Journal of Materials Science: Materials in Medicine

, Volume 22, Issue 12, pp 2641–2650 | Cite as

Enzymatic cross-linking of human recombinant elastin (HELP) as biomimetic approach in vascular tissue engineering

  • Sabrina Bozzini
  • Liliana Giuliano
  • Lina Altomare
  • Paola Petrini
  • Antonella Bandiera
  • Maria Teresa Conconi
  • Silvia Farè
  • Maria Cristina Tanzi


The use of polymers naturally occurring in the extracellular matrix (ECM) is a promising strategy in regenerative medicine. If compared to natural ECM proteins, proteins obtained by recombinant DNA technology have intrinsic advantages including reproducible macromolecular composition, sequence and molecular mass, and overcoming the potential pathogens transmission related to polymers of animal origin. Among ECM-mimicking materials, the family of recombinant elastin-like polymers is proposed for drug delivery applications and for the repair of damaged elastic tissues. This work aims to evaluate the potentiality of a recombinant human elastin-like polypeptide (HELP) as a base material of cross-linked matrices for regenerative medicine. The cross-linking of HELP was accomplished by the insertion of cross-linking sites, glutamine and lysine, in the recombinant polymer and generating ε-(γ-glutamyl) lysine links through the enzyme transglutaminase. The cross-linking efficacy was estimated by infrared spectroscopy. Freeze-dried cross-linked matrices showed swelling ratios in deionized water (≈2500%) with good structural stability up to 24 h. Mechanical compression tests, performed at 37°C in wet conditions, in a frequency sweep mode, indicated a storage modulus of 2/3 kPa, with no significant changes when increasing number of cycles or frequency. These results demonstrate the possibility to obtain mechanically resistant hydrogels via enzymatic crosslinking of HELP. Cytotoxicity tests of cross-linked HELP were performed with human umbilical vein endothelial cells, by use of transwell filter chambers for 1–7 days, or with its extracts in the opportune culture medium for 24 h. In both cases no cytotoxic effects were observed in comparison with the control cultures. On the whole, the results suggest the potentiality of this genetically engineered HELP for regenerative medicine applications, particularly for vascular tissue regeneration.


Storage Modulus Human Umbilical Vein Endothelial Cell Cytotoxicity Test Genipin Vascular Tissue Engineering 



This research was financed by Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR), Italy, under PRIN Project 2007, funds to Prof. MC Tanzi.


