Virtual bone reflecting microstructure ofreal bone

• Project/Area Number: 18592234
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Orthodontic/Pediatric dentistry
• Research Institution: Okayama University
• Principal Investigator: KAMIOKA Hiroshi Okayama University, Grad. Sch. of Medicine, Dentistry and Pharmaceutical Sciences, Associate Professor Dentistry (80253219)
• Co-Investigator(Kenkyū-buntansha): YAMAMOTO Teruko Tohoku Univ., Grad. Sch. of Dentistry, Professor (00127250)
• Project Period (FY): 2006 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥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)
• Keywords: Osteocvte / Ultra high voltage electron microscopy / tomography / Three dimensional reconstruction / Virtual bone / simulation / hone

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.

Research Products

• [Journal Article] Hormonal, pH, and Calcium regulation of connexin 43 mediated dye transfer in osteocytes in chick calvaria. (2008)
  • Author(s): Ishihara Y, Kamioka H, Honjo T, Ueda H, Takano-Yamamoto T, Yamashiro T
  • Journal Title: Journal of Bone and Mineral Reserch 23 Pages: 350-360
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] Hormonal, pH, and Calcium regulation of connexin 43 mediated dye transfer in osteocytes in chick calvaria (2008)
  • Author(s): Ishihara, Y., Kamioka, H., Honjo, T., Ueda, H., Takano-Yamamoto, T., Yamashiro, T
  • Journal Title: Journal of Bone and Mineral Research 23 Pages: 350-360
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Primary cultures of chick osteocytes retain functional gap junctions between osteocytes, and between osteocytes and osteoblasts (2007)
  • Author(s): Kamioka H, Ishihara Y, Ris H, Murshid SA, Sugawara Y, Takano-Yamamoto T, Lim S
  • Journal Title: Microscopy and Microanalysis 13 Pages: 108-117
  • NAID: 120002311469
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] Actin and microtubule cytoskeletons of the processes of 3D-cultured MC3T3-El cells and osteocytes (2007)
  • Author(s): Murshid SA, Kamioka H, Ishihara Y, Ando R, Sugawara Y, Takano-Yamamoto T
  • Journal Title: Journal of Bone and Mineral Metabolism 25 Pages: 151-158
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] 骨細胞の機能と力学負荷による骨代謝調節 (2007)
  • Author(s): 手塚建一、上岡 寛
  • Journal Title: 医学のあゆみ 221 Pages: 76-80
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Primary cultures of chick osteocytes retain functional gap junctions between osteocytes, and between osteocytes and osteoblasts (2007)
  • Author(s): Kamioka, H., Ishihara, Y., Ris, H., Murshid, SA., Sugawara, Y., Takano-Yamamoto, T., Lim, S
  • Journal Title: Microscopy and Microanalysis 13 Pages: 108-117
  • NAID: 120002311469
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Actin and microtubule cytoskeletons of the processes of 3D-cultured MC3T3-E1 cells and osteocytes (2007)
  • Author(s): Murshid, SA., Kamioka, H., Ishihara, Y., Ando, R., Sugawam, Y., Takano-Yamamoto, T
  • Journal Title: Journal of Bone and Mineral Metabolism 25 Pages: 151-158
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Mechanical Stress and Bone-from osteocyte biology to bone remodeling simulation (2007)
  • Author(s): Tezuka, K., Kamioka, H
  • Journal Title: Igaku no Ayumi 221 Pages: 76-80
  • NAID: 40015321596
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Primary cultures of chick osteocytes retain functional gap junctions between osteocytes, and between osteocytes and osteoblasts (2007)
  • Author(s): Kamioka H. Ishihara Y, Ris H, Murshid SA, Sugawara Y, Takano-Yamamoto T, Lim S
  • Journal Title: Microscopy and Microanalysis 13 Pages: 108-117
  • NAID: 120002311469
  • Peer Reviewed
