OSA's Digital Library

Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 19, Iss. 17 — Aug. 15, 2011
  • pp: 15996–16007

Two-photon induced collagen cross-linking in bioartificial cardiac tissue

Kai Kuetemeyer, George Kensah, Marko Heidrich, Heiko Meyer, Ulrich Martin, Ina Gruh, and Alexander Heisterkamp  »View Author Affiliations

Optics Express, Vol. 19, Issue 17, pp. 15996-16007 (2011)

View Full Text Article

Enhanced HTML    Acrobat PDF (3589 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



Cardiac tissue engineering is a promising strategy for regenerative therapies to overcome the shortage of donor organs for transplantation. Besides contractile function, the stiffness of tissue engineered constructs is crucial to generate transplantable tissue surrogates with sufficient mechanical stability to withstand the high pressure present in the heart. Although several collagen cross-linking techniques have proven to be efficient in stabilizing biomaterials, they cannot be applied to cardiac tissue engineering, as cell death occurs in the treated area. Here, we present a novel method using femtosecond (fs) laser pulses to increase the stiffness of collagen-based tissue constructs without impairing cell viability. Raster scanning of the fs laser beam over riboflavin-treated tissue induced collagen cross-linking by two-photon photosensitized singlet oxygen production. One day post-irradiation, stress-strain measurements revealed increased tissue stiffness by around 40% being dependent on the fibroblast content in the tissue. At the same time, cells remained viable and fully functional as demonstrated by fluorescence imaging of cardiomyocyte mitochondrial activity and preservation of active contraction force. Our results indicate that two-photon induced collagen cross-linking has great potential for studying and improving artificially engineered tissue for regenerative therapies.

© 2011 OSA

OCIS Codes
(170.1610) Medical optics and biotechnology : Clinical applications
(190.4180) Nonlinear optics : Multiphoton processes
(260.5130) Physical optics : Photochemistry

ToC Category:
Medical Optics and Biotechnology

Original Manuscript: May 31, 2011
Revised Manuscript: July 15, 2011
Manuscript Accepted: July 18, 2011
Published: August 5, 2011