  1. 1.
    Rodriguez-Cabello JC, Martin L, Alonso M, Arias FJ, Testera AM. ‘‘Recombinamers’’ as advanced materials for the post-oil age. Polymers. 2009;50:5159–69.CrossRefGoogle Scholar
  2. 2.
    Tamerler C, Sarikaya M. Molecular biomimetics: nanothecnology and bionanotechnology using genetically engineered peptides. Phil Trans R Soc A. 2009;367:1705–26.CrossRefGoogle Scholar
  3. 3.
    Badylak SF, Freytes DO, Gilbert TW. Extracellular matrix as a biological scaffold material: structure and function. Acta Biomater. 2009;5(1):1–13.CrossRefGoogle Scholar
  4. 4.
    Ma PX. Biomimetic materials for tissue engineering. Adv Drug Deliv Rev. 2008;60(2):184–98.CrossRefGoogle Scholar
  5. 5.
    Langer R, Tirrell DA. Designing materials for biology and medicine. Nature. 2004;428:487–92.CrossRefGoogle Scholar
  6. 6.
    Mithieux SM, Rasko JEJ, Weiss AS. Synthetic elastin hydrogels derived from massive elastic assemblies of self-organized human protein monomers. Biomaterials. 2004;25:4921–7.CrossRefGoogle Scholar
  7. 7.
    Daamen WF, Veerkamp JH, van Hest JCM, van Kuppevelt TH. Elastin as a biomaterial for tissue engineering. Biomaterials. 2007;28:4378–98.CrossRefGoogle Scholar
  8. 8.
    Chow D, Nunalee ML, Lim DW, Simnick AJ, Chilkoti A. Peptide-based biopolymers in biomedicine and biotechnology. Mater Sci Eng R Rep. 2008;6(2):125–55.CrossRefGoogle Scholar
  9. 9.
    Floss DM, Schallau K, Rose-John S, Conrad U, Scheller J. Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application. Trends Biotechnol. 2010;28(1):37–45.CrossRefGoogle Scholar
  10. 10.
    Urry DW. Entropic elastic processes in protein mechanisms I. Elastic structure due to an inverse temperature transition and elasticity due to internal chain dynamics. J Protein Chem. 1988;7:1–34.CrossRefGoogle Scholar
  11. 11.
    Luan CH, Harris RD, Prasad KU, Urry DW. Differential scanning calorimetry studies of the inverse temperature transition of the polypentapeptide of elastin and its analogues. Biopolymers. 1990;29:1699–706.CrossRefGoogle Scholar
  12. 12.
    Sallach RE, Cui W, Wen J, Martinez A, Conticello VP, Chaikof EL. Elastin-mimetic protein polymers capable of physical and chemical crosslinking. Biomaterials. 2009;30:409–22.CrossRefGoogle Scholar
  13. 13.
    Jagur-Grodzinskia J. Polymeric gels and hydrogels for biomedical and pharmaceutical applications. Polym Adv Technol. 2010;21:27–47.Google Scholar
  14. 14.
    Kopecěk J. Smart and genetically engineered biomaterials and drug delivery systems. Eur J Pharm Sci. 2003;20:1–16.CrossRefGoogle Scholar
  15. 15.
    L’Heureux N, Dusserre N, Konig G, Victor B, Keire P, Wight TN, Chronos NA, Kyles AE, Gregory CR, Hoyt G, Robbins RC, McAllister TN. Human tissue-engineered blood vessels for adult arterial revascularization. Nat Med. 2006;12:361–5.CrossRefGoogle Scholar
  16. 16.
    Mitchell SL, Niklason LE. Requirements for growing tissue-engineered vascular grafts. Cardiovasc Pathol. 2003;12:59–64.CrossRefGoogle Scholar
  17. 17.
    Opitz F, Schenke-Layland K, Cohnert TU, Starcher B, Halbhuber KJ, Martin DP, Stock UA. Tissue engineering of aortic tissue: dire consequence of suboptimal elastic fiber synthesis in vivo. Cardiovasc Res. 2004;63:719–30.CrossRefGoogle Scholar
  18. 18.
    McMillan RA, Caran KL, Apkarian RP, Conticello VP. High-resolution topographic imaging of environmentally responsive, elastin-mimetic hydrogels. Macromolecules. 1999;32:9067–70.CrossRefGoogle Scholar
  19. 19.
    McHale MK, Setton LA, Chilkoti A. Synthesis and in vitro evaluation of enzymatically cross-linked elastin-like polypeptide gels for cartilaginous tissue repair. Tissue Eng. 2005;11(11–12):1768–79.CrossRefGoogle Scholar
  20. 20.
    Girotti A, Reguera J, Rodriguez-Cabello JC, Arias FJ, Alonso M, Testera AM. Design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for tissue engineering purposes. J Mater Sci Mater Med. 2004;15(4):479–84.CrossRefGoogle Scholar
  21. 21.
    Annabi N, Mithieux SM, Weiss AS, Dehghani F. The fabrication of elastin-based hydrogels using high pressure CO2. Biomaterials. 2009;30:1–7.CrossRefGoogle Scholar
  22. 22.
    Welsh ER, Tirrell DA. Engineering the extracellular matrix: a novel approach to polymeric biomaterials I. Control of the physical properties of artificial protein matrices designed to support adhesion of vascular endothelial cells. Biomacromolecules. 2000;1:23–30.CrossRefGoogle Scholar
  23. 23.
    Annabi N, Mithieux SM, Weiss AS, Dehghani F. Cross-linked open-pore elastic hydrogels based on tropoelastin, elastin and high pressure CO2. Biomaterials. 2010;31:1655–65.CrossRefGoogle Scholar
  24. 24.
    Martino M, Tamburro AM. Chemical synthesis of cross-linked poly(KGGVG), an elastin-like biopolymer. Biopolymers. 2001;59:29–37.CrossRefGoogle Scholar
  25. 25.
    Bellingham CM, Lillie MA, Gosline JM, Wright GM, Starcher BC, Bailey AJ, Woodhouse KA, Keeley FW. Recombinant human elastin polypeptides self-assemble into biomaterials with elastin-like properties. Biopolymers. 2003;70:445–55.CrossRefGoogle Scholar