• [Journal Article] Actin and microtubule cytoskeletons of the processes of 3D-cultured MC3T3-E1 cells and osteocytes (2007)
  • Author(s): Murshid SA, Kamioka H, Ishihara Y, Ando R, Sugawara Y, Takano-Yamamoto T
  • Journal Title: Journal of Bone and Mineral Metabolism 25 Pages: 151-158
  • Peer Reviewed
• [Journal Article] 骨微細構造を反映した仮想骨の作成 (2006)
  • Author(s): 上岡 寛、山本照子
  • Journal Title: 文科省ナノテクノロジー総合支援プロジェクト実績報告書 Pages: 82-83
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] 骨微細構造を反映した仮想骨の作成 (2006)
  • Author(s): 上岡 寛、手塚建一、山本照子
  • Journal Title: 超高圧電子顕微鏡センター年報 Pages: 107-110
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] 骨細胞における機械的刺激への応答能について (2006)
  • Author(s): 上岡 寛、菅原康代、Murshid A Sakhr、石原嘉人、本城正、山本照子
  • Journal Title: 生体医工学雑誌 44 Pages: 484-489
  • NAID: 110006249802
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Virtural bone reflecting microstructure of real bone (2006)
  • Author(s): Kamioka, H., Yamamoto, T
  • Journal Title: Report for Nanotechnology Support of the MEXT, Japan Pages: 82-83
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Virtural bone reflecting microstructure of real bone (2006)
  • Author(s): Kamioka, H., Tezuka, K., Yamamoto, T
  • Journal Title: Annual report of Ultra-high voltage electron microscope center Pages: 107-110
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Mechanosensitivity of osteocytes (2006)
  • Author(s): Kamioka, H., Sugawara, Y., Murshid, SA., Ishihara, Y., Honjo, T., Yamamoto, T
  • Journal Title: Medical and Biological Engineering Pages: 484-489
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Fluid shear stress induces less calcium response in a single primary osteocyte than in a single osteoblast : implication of different focal adhesion formation. (2006)
  • Author(s): Kamioka H, Sugawara Y, Murshid SA, Ishihara Y, Honjo T, Takano-Yamamoto T
  • Journal Title: Journal of Bone and Mineral Research 21(7) Pages: 1012-1021
• [Journal Article] Brain-derived neurotrophic factor-immunoreactive primary sensory neurons in the rat trigeminal ganglion and trigeminal sensory nuclei. (2006)
  • Author(s): Ichikawa H, Yabuuchi T, Jin HW, Terayama R, Yamaai T, Deguchi T, Kamioka H, Takano-Yamamoto T, Sugimoto T
  • Journal Title: Brain Research 1081(1) Pages: 113-118
• [Journal Article] αVβ3 integrin ligands enhance volume-sensitive calcium influx in mechanically stretched osteocytes (2006)
  • Author(s): Miyauchi A, Gotoh M, Kamioka H, Notoya K, Sekiya H, Takagi Y, Yoshimoto Y, Ishikawa H, Chihara K, Takano-Yamamoto T, Fujita T, Mikuni-Takagaki Y
  • Journal Title: Journal of Bone and Mineral Metabolism 24(6) Pages: 498-504
• [Journal Article] The alteration of a mechanical property of bone cells during the process of changing from osteoblasts to osteocytes.
  • Author(s): Sugawara Y, Ando R, Kamioka H, Murshid SA, Hashimoto K, Kataoka N, Tsujioka K, Ishihara Y, Yamashiro T. Takano-Yamamoto T
  • Journal Title: Bone (印刷中)
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] The alteration of a mechanical property of bone cells during the process of changing from osteoblasts to osteocytes
  • Author(s): Sugawara, Y., Ando, R., Kamioka, H., Murshid, SA., Hashimoto, K., Kataoka, N., Tsujioka, K., Ishihara, Y., Yamashiro, T., Takano-Yamamoto, T
  • Journal Title: Bone (in press)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Primary cultures of chick osteocytes retain functional gap junctions between osteocytes, and between osteocytes and osteoblasts
  • Author(s): Kamioka H, Ishihara Y, Ris H, Murshid SA, Sugawara Y, Takano-Yamamoto T, Lim S
  • Journal Title: Microscopy and Microanalysis (印刷中)
  • NAID: 120002311469
• [Journal Article] Actin and microtubule cytoskeletons of the processes of 3D-cultured MC3T3-E1 cells and osteocytes
