| Project/Area Number |
18592234
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Orthodontic/Pediatric dentistry
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| Research Institution | Okayama University |
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Principal Investigator |
KAMIOKA Hiroshi Okayama University, Grad. Sch. of Medicine, Dentistry and Pharmaceutical Sciences, Associate Professor Dentistry (80253219)
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| Co-Investigator(Kenkyū-buntansha) |
YAMAMOTO Teruko Tohoku Univ., Grad. Sch. of Dentistry, Professor (00127250)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,880,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥480,000)
Fiscal Year 2007: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Osteocvte / Ultra high voltage electron microscopy / tomography / Three dimensional reconstruction / Virtual bone / simulation / hone |
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| Research Abstract |
Aim: We applied electron tomography to the osteocytes in bone in order to make virtual bone.
Materials & Methods: Chick calvariae were desected from 16-day old chick embryo. The calvariae were treated by 1 mM collagenase and 5 mM EDTA and trimmed in 3x3-mm for further use. These sections were fixed with 3% paraformaldehyde for 24h, stained with 2% protargol solution containing copper beads for 48h as silver-staining, and embedded in paraffin. After these treatment, 2 μm sections were placed on fromvar coated cupper grids. For tomographic data collection, sections were dipped in a solution of colloidal gold for 1-2 min. Electron tomographic data collection was carried out with H-3000 ultra-high voltage EM (Hitachi) at 2 MV equipped with a 360 °-tilt specimen holder. Images were recorded on a Tietz Tem-Cam 4k x 4k-pixel F415S slow scan CCD camera (TVIPS) binned to 2k x 2k pixels. Alignment of the tilt series and reconstruction of 3D volumes from aligned images was performed by the weighted back-projection method by using a tomography software package IMOD.
Results: Stereo-pair image of osteocyte was taken by ultrahigh-voltage electron microscopy with tilt +/-7 °. Osteocyte clearly appeared with numerous processes and brunches. The tomograms were generated from two +1-60 ° tilt series of a 2.0 μm section of chick calvaria and the microgram were taken 2 ° intervals. We could observe the series with 0.3 μm interval in depth of osteocyte tomography. The final pixel size on the specimen was 8 nm. We could distinguish the osteocyte cell body and their processes with high-density dots of silver on the surface of cell membrane.
Conclusions: It is possible to observe silver-stained osteocytes in thick section (2 μm) by ultrahigh-voltage electron microscopy. In addition, high-resolution (8 nm) electron tomography was performed to the osteocytes in chick calvariae.
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