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


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.

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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.


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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).

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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.

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Correspondence to Nico A. J. M. Sommerdijk.

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The authors declare no competing financial interests.

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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).

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