  • Author(s): Murshid SA, Kamioka H, Ishihara Y, Ando R, Sugawara Y, Takano-Yamamoto T
  • Journal Title: Journal of Bone and Mineral Metabolism (印刷中)
• [Journal Article] 骨細胞における機械的刺激への応答能について
  • Author(s): 上岡 寛, 菅原康代, Murshid A Sakhr, 石原嘉人, 本城正, 山本照子
  • Journal Title: 生体医工学雑誌 解説特集 (印刷中)
  • NAID: 110006249802
• [Presentation] 超高電圧TEMトモグラフィーを用いた骨細胞周囲の高精細構造・応力解析 (2007)
  • Author(s): 上岡 寛、Murshid SA, 手塚建一、山本照子
  • Organizer: 第25回日本骨代謝学会 ミニシンポジウム
  • Place of Presentation: 大阪市
  • Year and Date: 2007-07-21
• [Presentation] 骨の機械的刺激への応答能を医工学的に解析するためにー (2007)
  • Author(s): 上岡 寛
  • Organizer: 岐阜大学大学院医学系研究科主催 特別講演
  • Place of Presentation: 岐阜市
  • Year and Date: 2007-04-26
  • Description: 「研究成果報告書概要(和文)」より
• [Presentation] Analysis of the mechanosensitivity of bone (2007)
  • Author(s): Kamioka, H
  • Organizer: Special lecture at Dept. of tissure and organ development reorganization and advanced medical science, Gifu Univ
  • Place of Presentation: Gifu
  • Year and Date: 2007-04-26
  • Description: 「研究成果報告書概要(欧文)」より
• [Presentation] 超高電圧TEMトモグラフィーを用いた骨細胞周囲の高詳細構造・応力解析 (2007)
  • Author(s): 上岡 寛、Murshid SA, 手塚建一、山本照子
  • Organizer: 第25回日本骨代謝学会ミニシンポジウム
  • Place of Presentation: 大阪市
  • Description: 「研究成果報告書概要(和文)」より
• [Presentation] Osteocyte in bone revealed by electron tomography. (2007)
  • Author(s): Kamioka H, Murshid SA, Ishihara Y, Ando R, Sugawara Y, Kajimura N, Hasegawa T, Takaoka A, Tanakano-Yamamoto T
  • Organizer: 16th International congress of the IFAA
  • Place of Presentation: Sapporo
  • Description: 「研究成果報告書概要(和文)」より
• [Presentation] 生骨組織中での細胞間コミュニケーションに影響する外的調節因子について (2007)
  • Author(s): 石原嘉人, 上岡寛、山本照子、山城隆
  • Organizer: 第25回日本骨代謝学会
  • Place of Presentation: 大阪市
  • Description: 「研究成果報告書概要(和文)」より
• [Presentation] Mechanosensitivity of a single osteocyte: Difference in cell process and cell body (2007)
  • Author(s): Aonuma Y, Adachi T, Tanaka M, Hojo M, Takano-Yamamoto T, Kamioka H
  • Organizer: Third Asian Pacific conference on biomechanics
  • Place of Presentation: 東京
  • Description: 「研究成果報告書概要(和文)」より
• [Presentation] Osteocyte in bone revealed by electron tomography (2007)
  • Author(s): Kamioka, H., Murshid, SA., Ishihara, Y., Ando, R., Sugawara, Y., Kajimura, N., Hasegawa, T., Takaoka, A., Tanakano-Yamamoto, T
  • Organizer: 16th International congress of the IFAA
  • Place of Presentation: Sapporo
  • Description: 「研究成果報告書概要(欧文)」より
• [Presentation] Mechanosensitivity of a single osteocyte : Difference in cell process and cell body (2007)
  • Author(s): Aonuma, Y., Adachi, T., Tanaka, M., Hojo, M., Takano-Yamamoto, T., Kamioka, H
  • Organizer: 3rd Asian Pacific conference on biomechanics
  • Place of Presentation: Tokyo
  • Description: 「研究成果報告書概要(欧文)」より
• [Presentation] Microstructure and microstrain analysis using ultra-high voltage electron microscope (2007)
  • Author(s): Kamioka, H., Murshid, SA., Tezuka, K., Yamamoto, T
  • Organizer: The 22nd Annual meeting of the Japanese society for bone and mineral research
  • Place of Presentation: Osaka
  • Description: 「研究成果報告書概要(欧文)」より
• [Presentation] Regulator of cell-cell communication among osteocytes in bone (2007)
  • Author(s): Ishihara, Y., Kamioka, H., Yamamoto, T., Yamashiro, T
  • Organizer: The 22nd Annual meeting of the Japanese society for bone and mineral research
  • Place of Presentation: Osaka
  • Description: 「研究成果報告書概要(欧文)」より
• [Book] Interface Oral Health Science 2007 Less response of osteocyte than osteoblasts to mechanical force: Implication of different focal adhesion formation. (2007)
  • Author(s): Takano-Yamamoto T, Kamioka H, Sugawara Y
  • Publisher: SPRINGER Ed: Watanabe M, Okuno 0