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The recontsruction of periodontal ligament on artificial dental roots

Research Project

Project/Area Number 07557368
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section展開研究
Research Field Conservative dentistry
Research InstitutionTohoku Univ.

Principal Investigator

OKUDA Reiichi  Tohoku Univ., School of Dentistry Professor, 歯学部, 教授 (80005024)

Co-Investigator(Kenkyū-buntansha) IWAMATSU Yoko  Tohoku Univ., Dental Hospital Assistant Professor, 歯学部附属病院, 助手 (50261524)
SHIMIZU Yoshinobu  Tohoku Univ., School of Dentistry Associate Professor, 歯学部, 助教授 (20005078)
MOTEGI Katsutoshi  Tohoku Univ., School of Dentistry Professor, 歯学部, 教授 (60013984)
WATANABE Makoto  Tohoku Univ., School of Dentistry Professor, 歯学部, 教授 (80091768)
KAGAYAMA Manabu  Tohoku Univ., School of Dentistry Professor, 歯学部, 教授 (60004610)
田上 篤  東北大学, 歯学部・付属病院, 助手 (00236374)
Project Period (FY) 1995 – 1997
Project Status Completed (Fiscal Year 1997)
Budget Amount *help
¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1996: ¥2,400,000 (Direct Cost: ¥2,400,000)
KeywordsArtificial dental root / Titanium / Periodontal Ligament cells / Reconstruction of periodontal ligament / cementum / Epithelial rests of Malassez / 立体培養 / 歯根膜細胞構築 / マラッセの上皮遺残
Research Abstract

1.Cultured cells
1) The fibroblasts derived from periodontal ligament suppressed the apparence of osteoclast-like cells when co-cultured with peripheral blood lymphocytes/monocytes unlike fibroblasts from gingiva and osteoblasts.
2) Growth and differentiation (ALPase activity) of the cloned fibroblasts in explant cultures of human periodontal ligament were studied. Their individual clones had various characterizations.
3) When the explants of human periodontal ligament were cultured by serum-free MCDB153 medium after attachment to the substrate, the epithelial cells that were immuno-positive for the anti-cytokeratin antibody were observed like as pave stone in morphology. Their growth and differentiation were different from those of human gingiva.
2.Extracellular matrices during the formation of periodontal ligament
1) There was a correlation between ALPase activity of fibroflasts and the morphology of cells, fibers and Hertwig's epithelial root sheath during the formation of periodontal li … More gament.
2) Cementoblasts that were forming cementum showed positive reaction of anti-osteocalcin antibody as well as osteoblasts and odontoblasts.
3) The reaction of anti-glycosaminoglycans antibody was shown in the low calcified cellular cementum and it was changed by aging.
3.Functions of various cells in periodontal ligament
1) Fibroblasts in periodontal ligament showed ACPase activity and the positive reaction of anti-F4/80 and MOMA-2 antibody and they were changed by the occlusal function.
2) Morphological charateristics and distribution of epithelial rests of Malassez were elucidated by immunohistochemical methods in vivo. The epithelial cells were demonstrated the same distribution of cementoblasts and located near the area synthesized much quantities of cementum by vital staining method, and they would participate in cementogenesis and synthesizing cementum. Furthermore, in these epithelial cells, the incorporation of BrdU was not observed, but the reaction of bcl-2 protein considered as one of the suppressors of appoptosis was observed by immunohistochemical methods.
3) Cementocytes were suggested to participate in secondaly mineralization of cellular cementum and homeostasis of the width of periodontal ligament. Less

Report

(4 results)
  • 1997 Annual Research Report   Final Research Report Summary
  • 1996 Annual Research Report
  • 1995 Annual Research Report
  • Research Products

    (80 results)

All Other

All Publications (80 results)

