Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors

Nature Materials volume 9, pages 1004–1009 (2010)Cite this article

Subjects

Abstract

Bone is a composite material in which collagen fibrils form a scaffold for a highly organized arrangement of uniaxially oriented apatite crystals1,2. In the periodic 67 nm cross-striated pattern of the collagen fibril3,4,5, the less dense 40-nm-long gap zone has been implicated as the place where apatite crystals nucleate from an amorphous phase, and subsequently grow6,7,8,9. This process is believed to be directed by highly acidic non-collagenous proteins6,7,9,10,11; however, the role of the collagen matrix12,13,14 during bone apatite mineralization remains unknown. Here, combining nanometre-scale resolution cryogenic transmission electron microscopy and cryogenic electron tomography15 with molecular modelling, we show that collagen functions in synergy with inhibitors of hydroxyapatite nucleation to actively control mineralization. The positive net charge close to the C-terminal end of the collagen molecules promotes the infiltration of the fibrils with amorphous calcium phosphate (ACP). Furthermore, the clusters of charged amino acids, both in gap and overlap regions, form nucleation sites controlling the conversion of ACP into a parallel array of oriented apatite crystals. We developed a model describing the mechanisms through which the structure, supramolecular assembly and charge distribution of collagen can control mineralization in the presence of inhibitors of hydroxyapatite nucleation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Cryo-electron tomography of a collagen fibril mineralized in the presence of 10 μg ml−1 of pAsp for 72 h and stained with uranyl acetate.
Figure 2: CryoTEM images of collagen at different stages of mineralization in the presence of 10 μg ml−1 of pAsp.
Figure 3: Uranyl acetate map of the different stages of collagen mineralization in the presence of 10 μg ml−1 of pAsp.
Figure 4: Analysis of the mass density and electrostatic potential energy of a microfibril, based on the crystal structure 5.
Figure 5: Analysis of calcium phosphate precipitation in the absence and presence of pAsp.

Similar content being viewed by others

References

  1. Hulmes, D. J. S., Wess, T. J., Prockop, D. J. & Fratzl, P. Radial packing, order, and disorder in collagen fibrils. Biophys. J. 68, 1661–1670 (1995).

    Article  CAS  Google Scholar 

  2. Traub, W., Arad, T. & Weiner, S. 3-dimensional ordered distribution of crystals in turkey tendon collagen-fibers. Proc. Natl Acad. Sci. USA 86, 9822–9826 (1989).

    Article  CAS  Google Scholar 

  3. Hodge, A. J. & Petruska, J. A. in Aspects of Protein Structure (ed. Ramachandran, G. N.) 289–300 (Academic, 1963).

    Google Scholar 

  4. Miller, A. Collagen: The organic matrix of bone. Phil. Trans. R. Soc. B 304, 455–477 (1984).

    Article  CAS  Google Scholar 

  5. Orgel, J. P. R. O., Irving, T. C., Miller, A. & Wess, T. J. Microfibrillar structure of type I collagen in situ. Proc. Natl Acad. Sci. USA 103, 9001–9005 (2006).

    Article  CAS  Google Scholar 

  6. Glimcher, M. J. & Muir, H. Recent studies of the mineral phase in bone and its possible linkage to the organic matrix by protein-bound phosphate bonds. Phil. Trans. R. Soc. B 304, 479–508 (1984).

    Article  CAS  Google Scholar 

  7. Landis, W. J., Song, M. J., Leith, A., Mcewen, L. & Mcewen, B. F. Mineral and organic matrix interaction in normally calcifying tendon visualized in 3 dimensions by high-voltage electron-microscopic tomography and graphic image-reconstruction. J. Struct. Biol. 110, 39–54 (1993).

    Article  CAS  Google Scholar 

  8. Mahamid, J. et al. Mapping amorphous calcium phosphate transformation into crystalline mineral from the cell to bone in zebrafish fin rays. Proc. Natl Acad. Sci. USA 107, 6316–6321 (2010).

