Advertisement

Assessment of cell viability in a three-dimensional enzymatically cross-linked collagen scaffold

  • Y. Garcia
  • R. Collighan
  • M. Griffin
  • A. PanditEmail author
Article

Abstract

Microbial transglutaminase (mTGase) is an enzyme that introduces a covalent bond between peptide bound glutamine and lysine residues. Proteins cross-linked in this manner are often more resistant to proteolytic degradation and show increased tensile strength. This study evaluates the effects of mTGase mediated cross-linking of collagen on the cellular morphology, behaviour and viability of murine 3T3 fibroblasts following their seeding into collagen scaffolds. Additionally, cell mediated scaffold contraction, porosity and level of cross-linking of the scaffold has been analysed using image analysis software, scanning electron microscopy (SEM), colorimetric assays, and Fourier transform infrared spectroscopy (FTIR). We demonstrate that the biocompatibility and cellular morphology, when comparing cultures of fibroblasts integrated in mTGase cross-linked collagen scaffolds with the native collagen counterparts, remained unaffected. It has been also elicited that the structural characteristics of collagen have been preserved while introducing enzymatically resistant covalent bonds.

Keywords

Collagen Scaffold Olympus BX51 Fluorescent Microscope Microbial Transglutaminase Triple Helix Structure TNBS Assay 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

This experimental work has been funded by The Irish Higher Education Authority’s Programme for Research in third Level Institutions, Enterprise Ireland Research Innovation Fund and EPSRC (grant reference GR/S21755/02). Special thanks are due to Damien O’Halloran and Michael Ball for all the help provided in the laboratory work and Afshin Samali and Eva Szegezdi for their valuable comments on apoptosis evaluation.

