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Bone matrix calcification during embryonic and postembryonic rat calvarial development assessed by SEM–EDX spectroscopy, XRD, and FTIR spectroscopy

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Abstract

Bone mineral is constituted of biological hydroxyapatite crystals. In developing bone, the mineral crystal matures and the Ca/P ratio increases. However, how an increase in the Ca/P ratio is involved in maturation of the crystal is not known. The relationships among organic components and mineral changes are also unclear. The study was designed to investigate the process of calcification during rat calvarial bone development. Calcification was evaluated by analyzing the atomic distribution and concentration of Ca, P, and C with scanning electron microscopy (SEM)–energy-dispersive X-ray (EDX) spectroscopy and changes in the crystal structure with X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. Histological analysis showed that rat calvarial bone formation started around embryonic day 16. The areas of Ca and P expanded, matching the region of the developing bone matrix, whereas the area of C became localized around bone. X-ray diffraction and FTIR analysis showed that the amorphous-like structure of the minerals at embryonic day 16 gradually transformed into poorly crystalline hydroxyapatite, whereas the proportion of mineral to protein increased until postnatal week 6. FTIR analysis also showed that crystallization of hydroxyapatite started around embryonic day 20, by which time SEM–EDX spectroscopy showed that the Ca/P ratio had increased and the C/Ca and C/P ratios had decreased significantly. The study suggests that the Ca/P molar ratio increases and the proportion of organic components such as proteins of the bone matrix decreases during the early stage of calcification, whereas crystal maturation continues throughout embryonic and postembryonic bone development.

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Acknowledgment

This work was supported in part by Grants-in-424 Aid (24792170, 23592694, 23106010, 25932006, 25670829, 26293417) from the Japan Society for the Promotion of Science.

Conflict of interest

The authors declare they have no conflict of interest.

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Authors and Affiliations

  1. Division of Craniofacial Development and Regeneration, Tohoku University Graduate School of Dentistry, Sendai, 980-8575, Japan

    Akiko Henmi, Yasuto Mikami & Yasuyuki Sasano

  2. Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai, Japan

    Hiroshi Okata

  3. Division of Craniofacial Function Engineering, Tohoku University Graduate School of Dentistry, Sendai, Japan

    Takahisa Anada, Mariko Yoshinari & Osamu Suzuki

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  1. Akiko Henmi
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  2. Hiroshi Okata
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  3. Takahisa Anada
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  4. Mariko Yoshinari
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  5. Yasuto Mikami
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  6. Osamu Suzuki
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  7. Yasuyuki Sasano
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Correspondence to Yasuyuki Sasano.

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Henmi, A., Okata, H., Anada, T. et al. Bone matrix calcification during embryonic and postembryonic rat calvarial development assessed by SEM–EDX spectroscopy, XRD, and FTIR spectroscopy. J Bone Miner Metab 34, 41–50 (2016). https://doi.org/10.1007/s00774-014-0647-x

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  • DOI: https://doi.org/10.1007/s00774-014-0647-x

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