    Article  CAS  Google Scholar 

  9. Traub, W., Arad, T. & Weiner, S. Origin of mineral crystal-growth in collagen fibrils. Matrix 12, 251–255 (1992).

    Article  CAS  Google Scholar 

  10. George, A. & Veis, A. Phosphorylated proteins and control over apatite nucleation, crystal growth, and inhibition. Chem. Rev. 108, 4670–4693 (2008).

    Article  CAS  Google Scholar 

  11. Maitland, M. E. & Arsenault, A. L. A correlation between the distribution of biological apatite and amino-acid-sequence of type-I collagen. Calcif. Tissue Int. 48, 341–352 (1991).

    Article  CAS  Google Scholar 

  12. Berthet-Colominas, C., Miller, A. & White, S. W. Structural study of the calcifying collagen in turkey leg tendons. J. Mol. Biol. 134, 431–445 (1979).

    Article  CAS  Google Scholar 

  13. Katz, E. P. & Li, S. Structure and function of bone collagen fibrils. J. Mol. Biol. 80, 1–15 (1973).

    Article  CAS  Google Scholar 

  14. Landis, W. J. & Silver, F. H. Mineral deposition in the extracellular matrices of vertebrate tissues: Identification of possible apatite nucleation sites on type I collagen. Cells Tissues Organs 189, 20–24 (2009).

    Article  CAS  Google Scholar 

  15. Pouget, E. M. et al. The initial stages of template-controlled CaCO3 formation revealed by cryo-TEM. Science 323, 1455–1458 (2009).

    Article  CAS  Google Scholar 

  16. Stetlerstevenson, W. G. & Veis, A. Type-I collagen shows a specific binding-affinity for bovine dentin phosphophoryn. Calcif. Tissue Int. 38, 135–141 (1986).

    Article  CAS  Google Scholar 

  17. Stetlerstevenson, W. G. & Veis, A. Bovine dentin phosphophoryn—calcium-ion binding-properties of a high-molecular-weight preparation. Calcif. Tissue Int. 40, 97–102 (1987).

    Article  CAS  Google Scholar 

  18. Deshpande, A. S. & Beniash, E. Bioinspired synthesis of mineralized collagen fibrils. Cryst. Growth Des. 8, 3084–3090 (2008).

    Article  CAS  Google Scholar 

  19. Olszta, M. J. et al. Bone structure and formation: A new perspective. Mater. Sci. Eng. R. 58, 77–116 (2007).

    Article  Google Scholar 

  20. Price, P. A., Toroian, D. & Lim, J. E. Mineralization by inhibitor exclusion: The calcification of collagen with fetuin. J. Biol. Chem. 284, 17092–17101 (2009).

    Article  CAS  Google Scholar 

  21. Fratzl, P., Fratzl-Zelman, N. & Klaushofer, K. Collagen packing and mineralization—an X-ray-scattering investigation of turkey leg tendon. Biophys. J. 64, 260–266 (1993).

    Article  CAS  Google Scholar 

  22. Beniash, E., Traub, W., Veis, A. & Weiner, S. A transmission electron microscope study using vitrified ice sections of predentin: Structural changes in the dentin collagenous matrix prior to mineralization. J. Struct. Biol. 132, 212–225 (2000).

    Article  CAS  Google Scholar 

  23. Hodge, A. J. & Schmitt, F. O. The charge profile of the tropocollagen macromolecule and the packing arrangement in native-type collagen fibrils. Proc. Natl Acad. Sci. USA 46, 186–197 (1960).

    Article  CAS  Google Scholar 

  24. Chapman, J. A., Tzaphlidou, M., Meek, K. M. & Kadler, K. E. The collagen fibril—a model system for studying the staining and fixation of a protein. Electron Microsc. Rev. 3, 143–182 (1990).

    Article  CAS  Google Scholar 

  25. Jee, S. S., Culver, L., Li, Y. P., Douglas, E. P. & Gower, L. B. Biomimetic mineralization of collagen via an enzyme-aided PILP process. J. Cryst. Growth 312, 1249–1256 (2010).