  26. 26.
    Vieth S, Bellingham CM, Keeley FW, Hodge SM, Rousseau D. Microstructural and tensile properties of elastin-based polypeptides crosslinked with genipin and pyrroloquinoline quinone. Biopolymers. 2007;85(3):199–206.CrossRefGoogle Scholar
  27. 27.
    Di Zio K, Tirrell DA. Mechanical properties of artificial protein matrices engineered for control of cell and tissue behavior. Macromolecules. 2003;36:1553–8.CrossRefGoogle Scholar
  28. 28.
    McMillan RA, Conticello VP. Synthesis and characterization of elastin-mimetic protein gels derived from a well-defined polypeptide precursor. Macromolecules. 2000;33:4809–21.CrossRefGoogle Scholar
  29. 29.
    Trabbic-Carlson K, Setton LA, Chilkoti A. Swelling and mechanical behaviors of chemically cross-linked hydrogels of elastin-like polypeptides. Biomacromolecules. 2003;4:572–80.CrossRefGoogle Scholar
  30. 30.
    Lee J, Macosko CW, Urry DW. Elastomeric polypentapeptides cross-linked into matrixes and fibers. Biomacromolecules. 2001;2:170–9.CrossRefGoogle Scholar
  31. 31.
    Lim DW, Nettles DL, Setton LA, Chilkoti A. In Situ cross-linking of elastin-like polypeptide block copolymers for tissue repair. Biomacromolecules. 2008;9:222–30.CrossRefGoogle Scholar
  32. 32.
    Lim DW, Nettles DL, Setton LA, Chilkoti A. Rapid cross-linking of elastin-like polypeptides with (hydroxymethyl) phosphines in aqueous solution. Biomacromolecules. 2007;8:1463–70.CrossRefGoogle Scholar
  33. 33.
    Nowatzki PJ, Tirrell DA. Physical properties of artificial extracellular matrix protein films prepared by isocyanate crosslinking. Biomaterials. 2004;25:1261–7.CrossRefGoogle Scholar
  34. 34.
    Martin L, Alonso M, Girotti A, Arias FJ, Rodrıguez-Cabello JC. Synthesis and characterization of macroporous thermosensitive hydrogels from recombinant elastin-like polymers. Biomacromolecules. 2009;10:3015–22.CrossRefGoogle Scholar
  35. 35.
    Hrabchak C, Rouleau J, Moss I, Woodhouse K, Akens M, Bellingham C, Keeley F, Dennis M, Yee A. Assessment of biocompatibility and initial evaluation of genipin cross-linked elastin-like polypeptides in the treatment of an osteochondral knee defect in rabbits. Acta Biomater. 2010;6:2108–15.CrossRefGoogle Scholar
  36. 36.
    Leach JB, Wolinsky JB, Stone PJ, Wong JY. Crosslinked α-elastin biomaterials: towards a processable elastin mimetic scaffold. Acta Biomater. 2005;1:155–64.CrossRefGoogle Scholar
  37. 37.
    Nagapudi K, Brinkman WT, Leisen JE, Huang L, McMillan RA, Apkarian RP, Conticello VP, Chaikof EL. Photomediated solid-state cross-linking of an elastin-mimetic recombinant protein polymer. Macromolecules. 2002;35:1730–7.CrossRefGoogle Scholar
  38. 38.
    Fujimoto M, Okamoto K, Furuta M. Preparation of alpha-elastin nanoparticles by gamma irradiation. Radiat Phys chem. 2009;78:1046–8.CrossRefGoogle Scholar
  39. 39.
    Garcia Y, Hemantkumar N, Collighan R, Griffin M, Rodriguez-Cabello JC, Abhay P. In vitro characterization of a collagen scaffold enzymatically cross-linked with a tailored elastin-like polymer. Tissue Eng A. 2009;15(4):887–99.CrossRefGoogle Scholar
  40. 40.
    Bandiera A, Taglienti A, Micali F, Pani B, Tamaro M, Crescenzi V, Manzini G. Expression and characterization of human elastin repeat based temperature responsive protein polymers for biotechnological purposes. Biotechnol Appl Biochem. 2005;42(3):247–56.CrossRefGoogle Scholar
  41. 41.
    Martinuzzi M, Marchetti S, Bandiera A, Farè S, Tanzi MC. Towards molecular farming of a human elastin-like polymer in plants, society for Biomaterials 2011 annual meeting: Animating materials, April 13–16, 2011 Orlando, Florida, accepted for oral presentation.Google Scholar
  42. 42.
    Bandiera A. 3D matrices of human elastin-like polypeptides and method of preparation thereof, WO/2010/119420, International Application No.: PCT/IB2010/051642, publication date 21.10.2010, applicant: Università degli Studi di Trieste, Italy.Google Scholar
  43. 43.
    Socrates G. Infrared and raman characteristic group frequencies: tables and charts. 3rd Ed. London: Wiley; 2004.Google Scholar
  44. 44.
    Hoffman AS. Hydrogels for biomedical applications. Adv Drug Deliv Rev. 2002;43:3–12.CrossRefGoogle Scholar
  45. 45.
    Pankajakshan D, Agrawal DK. Scaffolds in tissue engineering of blood vessels. Can J Physiol Pharmacol. 2010;88(9):855–73.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Sabrina Bozzini
    • 1
  • Liliana Giuliano
    • 1
  • Lina Altomare
    • 1
  • Paola Petrini
    • 1
  • Antonella Bandiera
    • 2
  • Maria Teresa Conconi
    • 3
  • Silvia Farè
    • 1
  • Maria Cristina Tanzi
    • 1
  1. 1.Bioengineering Department, Biomaterials LaboratoryPolitecnico di MilanoMilanItaly
  2. 2.Department of Life SciencesUniversità degli Studi di TriesteTriesteItaly
  3. 3.Department of Pharmaceutical SciencesUniversità degli Studi di PadovaPadovaItaly

Personalised recommendations