References

  1. 1.
    C. H. LEE, A. SINGLA and Y. LEE, Int. J. Pharm. 221 (2001) 1CrossRefGoogle Scholar
  2. 2.
    M. D. FERNANDEZ-DIAZ, P. MONTERO and M. C. GOMEZ-GUILLEN, Food Chem. 74 (2001) 161CrossRefGoogle Scholar
  3. 3.
    W. FRIESS, Eur. J. Pharm. Biopharm. 45 (1998) 113CrossRefGoogle Scholar
  4. 4.
    D. Y. S. CHAU, R. J. COLLIGHAN, E. A. M. VERDERIO, V. L. ADDY and M. GRIFFIN, Biomaterials 26 (2005) 6518CrossRefGoogle Scholar
  5. 5.
    B. ECKES, P. ZIGRINO, D. KESSLER, O. HOLTKOTTER, P. SHEPHARD, C. MAUCH and T. KRIEG, Matrix. Biol. 19 (2000) 325CrossRefGoogle Scholar
  6. 6.
    F. GRINNELL, Trends Cell Biol. 10 (2000) 362CrossRefGoogle Scholar
  7. 7.
    M. GRIFFIN, R. CASADIO and C. M. BERGAMINI, Biochem. J. 368 (2002) 377CrossRefGoogle Scholar
  8. 8.
    R. SHARMA, P. C. LORENZEN and K. B. QVIST, Int. Dairy J. 11 (2001) 785CrossRefGoogle Scholar
  9. 9.
    M. MOTOKI and K. SEGURO, Trends Food Sci. Technol. 9 (1998) 204CrossRefGoogle Scholar
  10. 10.
    P. GRENARD, S. BRESSON-HADNI, S. EL ALAOUI, M. CHEVALLIER, D. A. VUITTON and S. RICARD-BLUM, J. Hepatol. 35 (2001) 367CrossRefGoogle Scholar
  11. 11.
    S. ALLEN, J. SOTOS, M. J. SYLTE and C. J. CZUPRYNSKI, Clin. Diagn. Lab. Immunol. 8 (2001) 460CrossRefGoogle Scholar
  12. 12.
    W. A. BUBNIS and C. M. OFNER 3rd, Anal. Biochem. 207 (1992) 129CrossRefGoogle Scholar
  13. 13.
    C. M. OFNER 3rd and W. A. BUBNIS, Pharm. Res. 13 (1996) 1821CrossRefGoogle Scholar
  14. 14.
    A. KAMINSKA and A. SIONKOWSKA, Polym. Degrad. Stab. 51 (1996) 15CrossRefGoogle Scholar
  15. 15.
    X. H. WANG, D. P. LI, W. J. WANG, Q. L. FENG, F. Z. CUI, Y. X. XU, X. H. SONG and M. VAN DER WERF, Biomaterials 24 (2003) 3213CrossRefGoogle Scholar
  16. 16.
    D. GOPINATH, M. R. AHMED, K. GOMATHI, K. CHITRA, P. K. SEHGAL and R. JAYAKUMAR, Biomaterials 25 (2004) 1911CrossRefGoogle Scholar
  17. 17.
    K. W. NG, D. T. LEONG and D. W. HUTMACHER, Tissue Eng. 11 (2005) 182CrossRefGoogle Scholar
  18. 18.
    P. FRIEDL and E.-B. BRÖCKER, Cell Mol. Life Sci. 57 (2000) 41CrossRefGoogle Scholar
  19. 19.
    C. A. LAMBERT, A. C. COLIGE, C. MUNAUT, C. M. LAPIERE and B. V. NUSGENS, Matrix Biol. 20 (2001) 397CrossRefGoogle Scholar
  20. 20.
    F. GRINNELL, Trends Cell Biol. 13 (2003) 264CrossRefGoogle Scholar
  21. 21.
    M. CHIQUET, A. S. RENEDO, F. HUBER and M. FLUCK, Matrix Biol. 22 (2003) 73CrossRefGoogle Scholar
  22. 22.
    B. CHEVALLAY, N. ABDUL-MALAK and D. HERBAGE, J. Biomed. Mater. Res. 49 (2000) 448CrossRefGoogle Scholar
  23. 23.
    T. L. LEE, Y. C. LIN, K. MOCHITATE and F. GRINNELL, J. Cell Sci. 105(Pt 1) (1993) 167Google Scholar
  24. 24.
    R. T. PRAJAPATI, B. CHAVALLY-MIS, D. HERBAGE, M. EASTWOOD and R. A. BROWN, Wound Repair Regen. 8 (2000) 226CrossRefGoogle Scholar
  25. 25.
    M. CHIQUET, Matrix Biol. 18 (1999) 417CrossRefGoogle Scholar
  26. 26.
    F. GRINNELL, C. H. HO, E. TAMARIZ, D. J. LEE and G. SKUTA, Mol. Biol. Cell 14 (2003) 384CrossRefGoogle Scholar
  27. 27.
    E. TAMARIZ and F. GRINNELL, Mol. Biol. Cell 13 (2002) 3915CrossRefGoogle Scholar
  28. 28.
    M. EASTWOOD, V. C. MUDERA, D. A. MCGROUTHER, R. A. BROWN, Cell Motil. Cytoskeleton 40 (1998) 13CrossRefGoogle Scholar
  29. 29.
    M. M. NOCIARI, A. SHALEV, P. BENIAS and C. RUSSO, J. Immunol. Methods 213 (1998) 157CrossRefGoogle Scholar
  30. 30.
    L. LORAND and R. M. GRAHAM, Nat. Rev. Mol. Cell Biol. 4 (2003) 140CrossRefGoogle Scholar
  31. 31.
    K. YOKOYAMA, N. NIO and Y. KIKUCHI, Appl. Microbiol. Biotechnol. 64 (2004) 447CrossRefGoogle Scholar
  32. 32.
    B. BRODSKY and J. A. M. RAMSHAW, Matrix Biol. 15 (1997) 545CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Y. Garcia
    • 1
  • R. Collighan
    • 2
  • M. Griffin
    • 2
  • A. Pandit
    • 1
    • 3
    Email author
  1. 1.National Centre for Biomedical Engineering ScienceNational University of IrelandGalwayIreland
  2. 2.School of Life and Health SciencesAston UniversityBirminghamUK
  3. 3.Department of Mechanical and Biomedical EngineeringNational University of IrelandGalwayIreland

Personalised recommendations