  • [Publications] IwamatsuY. et al.: "On the developing periodotal ligament of rats,using a new specimen prepatation method for SEM in comparison with histochemistry" Acta Anatomica Nipponica. 70(4). 322-329 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Tsuboi A. et al.: "Rubber hardness discrimination and associated bite force in implant patients" J.Dent Res. 74. 494- (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 許 重人 ら: "インプラント管じゃのゴム硬さ弁別と咬合力" 補綴誌. 39(93s). 135- (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kagayama M.et al.: "Time and position-spetion-specific expression of glycosaminoglycans in rat molar cementum related to physiological tooth movement" J.Periodont Res. 30. 285-289 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 金田 孝ニ ら: "中間セメント質にみられる細胞とアポトーシスの関係" 日本解剖学会誌. 70(S). 127- (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] ShimizuY. et al: "Suppression of osteoclast-like cell formation by periodontal ligament cells" J.Bone Miner Metab. 14(2). 65-72 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kagayama M.et al.: "Localization of glycosaminoglycans in periodontal ligament during physiological and experimental tooth movement" J.periodont Res.31. 229-234 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kagayama M.et al.: "An improved mounting method for observation of thick specimens using confocal microscopy" Biotechnic and Histochemistry. 71(5). 231-233 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 下西 充 ら: "培養ヒト歯根膜由来上皮細胞の免疫組織化学的特性" 日本歯科保存学雑誌. 39(s). 36- (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 加賀山 学 ら: "ラット臼歯におけるセメント質形成" 歯科基礎医学会雑誌. 38(s). 114- (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 清水 義信 ら: "ヒト末梢血リンパ球・単球の培養中にみられるTRAP陽性多核巨細胞について" 歯科基礎医学会雑誌. 38(s). 447- (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kagayama M. et al.: "Confocal microscopy of cementocytes and their lacunae and canaliculi in rat molars." Anat Embryol. 195. 491-496 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kagayama M. et al.: "Expression of osteocalcin in cementoblasts forming acellular cementum" J.Periodont Res.32. 273-278 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 清水 義信 ら: "In vitro培養したヒト末梢血リンパ球/単球から形成された破骨細胞様細胞と骨吸収窩" Jpn J. Oral Biol. 39(1). 55-63 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Shimizu Y. et al.: "Resorbable bioactive glass and resorbable hydroxyapatite in bone remodeling" Implant Dentistry. 6(3). 269-274 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 清水 義信 ら: "骨改造に対する生体活性ガラス、ハイドロキシアパタイトおよび脱灰凍結関そう人骨粉の作用" 生体材料. 15(3). 5-14 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 平田 政嗣 ら: "ラット臼歯歯根膜における上皮遺残の分布-酵素・免疫組織化学的研究" 日本歯科保存学雑誌. 40(s). 60- (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 金田 孝ニ ら: "歯根膜の発生過程におけるマクロファージ系細胞の分布変化" 歯科基礎学会雑誌. 39(s). 103- (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 下西 充: "培養ヒト歯根膜由来上皮細胞の免疫組織化学的特性" 日本歯科保存学雑誌. 40(4). 985-993 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 岩松 洋子 ら: "歯冠削除のマウス臼歯歯根膜における抗体の出現" 歯科基礎医学会雑誌. 39(s). 104- (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 平田 政嗣 ら: "マラッセの上皮遺残におけるBrdU取りこみおよびbcl-2蛋白の発現" 歯科基礎医学会雑誌. 39(s). 104- (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 西浦 寛人: "咀嚼機構における歯根膜感覚情報の役割に関する研究" JADR. (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Iwamatsu Y.et al.: "On the developing periodontal ligament of rats, using a new specimen perparation method for SEM in comparison with histochemistry." Acta Anat Nipponica. 70 (4). 322-329 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Tsuboi A.et al.: "Rubber hardness discrimination and associated bite force in implant patients." J.Dent Res. 74 (s). 494 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kagayama M.et al.: "Time and position-specific expression of glycosaminoglycans in rat molar cementum related to physiological tooth movement." J.Periodon Res. 494,30. 