Virtual Issues
Vol. 6, Iss. 9 Virtual Journal for Biomedical Optics

Kai Kuetemeyer, George Kensah, Marko Heidrich, Heiko Meyer, Ulrich Martin, Ina Gruh, and Alexander Heisterkamp, "Two-photon induced collagen cross-linking in bioartificial cardiac tissue," Opt. Express 19, 15996-16007 (2011)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. WHO (World Health Organization), “Cardiovascular Diseases,” Fact Sheet Number 317, Geneva, Switzerland, January2011.
  2. W. H. Zimmermann, C. Fink, D. Kralisch, U. Remmers, J. Weil, and T. Eschenhagen, “Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes,” Biotechnol. Bioeng. 68, 106–114 (2000). [CrossRef] [PubMed]
  3. W. H. Zimmermann, M. Tiburcy, and T. Eschenhagen, “Cardiac tissue engineering: a clinical perspective,” Future Cardiol. 3, 435–445 (2007). [CrossRef] [PubMed]
  4. K. L. Kreutziger and C. E. Murry, “Engineered human cardiac tissue,” Pediatr. Cardiol. 32, 334–341 (2011). [CrossRef] [PubMed]
  5. B. Bhana, R. K. Iyer, W. L. Chen, R. Zhao, K. L. Sider, M. Likhitpanichkul, C. A. Simmons, and M. Radisic, “Influence of substrate stiffness on the phenotype of heart cells,” Biotechnol. Bioeng. 105, 1148–1160 (2010).
  6. L. Moeller, A. Krause, J. Dahlmann, I. Gruh, A. Kirschning, and G. Draeger, “Preparation and evaluation of hydrogel-composites from methacrylated hyaluronic acid, alginate, and gelatin for tissue engineering,” Int. J. Artif. Organs 34, 93–102 (2011). [CrossRef]
  7. A. Marsano, R. Maidhof, L. Q. Wan, Y. Wang, J. Gao, N. Tandon, and G. Vunjak-Novakovic, “Scaffold stiffness affects the contractile function of three-dimensional engineered cardiac constructs,” Biotechnol. Prog. 26, 1382–1390 (2010). [CrossRef] [PubMed]
  8. C. Fink, S. Ergun, D. Kralisch, U. Remmers, J. Weil, and T. Eschenhagen, “Chronic stretch of engineered heart tissue induces hypertrophy and functional improvement,” FASEB J. 14, 669–679 (2000). [PubMed]
  9. G. Kensah, I. Gruh, J. Viering, H. Schumann, J. Dahlmann, H. Meyer, D. Skvorc, A. Baer, P. Akhyari, A. Heisterkamp, A. Haverich, and U. Martin, “A novel miniaturized multimodal bioreactor for continuous in situ assessment of bioartificial cardiac tissue during stimulation and maturation,” Tissue Eng. Pt. C Methods 17, 463–473 (2011). [CrossRef]
  10. W. M. Elbjeirami, E. O. Yonter, B. C. Starcher, and J. L. West, “Enhancing mechanical properties of tissue-engineered constructs via lysyl oxidase crosslinking activity,” J. Biomed. Mater. Res. 66, 513–521 (2003). [CrossRef]
  11. T. S. Girton, T. R. Oegema, and R. T. Tranquillo, “Exploiting glycation to stiffen and strengthen tissue equivalents for tissue engineering,” J. Biomed. Mater. Res. 46, 87–92 (1999). [CrossRef] [PubMed]
  12. C. L. McIntosh, L. L. Michaelis, A. G. Morrow, S. B. Itscoitz, D. R. Redwood, and S. E. Epstein, “Atrioventricular valve replacement with the Hancock porcine xenograft: a five-year clinical experience,” Surgery 78, 768–775 (1975). [PubMed]
  13. H. Dardik, I. M. Ibrahim, R. Baier, S. Sprayregen, M. Levy, and I. I. Dardik, “Human umbilical cord. A new source for vascular prosthesis,” JAMA J. Am. Med. Assoc. 236, 2859–2862 (1976). [CrossRef]
  14. G. Wollensak, E. Spoerl, and T. Seiler, “Riboflavin/ultraviolet-A-induced collagen crosslinking for the treatment of keratoconus,” Am. J. Ophthalmol. 135, 620–627 (2003). [CrossRef] [PubMed]
  15. A. Jayakrishnan and S. R. Jameela, “Glutaraldehyde as a fixative in bioprostheses and drug delivery matrices,” Biomaterials 17, 471–484 (1996). [CrossRef] [PubMed]
  16. M. C. DeRosa and R. J. Crutchley, “Photosensitized singlet oxygen and its applications,” Coordin. Chem. Rev. 233–234, 351–371 (2002). [CrossRef]
  17. A. S. McCall, S. Kraft, H. F. Edelhauser, G. W. Kidder, R. R. Lundquist, H. E. Bradshaw, Z. Dedeic, M. J. C. Dionne, E. M. Clement, and G. W Conrad, “Mechanisms of corneal tissue cross-linking in response to treatment with topical riboflavin and long-wavelength ultraviolet radiation (UVA),” Invest. Ophthalmol. Visual Sci. 51, 129–138 (2010). [CrossRef]
