Skip to main content
SpringerLink
Log in

The cytoprotective capacity of processed human cardiac extracellular matrix

  • Tissue Engineering Constructs and Cell Substrates
  • Original Research
  • Published:
Journal of Materials Science: Materials in Medicine Aims and scope Submit manuscript

Abstract

Freshly isolated human cardiac extracellular matrix sheets (cECM) have been shown to support stem cell proliferation and tissue-specific lineage commitment. We now developed a protocol for standardized production of durable, bio-functional hcECM microparticles and corresponding hydrogel, and tested its cytoprotective effects on contractile cells subjected to ischemia-like conditions. Human ventricular myocardium was decellularized by a 3-step protocol, including Tris/EDTA, SDS and serum incubation (cECM). Following snap-freezing and lyophilization, microparticles were created and characterized by laser diffraction, dynamic image analysis (DIA), and mass spectrometry. Moreover, cECM hydrogel was produced by pepsin digestion. Baseline cell-support characteristics were determined using murine HL-1 cardiomyocytes, and the cytoprotective effects of ECM products were tested under hypoxia and glucose/serum deprivation. In cECM, glycoproteins (thrombospondin 1, fibronectin, collagens and nidogen-1) and proteoglycans (dermatopontin, lumican and mimecan) were preserved, but residual intracellular and blood-borne proteins were also detected. The median particle feret diameter was 66 μm (15–157 μm) by laser diffraction, and 57 μm (20–182 μm) by DIA with crystal violet staining. HL-1 cells displayed enhanced metabolic activity (39 ± 12 %, P < 0.05) and proliferation (16 ± 3 %, P < 0.05) when grown on cECM microparticles in normoxia. During simulated ischemia, cECM microparticles exerted distinct cytoprotective effects (MTS conversion, 240 ± 32 %; BrdU uptake, 45 ± 14 %; LDH release, −72 ± 7 %; P < 0.01, each). When cECM microparticles were solubilized to form a hydrogel, the cytoprotective effect was initially abolished. However, modifying the preparation process (pepsin digestion at pH 2 and 25 °C, 1 mg/ml final cECM concentration) restored the cytoprotective cECM activity. Extracellular matrix from human myocardium can be processed to yield standardized durable microparticles that exert specific cytoprotective effects on cardiomyocyte-like cells. The use of processed cECM may help to optimize future clinical-grade myocardial tissue engineering approaches.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Oberwallner B, Brodarac A, Anic P, Saric T, Wassilew K, Neef K, Choi YH, Stamm C. Human cardiac extracellular matrix supports myocardial lineage commitment of pluripotent stem cells. Eur J Cardio-Thorac Surg. 2015;47:416–25 (discussion 425).

    Article  Google Scholar 

  2. Oberwallner B, Brodarac A, Choi YH, Saric T, Anic P, Morawietz L, Stamm C. Preparation of cardiac extracellular matrix scaffolds by decellularization of human myocardium. J Biomed Mater Res Part A. 2014;102:3263–72.

    Article  Google Scholar 

  3. Dai W, Gerczuk P, Zhang Y, Smith L, Kopyov O, Kay GL, Jyrala AJ, Kloner RA. Intramyocardial injection of heart tissue-derived extracellular matrix improves postinfarction cardiac function in rats. J Cardiovasc Pharmacol Ther. 2013;18:270–9.

    Article  Google Scholar 

  4. Godier-Furnemont AF, Martens TP, Koeckert MS, Wan L, Parks J, Arai K, Zhang G, Hudson B, Homma S, Vunjak-Novakovic G. Composite scaffold provides a cell delivery platform for cardiovascular repair. Proc Natl Acad Sci USA. 2011;108:7974–9.

    Article  Google Scholar 

  5. Gui L, Chan SA, Breuer CK, Niklason LE. Novel utilization of serum in tissue decellularization. Tissue Eng Part C Methods. 2010;16:173–84.

    Article  Google Scholar 

  6. Seif-Naraghi SB, Salvatore MA, Schup-Magoffin PJ, Hu DP, Christman KL. Design and characterization of an injectable pericardial matrix gel: a potentially autologous scaffold for cardiac tissue engineering. Tissue Eng Part A. 2010;16:2017–27.

    Article  Google Scholar 

  7. Singelyn JM, DeQuach JA, Seif-Naraghi SB, Littlefield RB, Schup-Magoffin PJ, Christman KL. Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering. Biomaterials. 2009;30:5409–16.

    Article  Google Scholar 

  8. Zabel C, Klose J. High-resolution large-gel 2de. Methods Mol Biol. 2009;519:311–38.

    Article  Google Scholar 

  9. Zabel C, Klose J. Protein extraction for 2de. Methods Mol Biol. 2009;519:171–96.

    Article  Google Scholar 

  10. Hartl D, Rohe M, Mao L, Staufenbiel M, Zabel C, Klose J. Impairment of adolescent hippocampal plasticity in a mouse model for alzheimer’s disease precedes disease phenotype. PLoS One. 2008;3:e2759.

    Article  Google Scholar 

  11. Mie G. Beiträge zur optik trüber medien, speziell kolloidaler metallösungen. Ann Phys. 1908;4:377–445.

    Article  Google Scholar 

  12. Claycomb WC, Lanson NA Jr, Stallworth BS, Egeland DB, Delcarpio JB, Bahinski A, Izzo NJ Jr. Hl-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci USA. 1998;95:2979–84.

    Article  Google Scholar 

  13. Bader AM, Brodarac A, Klose K, Bieback K, Choi YH, Kurtz A, Stamm C. Mechanisms of paracrine cardioprotection by cord blood mesenchymal stromal cells. Eur J Cardio-Thorac Surg. 2014;45:983–92.

