Reconstitution of the Ventricular Endocardium Within Acellular Hearts

Abstract

There is a need for developing a living tissue-engineered whole heart for transplantation. One solution is to create acellular myocardial tissue scaffolds and seed them with autologous cells for full reconstitution. Our goal was to reconstitute the endocardial layer of both ventricular cavities and the septum surfaces of decellularized hearts. Whole rabbit hearts were decellularized using a biventricular perfusion system. We designed a rotational support system for the scaffolds and seeded the two cardiac cavities with human fibroblasts, collagen hydrogels, fibrin hydrogel, and human endothelial cells in a layer-by-layer fashion. Afterwards, the scaffold was subjected to in vitro conditioning in a purpose-designed bioreactor. Results showed that hydrogels infused onto most surfaces and pores of the scaffold. Seeded cells effectively adhered to many areas of the two ventricles while remaining active by secreting new matrix proteins. These results indicate that layer-by-layer deposition can aid in the reconstitution of the cardiac endocardium.

Lay Summary

There is a need for developing a living whole heart for transplantation. In this study, we developed a perfusion system to remove all cells from rabbit hearts, while leaving the connective tissue collagen fibers intact. We then developed a rotational bioreactor system to seed the hearts with human cells in a layer-by-layer fashion by suspending human fibroblasts and endothelial cells in fibrin and collagen gels as carriers. Using these systems, we successfully reconstituted an important internal layer of the heart, the endocardium, lining the cavities of the heart, with most cells remaining alive and active.

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Abbreviations

ABS:

Acrylonitrile butadiene styrene

EDTA:

Ethylenediaminetetraacetic acid

PBS:

Phosphate-buffered saline

EtOH:

Sodium dodecyl sulfate ethanol

RNase:

Ribonuclease

DNAse:

Deoxyribonuclease

DNA:

Deoxyribonucleic acid

DAPI:

4′,6-Diamidino-2-phenylindole

IHC:

Immunohistochemistry

hADSC:

Human adipose-derived stem cell

hAOEC:

Human aortic endothelial cell

DMEM:

Dulbecco’s modified eagle medium

FBS:

Fetal bovine serum

RPM:

Revolutions per minute

SEM:

Scanning electron microscopy

HMDS:

Hexamethyldisilazane

References

  1. 1.

    Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, et al. Executive summary: heart disease and stroke statistics-2016 update: a report from the American Heart Association. Circulation. 2016;133:447–54. https://doi.org/10.1161/CIR.0000000000000366.

    Article  Google Scholar 

  2. 2.

    Momtahan N, Sukavaneshvar S, Roeder BL, Cook AD. Strategies and processes to decellularize and recellularize hearts to generate functional organs and reduce the risk of thrombosis. Tissue Eng Part B Rev. 2015;21:115–32. https://doi.org/10.1089/ten.teb.2014.0192.

    Article  Google Scholar 

  3. 3.

    Transplant Trends. https://www.unos.org/data/transplant-trends/. Accessed 15 Dec 2017.

  4. 4.

    Sarig U, Machluf M. Engineering cell platforms for myocardial regeneration. Expert Opin Biol Ther. 2011;11:1055–77. https://doi.org/10.1517/14712598.2011.578574.

    CAS  Article  Google Scholar 

  5. 5.

    Domenech M, Polo-Corrales L, Ramirez-Vick JE, Freytes DO. Tissue engineering strategies for myocardial regeneration: acellular versus cellular scaffolds? Tissue Eng Part B Rev. 2016;22:438–58. https://doi.org/10.1089/ten.teb.2015.0523.

    CAS  Article  Google Scholar 

  6. 6.

    Chen Q-Z, Harding SE, Ali NN, Lyon AR, Boccaccini AR. Biomaterials in cardiac tissue engineering: ten years of research survey. Mater Sci Eng R. 2008;59:1–37. https://doi.org/10.1016/j.mser.2007.08.001.

    CAS  Article  Google Scholar 

  7. 7.

    Arnal-Pastor M, Chachques JC, Pradas MM, Vallés-Lluch A. Biomaterials for cardiac tissue engineering. In: Regenerative medicine and tissue engineering; 2013. p. 275–323.

    Google Scholar 

  8. 8.

    Perea-Gil I, Uriarte JJ, Prat-Vidal C, Gálvez-Montón C, Roura S, Llucià-Valldeperas A, et al. In vitro comparative study of two decellularization protocols in search of an optimal myocardial scaffold for recellularization. Am J Transl Res. 2015;7:558–73.

    CAS  Google Scholar 

  9. 9.