285-289 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kindaichi K.et al.: "The relationship of cells in the intermediated cementum and apoptosis." Acta Anat Nipponica. 70 (s). 127 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Shimizu Y.et al.: "Suppression of osteoclast-like cell formation by periodontal ligament cells." J.Bone Miner Metab. 14 (2). 65-72 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kagayama M.et al.: "Localization of glycosaminoglycans in periodontal ligament during physiological and experimental tooth movement." J.Periodont Res.31. 229-234 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kagayama M.et al.: "An improved mounting method for observation of think specimens using confocal microscopy." Biotech and Histochem. 71 (5). 231-233 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Shimonishi M.et al.: "Immunohistochemical properties of cultured epithelial cells from human periodontal ligaments." Jpn J.Conserv.Dent. 39 (s). 36 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kagayama M.et al.: "The cementum formation of rat molars." Jpn J.Oral Biol.38 (s). 114 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Shimizu Y.et al.: "Formations of osteoclast-like cells and pits in in vitro culture of peripheral blood lymphocytes/monocytes." Jpn J.Oral Biol.38 (s). 447 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kagayama M.et al.: "Confocal microscopy of cementocytes and their lacunae and canaliculi in rat molars." Anat Embryol. 195. 491-496 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kagayama M.et al.: "Expression of osteocalcin in cementoblasts forming acellular cementum." J.Periodont Res.32. 273-278 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Shimizu Y.et al.: "Formations of osteoclast-like cells and pits in in vitro culture of peripheral blood lymphocytes/monocytes." Jpn J.Oral Biol.39 (1). 55-63 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Shimizu Y.et al.: "Resorbable bioactive glass and resorbable hydroxyapatite in bone remodeling." Implant Dentistry. 6 (3). 269-274 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Shimizu Y.et al.: "Effect of bioactive glass, hydroxy apatite and demineralized freeze dried bone in bone remodeling." J.Jpn society for biomatesials.15 (3). 5-14 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Hirata M.et al.: "Distribution of the epithelial rests in the rat molar periodontal ligament. -Histochemical and immunohistochemical study-" Jpn J.Conserv.Dent.40 (s). 60 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kindaichi K.et al.: "Distribution of cells in macrophage family during the development of periodontal ligament." Jpn J.Oral Biol.39 (s). 103 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Shimonishi M.: "Properties of cultured epithelial cells from human periodontal libaments." Jpn J.Conserv.Dent.40 (4). 985-993 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Iwamatsu Y.et al.: "Distribution of anti-macrophage antibody positive cells in the mouse molar periodontal ligament with occlusal changing." Jpn J.Oral Biol.39 (s). 104 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Hirata M.et al.: "The incorporation of BrdU and the reaction of bcl-2 protein of epithelial rests of Malassez." Jpn J.Oral Biol.39 (s). 104 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Nishiura H.: "The role of periodontal mechanosensitive neurons in the masticatory system." JADR. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Iwamatsu Y.et al.: "On the developing Periodontal ligament of rats,using a new specimen preparation method for SEM in comparison with histochemistry" Acta Anatomica Nipponica. 70(4). 322-329 (1995)