    Article  Google Scholar 

  14. Badylak SF. The extracellular matrix as a biologic scaffold material. Biomaterials. 2007;28:3587–93.

    Article  Google Scholar 

  15. Eitan Y, Sarig U, Dahan N, Machluf M. Acellular cardiac extracellular matrix as a scaffold for tissue engineering: in-vitro cell support, remodeling and biocompatibility. Tissue Eng Part C Methods. 2009;16:671–83.

    Article  Google Scholar 

  16. Hata H, Bar A, Dorfman S, Vukadinovic Z, Sawa Y, Haverich A, Hilfiker A. Engineering a novel three-dimensional contractile myocardial patch with cell sheets and decellularised matrix. Eur J Cardiothorac Surg. 2010;38:450–5.

    Article  Google Scholar 

  17. Ott HC, Matthiesen TS, Goh S-K, Black LD, Kren SM, Netoff TI, Taylor DA. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med. 2008;14:213–21.

    Article  Google Scholar 

  18. Bayrak A, Tyralla M, Ladhoff J, Schleicher M, Stock UA, Volk HD, Seifert M. Human immune responses to porcine xenogeneic matrices and their extracellular matrix constituents in vitro. Biomaterials. 2010;31:3793–803.

    Article  Google Scholar 

  19. Brodarac A, Saric T, Oberwallner B, Mahmoodzadeh S, Neef K, Albrecht J, Burkert K, Oliverio M, Nguemo F, Choi YH, Neiss WF, Morano I, Hescheler J, Stamm C. Susceptibility of murine induced pluripotent stem cell-derived cardiomyocytes to hypoxia and nutrient deprivation. Stem Cell Res Ther. 2015;6:83.

    Article  Google Scholar 

  20. Hughes BG, Schulz R. Targeting mmp-2 to treat ischemic heart injury. Basic Res Cardiol. 2014;109:424.

    Article  Google Scholar 

  21. Takawale A, Fan D, Basu R, Shen M, Parajuli N, Wang W, Wang X, Oudit GY, Kassiri Z. Myocardial recovery from ischemia-reperfusion is compromised in the absence of tissue inhibitor of metalloproteinase 4. Circ Heart Fail. 2014;7:652–62.

    Article  Google Scholar 

  22. Hsu PL, Su BC, Kuok QY, Mo FE. Extracellular matrix protein ccn1 regulates cardiomyocyte apoptosis in mice with stress-induced cardiac injury. Cardiovasc Res. 2013;98:64–72.

    Article  Google Scholar 

  23. Law CH, Li JM, Chou HC, Chen YH, Chan HL. Hyaluronic acid-dependent protection in h9c2 cardiomyocytes: a cell model of heart ischemia-reperfusion injury and treatment. Toxicology. 2013;303:54–71.

    Article  Google Scholar 

  24. Kandasamy AD, Chow AK, Ali MA, Schulz R. Matrix metalloproteinase-2 and myocardial oxidative stress injury: beyond the matrix. Cardiovasc Res. 2010;85:413–23.

    Article  Google Scholar 

  25. Seif-Naraghi SB, Singelyn JM, Salvatore MA, Osborn KG, Wang JJ, Sampat U, Kwan OL, Strachan GM, Wong J, Schup-Magoffin PJ, Braden RL, Bartels K, DeQuach JA, Preul M, Kinsey AM, DeMaria AN, Dib N, Christman KL. Safety and efficacy of an injectable extracellular matrix hydrogel for treating myocardial infarction. Sci Transl Med. 2013;5:173ra125.

    Article  Google Scholar 

Download references

Acknowledgments

We thank Dr. Katharina Wassilew for her help with preliminary experiments and Mrs. Anne Gale for copy-editing this manuscript. The work was supported with grants from the Federal Ministry of Education and Research and the federal states of Berlin and Brandenburg (FKZ 1315848A, FKZ 13GW0099).

Author information

Author notes

    Authors and Affiliations

    1. Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité – Universitätsmedizin Berlin, Berlin, Germany

      Benjamin Kappler, Petra Anic, Matthias Becker, Kristin Klose, Oliver Klein, Barbara Oberwallner & Christof Stamm

    2. Department of Cardiac and Thoracic Surgery, Heart Center of the University, University of Cologne, Cologne, Germany

      Yeong-Hoon Choi

    3. DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, Berlin, Germany

      Andreas Bader, Volkmar Falk & Christof Stamm

    4. Deutsches Herzzentrum Berlin (DHZB), Augustenburger Platz 1, 13353, Berlin, Germany

      Andreas Bader, Volkmar Falk & Christof Stamm

    Authors
    1. Benjamin Kappler
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Petra Anic
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Matthias Becker
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Andreas Bader
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Kristin Klose
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Oliver Klein
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Barbara Oberwallner
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Yeong-Hoon Choi
      View author publications

      You can also search for this author in PubMed Google Scholar

    9. Volkmar Falk
      View author publications

      You can also search for this author in PubMed Google Scholar

    10. Christof Stamm
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Christof Stamm.

    Additional information

    Benjamin Kappler and Petra Anic have contributed equally to this work.

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Kappler, B., Anic, P., Becker, M. et al. The cytoprotective capacity of processed human cardiac extracellular matrix. J Mater Sci: Mater Med 27, 120 (2016). https://doi.org/10.1007/s10856-016-5730-5

    Download citation

    • Received:

    • Accepted:

    • Published:

    • DOI: https://doi.org/10.1007/s10856-016-5730-5

    Keywords

    Search

    Navigation