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Journal of Electron Microscopy Advance Access originally published online on February 18, 2009
Journal of Electron Microscopy 2009 58(2):55-65; doi:10.1093/jmicro/dfp008
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Warfarin administration disrupts the assembly of mineralized nodules in the osteoid

Norio Amizuka1,*, Minqi Li1, Kuniko Hara2, Masatoshi Kobayashi2, Paulo H. L. de Freitas1, Sobhan Ubaidus1, Kimimitsu Oda1,3 and Yasuhiro Akiyama2

1 Center for Transdisciplinary Research, Niigata University, Niigata
2 Pharmacological Evaluation Section, Eisai Co., Ltd, Tokyo
3 Division of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Japan

* To whom correspondence should be addressed. E-mail: amizuka{at}dent.niigata-u.ac.jp

This study aimed to elucidate the ultrastructural role of Gla proteins in bone mineralization by means of a warfarin-administration model. Thirty-six 4-week-old male F344 rats received warfarin (warfarin group) or distilled water (control group), and were fixed after 4, 8 and 12 weeks with an aldehyde solution. Tibiae and femora were employed for histochemical analyses of alkaline phosphatase, osteocalcin and tartrate-resistant acid phosphatase, and for bone histomorphometry and electron microscopy. After 4, 8 and 12 weeks, there were no marked histochemical and histomorphometrical differences between control and warfarin groups. However, osteocalcin immunoreactivity was markedly reduced in the warfarin-administered bone. Mineralized nodules and globular assembly of crystalline particles were seen in the control osteoid. Alternatively, warfarin administration resulted in crystalline particles being dispersed throughout the osteoid without forming mineralized nodules. Immunoelectron microscopy unveiled lower osteocalcin content in the warfarin-administered osteoid, which featured scattered crystalline particles, whereas osteocalcin was abundant on the normally mineralized nodules in the control osteoid. In summary, Gla proteins appear to play a pivotal role in the assembly of mineralized nodules.

Keywords     osteoblast, osteocalcin, mineralization, mineralized nodule, warfarin, immunoelectron microscopy

Received      4 November 2008, accepted 26 January 2009


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