Abstract
Cardiovascular diseases are the leading cause of morbidity and mortality throughout the world underlining the importance of efficient treatments including disease modeling and drug discovery by cardiac tissue engineering. However, the predictive power of these applications is currently limited by the immature state of the cardiomyocytes. Here, we developed gelatin hydrogels chemically crosslinked by genipin, a biocompatible crosslinker, as cell culture scaffolds. Neonatal rat cardiomyocytes appear synchronous beating within 2 days after seeding on hydrogels. Furthermore, we applied the electrical stimulation as a conditioning treatment to promote the maturation of cardiomyocytes cultured on the hydrogels. Our results show that electrical stimulation improves the organization of sarcomeres, establishment of gap junctions, calcium-handling capacity and propagation of pacing signals, thereby, increase the beating velocity of cardiomyocytes and responsiveness to external pacing. The above system can be applied in promoting physiological function maturation of engineered cardiac tissues, exhibiting promising applications in cardiac tissue engineering and drug screening.
Similar content being viewed by others
References
Ogle BM, Bursac N, Domian I, Huang NF, Menasché P, Murry CE, Pruitt B, Radisic M, Wu JC, Wu SM, Zhang J, Zimmermann W-H, Vunjak-Novakovic G (2016) Distilling complexity to advance cardiac tissue engineering. Sci Transl Med 8(342):342ps313. https://doi.org/10.1126/scitranslmed.aad2304
Siu CW, Tse HF (2012) Cardiac regeneration: messages from CADUCEUS. Lancet 379:870–871. https://doi.org/10.1016/S0140-6736(12)60236-0
Tiburcy M, Zimmermann W (2014) Modeling myocardial growth and hypertrophy in engineered heart muscle. Trends Cardiovas Med 24:7–13. https://doi.org/10.1016/j.tcm.2013.05.003
Camci-Unal G, Annabi N, Dokmeci MR, Liao R, Khademhosseini A (2014) Hydrogels for cardiac tissue engineering. Npg Asia Mater 6:e99. https://doi.org/10.1038/am.2014.19
Peña B, Laughter M, Jett S, Rowland TJ, Taylor MRG, Mestroni L, Park D (2018) Injectable hydrogels for cardiac tissue engineering. Macromol Biosci 18:1800079. https://doi.org/10.1002/mabi.201800079
Navaei A, Truong D, Heffernan J, Cutts J, Brafman D, Sirianni RW, Vernon B, Nikkhah M (2016) PNIPAAm-based biohybrid injectable hydrogel for cardiac tissue engineering. Acta Biomater 32:10–23. https://doi.org/10.1016/j.actbio.2015.12.019
Fang Y, Zhang T, Song Y, Sun W (2020) Assessment of various crosslinking agents on collagen/chitosan scaffolds for myocardial tissue engineering. Biomed Mater 15:45003. https://doi.org/10.1088/1748-605X/ab452d
Nazir R, Bruyneel A, Carr C, Czernuszka J (2019) Collagen type I and hyaluronic acid based hybrid scaffolds for heart valve tissue engineering. Biopolymers 110(8):e23278. https://doi.org/10.1002/bip.23278
Khalil S, Sun W (2009) Bioprinting endothelial cells with alginate for 3D tissue constructs. J Biomech Eng 131:111002. https://doi.org/10.1115/1.3128729
Dvir T, Timko BP, Brigham MD, Naik SR, Karajanagi SS, Levy O, Jin H, Parker KK, Langer R, Kohane DS (2011) Nanowired three-dimensional cardiac patches. Nat Nanotechnol 6:720–725. https://doi.org/10.1038/nnano.2011.160
Kharaziha M, Shin SR, Nikkhah M, Topkaya SN, Masoumi N, Annabi N, Dokmeci MR, Khademhosseini A (2014) Tough and flexible CNT–polymeric hybrid scaffolds for engineering cardiac constructs. Biomaterials 35:7346–7354. https://doi.org/10.1016/j.biomaterials.2014.05.014
McCain ML, Agarwal A, Nesmith HW, Nesmith AP, Parker KK (2014) Micromolded gelatin hydrogels for extended culture of engineered cardiac tissues. Biomaterials 35:5462–5471. https://doi.org/10.1016/j.biomaterials.2014.03.052
Gao L, Gan H, Meng Z, Gu R, Wu Z, Zhang L, Zhu X, Sun W, Li J, Zheng Y et al (2014) Effects of genipin cross-linking of chitosan hydrogels on cellular adhesion and viability. Colloids Surf, B 117:398–405. https://doi.org/10.1016/j.colsurfb.2014.03.002
