contributions

K.K.P. supervised the research. S.C., L.A.M. and K.K.P. conceived and designed the study. S.C. developed the fabrication method of the FIG ink, designed and performed 3D printing experiments, analysed data, organized figures and wrote the paper. K.Y.L. performed the optical mapping experiments. H.C. and M.M.P. fabricated the gelatin fibres. K.Y.L., S.L.K., J.F.Z., M.M.P. and A.R.B. analysed data. S.C., S.L.K., Q.J. and H.A.M.A. performed the NRVM harvest with animal protocols. S.C., S.L.K., A.-C.H., X.L. and W.T.P. differentiated hiPSC into cardiomyocytes and cultured hiPSC-CM. R.G. and C.R. assisted in the fabrication of the FIG ink and 3D-printed scaffolds. All authors discussed the results and contributed to the writing of the manuscript.

Corresponding author

Correspondence to Kevin Kit Parker.

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Competing interests

Harvard College and Sogang University are patent applicants for one pending utility patent, application number 16/756,214, with L.A.M. and K.K.P. included among the inventors. This patent application describes three-dimensional biological tissue formation using macromolecular-fibre-infused additive manufacturing inks. The remaining authors declare no competing interests.

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Nature Materials thanks Wolfram Zimmermann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–12, note and references.

Reporting Summary

Supplementary Video 1

Direct 3D printing of ventricle-shaped scaffolds without using supporting materials.

Supplementary Video 2

Beating hiPSC-CMs after the differentiation process is finished.

Supplementary Video 3

Optical mapping images for the Ca2+ transient propagation of NRVM tissues cultured on the printed Gel–Alg hydrogel (left) and FIG scaffolds (right). A 1 Hz point electrical stimulation was applied at the left top corner of each tissue.

Supplementary Video 4

Optical mapping images for the Ca2+ transient propagation of hiPSC-CM tissues cultured on the printed FIG scaffolds under 1 Hz (left) and 2 Hz (right) point electrical stimulation at the left top corner.

Supplementary Video 5

Optical mapping images for the Ca2+ wave propagation occurred through the printed FIG patterns, showing sequential muscle activation.

Supplementary Video 6

Contraction of NRVM tissue on the 3D-printed rectangular scaffolds printed in the parallel, angled and perpendicular directions to the long side of the scaffold geometry, showing different contractile motions—rolling, twisting and bending, respectively—under 0.5 Hz field electrical stimulation.

Supplementary Video 7

Spontaneous contraction of 3D in vitro hiPSC-CM ventricle model in the RPMI/B27(–) media.

Supplementary Video 8

Spontaneous contraction of 3D in vitro NRVM ventricle model in Tyrode’s solution.

Supplementary Video 9

Ca2+ transient propagation from a 3D in vitro NRVM ventricle model under 1 Hz point electrical stimulation. The point stimulation was applied where the propagation started.

Supplementary Video 10

Spontaneous Ca2+ transient propagation from a 3D in vitro hiPSC-CM ventricle model observed at the top, middle and bottom planes.

Supplementary Video 11

Deformation mapping analysis from a 3D in vitro hiPSC-CM ventricle model. Movement of fluorescent beads adhered on the ventricle (left panel) was analysed during the systole and diastole cycle of the 3D in vitro ventricle tissue model (right panel).

Supplementary Video 12

Contractility assessment of a 3D in vitro hiPSC-CM ventricle tissue model using PIV analysis. Bright-field micrograph of a 3D in vitro hiPSC-CM ventricle model (left) with a green region of interest box (left), fluorescent bead movement from the corresponding region of interest (top, right) and phase-averaged velocity map from the region of interest (bottom, right).

Supplementary Code

G-code files used for the 3D-printed structures in Fig. 1.

Source data

Source Data Fig. 1

Source data for the plots in Fig. 1.

Source Data Fig. 2

Source data for the plots in Fig. 2.

Source Data Fig. 3

Source data for the plots in Fig. 3.

Source Data Fig. 4

Source data for the plots in Fig. 4.

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Choi, S., Lee, K.Y., Kim, S.L. et al. Fibre-infused gel scaffolds guide cardiomyocyte alignment in 3D-printed ventricles. Nat. Mater. 22, 1039–1046 (2023). https://doi.org/10.1038/s41563-023-01611-3

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