  18. G. Wollensak, E. Spoerl, M. Wilsch, and T. Seiler, “Keratocyte apoptosis after corneal collagen cross-linking using riboflavin/UVA treatment,” Cornea 23, 43–49 (2004). [CrossRef] [PubMed]
  19. T. Tanabe, M. Oyamada, K. Fujita, P. Dai, H. Tanaka, and T. Takamatsu, “Multiphoton excitationevoked chromophore assisted laser inactivation using green fluorescent protein,” Nat. Methods 2, 503–505 (2005). [CrossRef] [PubMed]
  20. K. Koenig, I. Riemann, P. Fischer, and K. H. Halbhuber, “Intracellular nanosurgery with near infrared femtosecond laser pulses,” Cell Mol. Biol. (Paris) 45, 195–201 (1999).
  21. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990). [CrossRef] [PubMed]
  22. D. Warther, S. Gug, A. Specht, F. Bolze, J. F. Nicoud, A. Mourot, and M. Goeldner, “Two-photon uncaging: new prospects in neuroscience and cellular biology,” Bioorgan. Med. Chem. 18, 7753–7758 (2010). [CrossRef]
  23. K. Kuetemeyer, R. Rezgui, H. Lubatschowski, and A. Heisterkamp, “Influence of laser parameters and staining on femtosecond laser-based intracellular nanosurgery,” Biomed. Opt. Express 1, 587–597 (2010). [CrossRef]
  24. P. K. Frederiksen, M. Jorgensen, and P. R. Ogilby, “Two-photon photosensitized production of singlet oxygen,” J. Am. Chem. Soc. 123, 1215–1221 (2001). [CrossRef] [PubMed]
  25. K. Koenig, “Multiphoton microscopy in life sciences,” J. Microsc. 200, 83–104 (2000). [CrossRef]
  26. A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80, 2029–2036 (2001). [CrossRef] [PubMed]
  27. S. Kalies, K. Kuetemeyer, and A. Heisterkamp, “Mechanisms of high-order photobleaching and its relationship to intracellular ablation,” Biomed. Opt. Express 2, 805–816 (2011). [CrossRef] [PubMed]
  28. A. Vogel, J. Noack, G. Huettman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B 81, 1015–1047 (2005). [CrossRef]
  29. G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms, and T. Schmidt, “Two-photon exciation action cross-sections of the autofluorescent proteins,” Chem. Phys. Lett. 350, 71–77 (2001). [CrossRef]
  30. W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100, 7075–7080 (2003). [CrossRef] [PubMed]
  31. R. A. Lorbeer, M. Heidrich, C. Lorbeer, D. F. Ramirez-Ojeda, G. Bicker, H. Meyer, and A. Heisterkamp, “Highly efficient 3D fluorescence microscopy with a scanning laser optical tomograph,” Opt. Express 19, 5419–5430 (2011). [CrossRef] [PubMed]
  32. M. H. Niemz, Laser-Tissue Interactions: Fundamentals and Applications (Springer, 2007).
  33. B. A. Roeder, K. Kokini, J. E. Sturgis, J. P. Robinson, and S. L. Voytik-Harbin, “Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure,” J. Biomech. Eng. 124, 214–222 (2002). [CrossRef] [PubMed]
  34. G. Wollensak, E. Spoerl, and T. Seiler, “Stress-strain measurements of human and porcine corneas after riboflavinultraviolet-A-induced cross-linking,” J. Cataract Refractive Surg. 29, 1780–1785 (2003). [CrossRef]
  35. M. Eghbali and K. T. Weber, “Collagen and the myocardium: fibrillar structure, biosynthesis and degradation in relation to hypertrophy and its regression,” Mol. Cell. Biochem. 96, 1–14 (1990). [CrossRef] [PubMed]
  36. C. Xu and W. W. Webb “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996). [CrossRef]
  37. B. P. Yu, “Cellular defenses against damage from reactive oxygen species,” Physiol. Rev. 74, 139–162 (1994). [PubMed]
  38. Z. H. Syedain, J. Bjork, L. Sando, and R. T. Tranquillo, “Controlled compaction with ruthenium-catalyzed photochemical cross-linking of fibrin-based engineered connective tissue,” Biomaterials 30, 6695–6701 (2009). [CrossRef] [PubMed]
  39. A. Vogel and V. Venugopalan, “Mechanisms of pulsed laser ablation of biological tissues,” Chem. Rev. 103, 577–644 (2003). [CrossRef] [PubMed]
  40. G. M. Fomovsky, J. R. Macadangdang, G. Ailawadi, and J. W. Holmes, “Model-based design of mechanical therapies for myocardial infarction,” J. Cardiovasc. Transl. Res. 4, 82–91 (2011). [CrossRef]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited