Amino Acids

, Volume 46, Issue 7, pp 1751–1761 | Cite as

Cross-linking of collagen I by tissue transglutaminase provides a promising biomaterial for promoting bone healing

  • Dario Fortunati
  • David Yi San Chau
  • Zhuo Wang
  • Russell John Collighan
  • Martin GriffinEmail author
Original Article


Transglutaminases (TGs) stabilize proteins by the formation of ε(γ-glutamyl)lysine cross-links. Here, we demonstrate that the cross-linking of collagen I (COL I) by tissue transglutaminase (TG2) causes an alteration in the morphology and rheological properties of the collagen fibers. Human osteoblasts (HOB) attach, spread, proliferate, differentiate and mineralize more rapidly on this cross-linked matrix compared to native collagen. When seeded on cross-linked COL I, HOB are more resistant to the loss of cell spreading by incubation with RGD containing peptides and with α1, α2 and β1 integrin blocking antibodies. Following adhesion on cross-linked collagen, HOB show increased phosphorylation of the focal adhesion kinase, and increased expression of β1 and β3 integrins. Addition of human bone morphogenetic protein to HOB seeded on TG2 cross-linked COL I enhanced the expression of the differentiation marker bone alkaline phosphatase when compared to cross-linked collagen alone. In summary, the use of TG2-modified COL I provides a promising new scaffold for promoting bone healing.


Tissue transglutaminase Cross-linking Collagen I HOB Integrins 





Tissue transglutaminase


Human osteoblasts

Collagen I



Focal adhesion kinase


Protein kinase α


Human bone morphogenetic protein 7


Bone alkaline phosphatase


Extra cellular matrix


Matrix metalloproteinases


Atomic force microscopy






Glyceraldehyde 3-phosphate dehydrogenase




Vascular endothelial growth factor


Reverse transcription


Tissue inhibitors of MMPs








Silicate-substituted calcium phosphate





This work was financially supported by the European Community programme TRACKS where D. F. was a recipient of a Marie Curie postdoctoral Fellowship with contract MRTN-CT-2006-036032 and by the Grant EPSRC (GR/S21755/02). We would like to thank Professor John Mitchell and Dr Matthew Boyd (University of Nottingham) for their kind help with the COL I gel rheology. We would like to thank Mr. A. Parchure for the technical assistance with the β1 integrin detection.

Conflict of interest

No competing financial interest exists.


  1. Akimov SS, Belkin AM (2001) Cell surface tissue transglutaminase is involved in adhesion and migration of monocytic cells on fibronectin. Blood 98:1567–1576PubMedCrossRefGoogle Scholar
  2. Aravamudhan A, Ramos DM, Nip J, Subramanian A, James R, Harmon MD, Yu X, Kumbar SG (2013) Osteoinductive small molecules: growth factor alternatives for bone tissue engineering. Curr Pharm Des 19:3420–3428PubMedCrossRefGoogle Scholar
  3. Bergamini CM, Collighan RJ, Wang Z, Griffin M (2011) Structure and regulation of type 2 transglutaminase in relation to its physiological functions and pathological roles. Adv Enzymol Relat Areas Mol Biol 78:1–46PubMedGoogle Scholar
  4. Cameron K, Travers P, Chander C, Buckland T, Campion C, Noble B (2013) Directed osteogenic differentiation of human mesenchymal stem/precursor cells on silicate substituted calcium phosphate. J Biomed Mater Res A 101:13–22PubMedCrossRefGoogle Scholar
  5. Chan KL, Khankhel AH, Thompson RL, Coisman BJ, Wong KH, Truslow JG, Tien J (2013) Crosslinking of collagen scaffolds promotes blood and lymphatic vascular stability. J Biomed Mater Res A. doi: 10.1002/jbm.a.34990
  6. Chau DYS, Collighan RJ, Verderio EAM, Addy VL, Griffin M (2005) The cellular response to transglutaminase-cross-linked collagen. Biomaterials 26:6518–6529PubMedCrossRefGoogle Scholar
  7. Ferreira AM, Gentile P, Chiono V, Ciardelli G (2012) Collagen for bone tissue regeneration. Acta Biomater 8:3191–3200PubMedCrossRefGoogle Scholar
  8. Folk JE, Chung SI (1973) Molecular and catalytic properties of transglutaminases. Adv Enzymol Relat Areas Mol Biol 38:109–191PubMedGoogle Scholar
  9. Freund KF, Doshi KP, Gaul SL, Claremon DA, Remy DC, Baldwin JJ, Pitzenberger SM, Stern AM (1994) Transglutaminase inhibition by 2-[(2-oxopropyl)thio]imidazolium derivatives: mechanism of factor XIIIa inactivation. Biochemistry 33:10109–10119PubMedCrossRefGoogle Scholar
  10. Griffin M, Casadio R, Bergamini CM (2002) Transglutaminases: nature’s biological glues. Biochem J 368:377–396PubMedCentralPubMedCrossRefGoogle Scholar
  11. Heath DJ, Downes S, Verderio E, Griffin M (2001) Characterization of tissue transglutaminase in human osteoblast-like cells. J Bone Miner Res 16:1477–1485PubMedCrossRefGoogle Scholar
  12. Huang L, Haylor JL, Hau Z, Jones RA, Vickers ME, Wagner B, Griffin M, Saint RE, Coutts IG, El Nahas AM, Johnson TS (2009) Transglutaminase inhibition ameliorates experimental diabetic nephropathy. Kidney Int 76(4):383–394. doi: 10.1038/ki.2009.230 PubMedCrossRefGoogle Scholar
  13. Kotsakis P, Wang Z, Collighan RJ, Griffin M (2011) The role of tissue transglutaminase (TG2) in regulating the tumor progression of the mouse colon carcinoma CT26. Amino Acids 41:909–921PubMedCrossRefGoogle Scholar
  14. Kruger TE, Miller AH, Wang J (2013) Collagen scaffolds in bone sialoprotein-mediated bone regeneration. Sci World J 2013:812718CrossRefGoogle Scholar
  15. Leblanc A, Day N, Menard A, Keillor JW (1999) Guinea pig liver transglutaminase: a modified purification procedure affording enzyme with superior activity in greater yield. Protein Expr Purif 17:89–95PubMedCrossRefGoogle Scholar
  16. McKleroy W, Lee TH, Atabai K (2013) Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis. Am J Physiol Lung Cell Mol Physiol 304:L709–L721PubMedCentralPubMedCrossRefGoogle Scholar
  17. Mehta K, Kumar A, Kim HI (2010) Transglutaminase 2: a multi-tasking protein in the complex circuitry of inflammation and cancer. Biochem Pharmacol 80:1921–1929PubMedCrossRefGoogle Scholar
  18. Olsen KC, Sapinoro RE, Kottmann RM, Kulkarni AA, Iismaa SE, Johnson GV, Thatcher TH, Phipps RP, Sime PJ (2011) Transglutaminase 2 and its role in pulmonary fibrosis. Am J Respir Crit Care Med 184:699–707PubMedCentralPubMedCrossRefGoogle Scholar
  19. Orlandi A, Oliva F, Taurisano G, Candi E, Di Lascio A, Melino G, Spagnoli G, Tarantino U (2009) Transglutaminase-2 differently regulates cartilage destruction and osteophyte formation in a surgical model of osteoarthritis. Amino Acids 36:755–763PubMedCrossRefGoogle Scholar
  20. Parsons JT (2003) Focal adhesion kinase: the first ten years. Cell Sci. 116:1409–1416CrossRefGoogle Scholar
  21. Piechocka IK, van Oosten ASG, Breuls RGM, Koenderink GH (2011) Rheology of heterotypic collagen networks. Biomacromolecules 12:2797–2805PubMedCrossRefGoogle Scholar
  22. Piercy-Kotb SA, Mousa A, Al-Jallad HF, Myneni VD, Chicatun F, Nazhat SN, Kaartinen MT (2012) Factor XIIIA transglutaminase expression and secretion by osteoblasts is regulated by extracellular matrix collagen and the MAP kinase signaling pathway. J Cell Physiol 227:2936–2946PubMedCrossRefGoogle Scholar
  23. Plant AL, Bhadriraju K, Spurlin TA, Elliott JT (2009) Cell response to matrix mechanics: focus on collagen. Biochim Biophys Acta 1793:893–902PubMedCrossRefGoogle Scholar
  24. Rodgers SD, Marascalchi BJ, Grobelny BT, Smith ML, Samadani U (2013) Revision surgery after interbody fusion with rhBMP-2: a cautionary tale for spine surgeons. J Neurosurg Spine 18:582–587PubMedCrossRefGoogle Scholar
  25. Tarantino U, Ferlosio A, Arcuri G, Spagnoli LG, Orlandi A (2013) Transglutaminase 2 as a biomarker of osteoarthritis: an update. Amino Acids 44:199–207PubMedCrossRefGoogle Scholar
  26. Telci D, Wang Z, Li X, Verderio EA, Humphries MJ, Baccarini M, Basaga H, Griffin M (2008) Fibronectin tissue transglutaminase matrix rescues RGD-impaired cell adhesion through syndecan-4 and beta1 integrin co-signaling. J Biol Chem 283:20937–20947PubMedCentralPubMedCrossRefGoogle Scholar
  27. Wang Z, Griffin M (2012) TG2, a novel extracellular protein with multiple functions. Amino Acids 42:939–949PubMedCrossRefGoogle Scholar
  28. Wang Z, Griffin M (2013) The role of TG2 in regulating S100A4-mediated mammary tumor cell migration. PLoS One 8:e57017PubMedCentralPubMedCrossRefGoogle Scholar
  29. Wang Z, Collighan RJ, Gross SR, Danen EH, Orend G, Telci D, Griffin M (2010) RGD-independent cell adhesion via a tissue transglutaminase-fibronectin matrix promotes fibronectin fibril deposition and requires syndecan-4/2 and α5β1 integrin co-signaling. J Biol Chem 285:40212–40229PubMedCentralPubMedCrossRefGoogle Scholar
  30. Wang Z, Telci D, Griffin M (2011) Importance of syndecan-4 and syndecan-2 in osteoblast cell adhesion and survival mediated by a tissue transglutaminase-fibronectin complex. Exp Cell Res 317:367–381PubMedCrossRefGoogle Scholar
  31. Wang Z, Collighan RJ, Pytel K, Rathbone DL, Li X, Griffin M (2012) Characterization of heparin-binding site of tissue transglutaminase: its importance in cell surface targeting, matrix deposition, and cell signaling. J Biol Chem 287:13063–13083PubMedCentralPubMedCrossRefGoogle Scholar
  32. Wang Z, Perez M, Caja S, Melino G, Johnson TS, Lindfors K, Griffin M (2013) A novel extracellular role for tissue transglutaminase in matrix-bound VEGF-mediated angiogenesis. Cell Death Dis 4:e808PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Dario Fortunati
    • 1
  • David Yi San Chau
    • 1
  • Zhuo Wang
    • 1
  • Russell John Collighan
    • 1
  • Martin Griffin
    • 1
    Email author
  1. 1.School of Life and Health SciencesAston UniversityBirminghamUK

Personalised recommendations