    Article  CAS  Google Scholar 

  26. Rochette, C. N. et al. A shielding topology stabilizes the early stage protein-mineral complexes of fetuin-a and calcium phosphate: A time-resolved small-angle X-ray study. Chembiochem 10, 735–740 (2009).

    Article  CAS  Google Scholar 

  27. Toroian, D., Lim, J. E. & Price, P. A. The size exclusion characteristics of type I collagen—implications for the role of noncollagenous bone constituents in mineralization. J. Biol. Chem. 282, 22437–22447 (2007).

    Article  CAS  Google Scholar 

  28. Kawska, A., Hochrein, O., Brickmann, A., Kniep, R. & Zahn, D. The nucleation mechanism of fluorapatite-collagen composites: Ion association and motif control by collagen proteins. Angew. Chem. Int. Ed. 47, 4982–4985 (2008).

    Article  CAS  Google Scholar 

  29. He, G. & George, A. Dentin matrix protein 1 immobilized on type I collagen fibrils facilitates apatite deposition in vitro. J. Biol. Chem. 279, 11649–11656 (2004).

    Article  CAS  Google Scholar 

  30. He, G. et al. Spatially and temporally controlled biomineralization is facilitated by interaction between self-assembled dentin matrix protein 1 and calcium phosphate nuclei in solution. Biochemistry 44, 16140–16148 (2005).

    Article  CAS  Google Scholar 

  31. Posner, A. S. & Betts, F. Synthetic amorphous calcium phosphate and its relation to bone mineral structure. Acc. Chem. Res. 8, 273–281 (1975).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank G. Falini (University of Bologna, Italy) for kindly providing the horse tendon collagen; L. B. Gower (University of Florida, Florida, USA) for a critical review of the manuscript; S. Weiner (Weizmann Institute of Science, Israel) and J. P. R. O. Orgel (Illinois Institute of Technology, Illinois, US) for helpful discussions; and J. van Roosmalen (Eindhoven University of Technology, The Netherlands) for his help with the tomography reconstructions. Supported by the Dutch Science Foundation, NWO, The Netherlands and by the European Community (FP6, project code NMP4-CT-2006-033277 TEM-PLANT).

Author information

Authors and Affiliations

  1. Laboratory of Materials and Interface Chemistry and Soft Matter CryoTEM Unit, Eindhoven University of Technology, 5600 MB Eindhoven, PO Box 513, The Netherlands

    Fabio Nudelman, Paul H. H. Bomans, Heiner Friedrich, Laura J. Brylka, Gijsbertus de With & Nico A. J. M. Sommerdijk

  2. Department of Biomedical Engineering, Biomodeling and Bioinformatics, Eindhoven University of Technology, 5600 MB Eindhoven, PO Box 513, The Netherlands

    Koen Pieterse & Peter A. J. Hilbers

  3. Department of Oral Biology, University of Illinois, Chicago, Illinois 60612, USA

    Anne George

Authors
  1. Fabio Nudelman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Koen Pieterse
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anne George
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Paul H. H. Bomans
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Heiner Friedrich
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Laura J. Brylka
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Peter A. J. Hilbers
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Gijsbertus de With
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Nico A. J. M. Sommerdijk
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

F.N. carried out most experiments and co-wrote the manuscript. K.P. and P.A.J.H. carried out the molecular modelling. A.G. provided the C-DMP1 and the expertise in the work with the protein. L.J.B. contributed to the development of the mineralization experiments for cryoTEM. P.H.H.B. provided support with the cryoTEM. H.F. provided support with the tomographic reconstructions. G.W. and N.A.J.M.S. supervised the project and N.A.J.M.S. co-wrote the manuscript. All authors discussed the results and revised the manuscript.

Corresponding author

Correspondence to Nico A. J. M. Sommerdijk.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 3121 kb)

Supplementary Information

Supplementary Movie 1 (MOV 9215 kb)

Supplementary Information

Supplementary Movie 2 (MOV 7532 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nudelman, F., Pieterse, K., George, A. et al. The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors. Nature Mater 9, 1004–1009 (2010). https://doi.org/10.1038/nmat2875

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmat2875

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research