Macaya DJ, Hayakawa K, Arai K, Spector M (2013) Astrocyte infiltration into injectable collagen-based hydrogels containing FGF-2 to treat spinal cord injury. Biomaterials 34:3591–3602. https://doi.org/10.1016/j.biomaterials.2012.12.050
Zhang F, Zhang N, Meng H, Liu H, Lu Y, Liu C, Zhang Z, Qu K, Huang N (2019) Easy applied gelatin-based hydrogel system for long-term functional cardiomyocyte culture and myocardium formation. Acs Biomater Sci Eng 5:3022–3031. https://doi.org/10.1021/acsbiomaterials.9b00515
Matsa E, Burridge PW, Wu JC (2014) Human stem cells for modeling heart disease and for drug discovery. Sci Transl Med 6:236p–239p. https://doi.org/10.1126/scitranslmed.3008921
Bellin M, Marchetto MC, Gage FH, Mummery CL (2012) Induced pluripotent stem cells: the new patient? Nat Rev Mol Cell Bio 13:713–726. https://doi.org/10.1038/nrm3448
Feric NT, Radisic M (2016) Maturing human pluripotent stem cell-derived cardiomyocytes in human engineered cardiac tissues. Adv Drug Deliv Rev 96:110–134. https://doi.org/10.1016/j.addr.2015.04.019
Zimmermann WH, Fink C, Kralisch D, Remmers U, Weil J, Eschenhagen T (2000) Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes. Biotechnol Bioeng 68:106–114. https://doi.org/10.1002/(SICI)1097-0290(20000405)68:1<106::AID-BIT13>3.0.CO;2-3
Tandon N, Cannizzaro C, Chao PG, Maidhof R, Marsano A, Au HTH, Radisic M, Vunjak-Novakovic G (2009) Electrical stimulation systems for cardiac tissue engineering. Nat Protoc 4:155–173. https://doi.org/10.1038/nprot.2008.183
Zimmermann W, Melnychenko I, Wasmeier G, Didié M, Naito H, Nixdorff U, Hess A, Budinsky L, Brune K, Michaelis B et al (2006) Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts. Nat Med 12:452–458. https://doi.org/10.1038/nm1394
Holt E, Lunde PK, Sejersted OM, Christensen G (1997) Electrical stimulation of adult rat cardiomyocytes in culture improves contractile properties and is associated with altered calcium handling. Basic Res Cardiol 92:289–298. https://doi.org/10.1007/BF00788941
Chan Y, Ting S, Lee Y, Ng K, Zhang J, Chen Z, Siu C, Oh SKW, Tse H (2013) Electrical stimulation promotes maturation of cardiomyocytes derived from human embryonic stem cells. J Cardiovasc Transl 6:989–999. https://doi.org/10.1007/s12265-013-9510-z
Nunes SS, Miklas JW, Liu J, Aschar-Sobbi R, Xiao Y, Zhang B, Jiang J, Massé S, Gagliardi M, Hsieh A et al (2013) Biowire: a platform for maturation of human pluripotent stem cell–derived cardiomyocytes. Nat Methods 10:781–787. https://doi.org/10.1038/nmeth.2524
Kim SW, Kim HW, Huang W, Okada M, Welge JA, Wang Y, Ashraf M (2013) Cardiac stem cells with electrical stimulation improve ischaemic heart function through regulation of connective tissue growth factor and miR-378. Cardiovasc Res 100:241–251. https://doi.org/10.1093/cvr/cvt192
Tsang KMC, Annabi N, Ercole F, Zhou K, Karst DJ, Li F, Haynes JM, Evans RA, Thissen H, Khademhosseini A et al (2015) Facile one-step micropatterning using photodegradable gelatin hydrogels for improved cardiomyocyte organization and alignment. Adv Funct Mater 25:977–986. https://doi.org/10.1002/adfm.201403124
Tandon N, Marsano A, Maidhof R, Wan L, Park H, Vunjak-Novakovic G (2011) Optimization of electrical stimulation parameters for cardiac tissue engineering. J Tissue Eng Regen M 5:e115–e125. https://doi.org/10.1002/term.377
Ren K, Fourel L, Rouvière CG, Albiges-Rizo C, Picart C (2010) Manipulation of the adhesive behaviour of skeletal muscle cells on soft and stiff polyelectrolyte multilayers. Acta Biomater 6:4238–4248. https://doi.org/10.1016/j.actbio.2010.06.014
Mehrasa M, Asadollahi MA, Nasri-Nasrabadi B, Ghaedi K, Salehi H, Dolatshahi-Pirouz A, Arpanaei A (2016) Incorporation of mesoporous silica nanoparticles into random electrospun PLGA and PLGA/gelatin nanofibrous scaffolds enhances mechanical and cell proliferation properties. Mater Sci Eng, C 66:25–32. https://doi.org/10.1016/j.msec.2016.04.031