    Robertson MJ, Dries-Devlin JL, Kren SM, Burchfield JS, Taylor DA. Optimizing recellularization of whole decellularized heart extracellular matrix. PLoS One. 2014;9. https://doi.org/10.1371/journal.pone.0090406.

    Article  Google Scholar 

  10. 10.

    Weymann A, Patil NP, Sabashnikov A, Jungebluth P, Korkmaz S, Li S, et al. Bioartificial heart: a human-sized porcine model - the way ahead. PLoS One. 2014;9. https://doi.org/10.1371/journal.pone.0111591.

    Article  Google Scholar 

  11. 11.

    Ott HC, Matthiesen TS, Goh SK, Black LD, Kren SM, Netoff TI, et al. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med. 2008;14:213–21. https://doi.org/10.1038/nm1684.

    CAS  Article  Google Scholar 

  12. 12.

    Nagayo M. Zur normalen und pathologischen Histologie des Endocardium parietale. Berlin: G Fischer; 1909.

    Google Scholar 

  13. 13.

    Candiollo L. The fine structure of the endocardial endothelium. Z Zelforsch. 1963;61:486–92. https://doi.org/10.1177/0192513X12437708.

    CAS  Article  Google Scholar 

  14. 14.

    Melax H, Leeson TS. Fine structure of the endocrdium in adult rats. Cardiovasc Res. 1967;1:349–55. https://doi.org/10.1017/S1461145712000284.

    CAS  Article  Google Scholar 

  15. 15.

    Brutsaert DL. The endocardium. Annu Rev Physiol. 1989;51:263–73.

    CAS  Article  Google Scholar 

  16. 16.

    Brutsaert DL, Meulemans AL, Sipido KR, Sys SU. Effects of damaging the endocardial surface on the mechanical performance of isolated cardiac muscle. Circ Res. 1988;62:358–66.

    CAS  Article  Google Scholar 

  17. 17.

    Fisher ER, Davis ER. Observations concerning the pathogenesis of endocardial thickening in the adult heart. Am Heart J. 1958;56:553–61.

    CAS  Article  Google Scholar 

  18. 18.

    OKADA R. Clinicopathological study on the thickening of parietal endocardium in the adult heart. Jpn Heart J. 1961;2:220–55. https://doi.org/10.1536/ihj.2.220.

    CAS  Article  Google Scholar 

  19. 19.

    Hutchins GM, Vie SA. The progression of interstitial myocarditis to idiopathic endocardial fibroelastosis. Am J Pathol. 1971;66:483–96.

    Google Scholar 

  20. 20.

    Ng SLJ, Narayanan K, Gao S, Wan ACA. Lineage restricted progenitors for the repopulation of decellularized heart. Biomaterials. 2011;32:7571–80. https://doi.org/10.1016/j.biomaterials.2011.06.065.

    CAS  Article  Google Scholar 

  21. 21.

    Weymann A, Loganathan S, Takahashi H, Schies C, Claus B, Hirschberg K, et al. Development and evaluation of a perfusion decellularization porcine heart model. Circ J. 2011;75:852–60. https://doi.org/10.1253/circj.CJ-10-0717.

    Article  Google Scholar 

  22. 22.

    Schulte JB, Simionescu A, Simionescu DT. The acellular myocardial flap: a novel extracellular matrix scaffold enriched with patent microvascular networks and biocompatible cell niches. Tissue Eng Part C Methods. 2013;19:518–30. https://doi.org/10.1089/ten.TEC.2012.0536.

    CAS  Article  Google Scholar 

  23. 23.

    Deborde C, Simionescu DT, Wright C, Liao J, Sierad LN, Simionescu A. Stabilized collagen and elastin-based scaffolds for mitral valve tissue engineering. Tissue Eng Part A. 2016;22:1241–51. https://doi.org/10.1089/ten.tea.2016.0032.

    CAS  Article  Google Scholar 

  24. 24.

    Carvalho J, de CPH, Gomes DA, Goes AM. Characterization of decellularized heart matrices as biomaterials for regular and whole organ tissue engineering and initial in-vitro recellularization with ips cells. J Tissue Sci Eng. 2012;11. https://doi.org/10.4172/2157-7552.S11-002.Characterization.

  25. 25.

    Yasui H, Lee JK, Yoshida A, Yokoyama T, Nakanishi H, Miwa K, et al. Excitation propagation in three-dimensional engineered hearts using decellularized extracellular matrix. Biomaterials. 2014;35:7839–50. https://doi.org/10.1016/j.biomaterials.2014.05.080.

    CAS  Article  Google Scholar 

  26. 26.

