Advertisement

Reconstitution of the Ventricular Endocardium Within Acellular Hearts

  • Clayton Compton
  • Jessica Canavan
  • John Mcleod
  • Connor Prevost
  • Dan SimionescuEmail author
Article
  • 2 Downloads

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.

Keywords

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

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

Notes

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.).

Compliance with Ethical Standards

Conflict of Interest

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.

Animal Studies

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

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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.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.
  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.
  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.CrossRefGoogle 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.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.CrossRefGoogle Scholar
  15. 15.
    Brutsaert DL. The endocardium. Annu Rev Physiol. 1989;51:263–73.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle Scholar

Copyright information

© The Regenerative Engineering Society 2019

Authors and Affiliations

  1. 1.Clemson University ClemsonClemsonUSA

Personalised recommendations