Prabhakaran MP, Venugopal J, Kai D, Ramakrishna S (2011) Biomimetic material strategies for cardiac tissue engineering. Mater Sci Eng, C 31:503–513. https://doi.org/10.1016/j.msec.2010.12.017
Robertson C, Tran DD, George SC (2013) Concise review: maturation phases of human pluripotent stem cell-derived cardiomyocytes. Stem Cells 31:829–837. https://doi.org/10.1002/stem.1331
Zhang N, Stauffer F, Simona BR, Zhang F, Zhang Z, Huang N, Vörös J (2018) Multifunctional 3D electrode platform for real-time in situ monitoring and stimulation of cardiac tissues. Biosens Bioelectron 112:149–155. https://doi.org/10.1016/j.bios.2018.04.037
Godier-Furnémont AFG, Tiburcy M, Wagner E, Dewenter M, Lämmle S, El-Armouche A, Lehnart SE, Vunjak-Novakovic G, Zimmermann W (2015) Physiologic force-frequency response in engineered heart muscle by electromechanical stimulation. Biomaterials 60:82–91. https://doi.org/10.1016/j.biomaterials.2015.03.055
Ronaldson-Bouchard K, Ma SP, Yeager K, Chen T, Song L, Sirabella D, Morikawa K, Teles D, Yazawa M, Vunjak-Novakovic G (2018) Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature 556:239–243. https://doi.org/10.1038/s41586-018-0016-3
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (Grant No. 31871017), the Natural Science Foundation of Jiangsu Province, China (Grant No. BK20171352), the Southeast University-Nanjing Medical University Cooperative research project (2242019K3DN05), the Medical Science and Technology Development Foundation, Jiangsu Provincial Commission of Health and Family Planning, China (ZDRCA2016073), and the “111” Project (B17011, Ministry of Education of China).
Author information
Authors and Affiliations
Contributions
FZ participated in the experimental research, data analysis, writing and editing of the manuscript. KYQ and XPL performed cardiomyocyte harvest and culture. CML and LSO established the experimental setup for electrical stimulation and developed softwares for data analysis. KHW and XWW provided guidance on cell study and helped to revise the manuscript. NPH conducted the design of the work as well as the deep review, editing, guidance, and supervision. All authors have read and approved the article for publication.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that there is no conflict of interest.
Ethical approval
Cardiomyocytes were extracted from neonatal rat ventricles of two-day-old Sprague–Dawley rats. All related procedures were carried out as approved by the Institutional Animal Care and Use Committee (IACUC) of Southeast University.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supporting movie S1: Synchronously beating behavior and calcium transient of cardiomyocytes on the gelatin-based hydrogels at day 2 after cell seeding. (AVI 15053 kb)
Supporting movie S2: Cardiomyocytes cultured on the culture dishes over time. (AVI 14868 kb)
Supporting movie S3: Beating behavior of cardiomyocytes cultured on different substrates. (AVI 19331 kb)
Supporting movie S4: The electrical stimulation can modulate the beating behavior of cardiomyocytes. (AVI 26793 kb)
Supporting movie S5: Electrical stimulation promotes the elongation and alignment of cardiomyocytes. (AVI 34992 kb)
42242_2020_100_MOESM6_ESM.docx
The following files are available free of charge: Characterization of gelatin-based scaffolds and electrical stimulation device; Immunofluorescent staining of cardiac-specific proteins (Cx43: in red; α-actinin: in green) on cardiomyocytes and vimentin (in blue) on non-muscle cells including fibroblasts and endothelial cells, with and without electrical stimulation. (DOCX 2130 kb)
Rights and permissions
About this article
Cite this article
Zhang, F., Qu, K., Li, X. et al. Gelatin-based hydrogels combined with electrical stimulation to modulate neonatal rat cardiomyocyte beating and promote maturation. Bio-des. Manuf. 4, 100–110 (2021). https://doi.org/10.1007/s42242-020-00100-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s42242-020-00100-9