    Akhyari P, Aubin H, Gwanmesia P, Barth M, Hoffmann S, Huelsmann J, et al. The quest for an optimized protocol for whole-heart decellularization: a comparison of three popular and a novel Decellularization technique and their diverse effects on crucial extracellular matrix qualities. Tissue Eng Part C Methods. 2011;17:915–26. https://doi.org/10.1089/ten.tec.2011.0210.

    CAS  Article  Google Scholar 

  27. 27.

    Zhang G-W, Gu T-X, Guan X-Y, Sun X-J, Qi X, Li X-Y, et al. bFGF binding cardiac extracellular matrix promotes the repair potential of bone marrow mesenchymal stem cells in a rabbit model for acute myocardial infarction. Biomed Mater. 2015;10:065018. https://doi.org/10.1088/1748-6041/10/6/065018.

    CAS  Article  Google Scholar 

  28. 28.

    Crawford B, Koshy ST, Jhamb G, Woodford C, Thompson CM, Levy AS, et al. Cardiac decellularisation with long-term storage and repopulation with canine peripheral blood progenitor cells. Can J Chem Eng. 2012;90:1457–64. https://doi.org/10.1002/cjce.20670.

    CAS  Article  Google Scholar 

  29. 29.

    Lin B, Lu TY, Yang L. Hear the beat: decellularized mouse heart regenerated with human induced pluripotent stem cells. Expert Rev Cardiovasc Ther. 2014;12:135–7. https://doi.org/10.1586/14779072.2014.879039.

    CAS  Article  Google Scholar 

  30. 30.

    Wainwright JM, Czajka CA, Patel UB, Freytes DO, Tobita K, Gilbert TW, et al. Preparation of cardiac extracellular matrix from an intact porcine heart. Tissue Eng Part C Methods. 2010;16:525–32. https://doi.org/10.1089/ten.tec.2009.0392.

    CAS  Article  Google Scholar 

  31. 31.

    Lu TY, Lin B, Kim J, Sullivan M, Tobita K, Salama G, et al. Repopulation of decellularized mouse heart with human induced pluripotent stem cell-derived cardiovascular progenitor cells. Nat Commun. 2013;4:1–11. https://doi.org/10.1038/ncomms3307.

    CAS  Article  Google Scholar 

  32. 32.

    Alcon A, Bozkulak EC, Qyang Y. Regenerating functional heart tissue for myocardial repair. Cell Mol Life Sci. 2012;69:2635–56. https://doi.org/10.1016/j.neuron.2009.10.017.A.

    CAS  Article  Google Scholar 

  33. 33.

    Rao RR, Peterson AW, Ceccarelli J, Putnam AJ, Stegemann JP. Matrix composition regulates three-dimensional network formation by endothelial cells and mesenchymal stem cells in collagen/fibrin materials. Angiogenesis. 2012;15:253–64. https://doi.org/10.1007/s10456-012-9257-1.

    CAS  Article  Google Scholar 

  34. 34.

    Goo S, Joshi P, Sands G, Gerneke D, Taberner A, Dollie Q, et al. Trabeculae carneae as models of the ventricular walls: implications for the delivery of oxygen. J Gen Physiol. 2009;134:339–50. https://doi.org/10.1085/jgp.200910276.

    Article  Google Scholar 

Download references

Acknowledgements

The authors wish to thank Godly-Snell Research Center for providing rabbit hearts used in preliminary studies pertaining to this data. They would also like to acknowledge Advanced Materials and Research Laboratory for providing training and access to their scanning electron microscopes.

Sources of Funding

This project was funded by the Harriet and Jerry Dempsey Bioengineering Professorship Award (to D.S.).

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Correspondence to Dan Simionescu.

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The authors declare that they have no competing interests.

Human Subjects/Informed Consent Statement

No human studies were carried out by the authors for this article.

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No animal studies were carried out by the authors for this article.

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Future Work

Future studies will be geared towards improvements in seeding efficacy, long-term conditioning in the bioreactor, and assessment of biological functionality of the newly reconstituted endocardium. Using this system, we will also perfuse endothelial cells through the scaffold to reconstitute the coronary vasculature.

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Compton, C., Canavan, J., Mcleod, J. et al. Reconstitution of the Ventricular Endocardium Within Acellular Hearts. Regen. Eng. Transl. Med. 6, 90–100 (2020). https://doi.org/10.1007/s40883-019-00099-1

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Keywords

  • Stem cells
  • Tissue engineering
  • Acellular cardiac scaffolds
  • Bioreactors
  • Hydrogels
  • Layer-by-layer