    • Related Report
      1997 Annual Research Report
  • [Publications] Tsuboi A.et al.: "Rubber hardness discrimination and associated bite force in implant patients" J.Dent Res. 74. 494 (1995)

    • Related Report
      1997 Annual Research Report
  • [Publications] 許 重人ら: "インプラント患者のゴム硬さ弁別と咬合力" 補綴誌. 39(93s). 135 (1995)

    • Related Report
      1997 Annual Research Report
  • [Publications] Kagayama M.et al.: "Time and position-specific expression of glycosaminoglycans in rat moolar cementum related to physiological tooth movement" J.Periodont Res. 30. 285-289 (1995)

    • Related Report
      1997 Annual Research Report
  • [Publications] 金田一孝二ら: "中間セメント質にみられる細胞とアポトーシスの関係" 日本解剖学会誌. 70(s). 127 (1995)

    • Related Report
      1997 Annual Research Report
  • [Publications] Shimizu Y.et al: "Suppression of osteoclast-like cell formation by periodontal ligament cells" J.Bone Miner Metab. 14(2). 65-72 (1996)

    • Related Report
      1997 Annual Research Report
  • [Publications] Kagayama M.et al.: "Localization of glycosaminoglycans in periodontal ligament during physiological and experimental tooth movement" J.Periodont Res.31. 229-234 (1996)

    • Related Report
      1997 Annual Research Report
  • [Publications] Kagayama M.et al.: "An improved mounting method for observation of thick specimens using confocal microscopy" Biotechnic and Histochemistry. 71(5). 231-233 (1996)

    • Related Report
      1997 Annual Research Report
  • [Publications] 下西 充ら: "培養ヒト歯根膜由来上皮細胞の免疫組織化学的特性" 日本歯科保存学雑誌. 39(s). 36 (1996)

    • Related Report
      1997 Annual Research Report
  • [Publications] 加賀山 学ら: "ラット臼歯におけるセメント質形成" 歯科基礎医学会雑誌. 38(s). 114 (1996)

    • Related Report
      1997 Annual Research Report
  • [Publications] 清水 義信ら: "ヒト末梢血リンパ球・単球の培養中にみられるTRAP陽性多核巨細胞について" 歯科基礎医学会雑誌. 38(s). 447 (1996)

    • Related Report
      1997 Annual Research Report
  • [Publications] Kagayama M.et al.: "Confocal microscopy of cementocytes and their lacunae and canaliculi in rat molars." Anat Embryol. 195. 491-496 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Kagayama M.et al.: "Expression of osteocalcin in cementoblasts forming acellular cementum" J.Periodont Res.32. 273-278 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 清水 義信ら: "In vitro培養したヒト末梢血リンパ球/単球から形成された破骨細胞様細胞と骨吸収窩" Jpn J.Oral Biol.39(1). 55-63 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Shimizu Y.et al.: "Resorbable bioactive glass and resorbable hydroxyapatite in bone remodeling" Implant Dentistry. 6(3). 269-274 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 清水 義信ら: "骨改造に対する生体活性ガラス、ハイドロキシアパタイトおよび脱灰凍結乾燥人骨粉の作用" 生体材料. 15(3). 5-14 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 平田 政嗣ら: "ラット臼歯歯根膜における上皮遺残の分布-酵素・免疫組織化学的研究-" 日本歯科保存学雑誌. 40(s). 60 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 金田一 孝二ら: "歯根膜の発生過程におけるマクロファージ系細胞の分布変化" 歯科基礎医学会雑誌. 39(s). 103 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 下西 充: "培養ヒト歯根膜由来上皮細胞の免疫組織化学的特性" 日本歯科保存学雑誌. 40(4). 985-993 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 岩松 洋子ら: "歯冠削除時のマウス臼歯歯根膜における抗マクロファージ抗体の出現" 歯科基礎医学会雑誌. 39(s). 104 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 平田 政嗣ら: "マラッセの上皮遺残におけるBrdU取り込みおよびbcl-2蛋白の発現" 歯科基礎医学会雑誌. 39(s). 104 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 西浦 寛人: "咀嚼機構における歯根膜感覚情報の役割に関する研究" JADR. (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Iwamatsu Y. et al.: "On the developing periodontal ligament of rats, using a new specimen preparation method for SEM in comparison with histochemistry" Acta Anatomica Nipponica. 70. 322-329 (1995)

    • Related Report
      1996 Annual Research Report
  • [Publications] Tsuboi A. et al: "Rubber hardness discrimination and associated bite force in implant patients" J. Dent Res. 74. 494- (1995)

    • Related Report
      1996 Annual Research Report
  • [Publications] 許 重人ら: "インプラント患者のゴム硬さ弁別と咬合力" 補綴誌. 39(93S). 135- (1995)

    • Related Report
      1996 Annual Research Report
  • [Publications] Kagayama M. et al.: "Time and position-specific expression of glycosaminoglycans in rat molar cementum related to physiological tooth movement" J. Periodont Res. 30. 285-289 (1995)

    • Related Report
      1996 Annual Research Report
  • [Publications] 下西 充ら: "培養ヒト歯根膜由来上皮細胞の免疫組織化学的特性" 日本歯科保存学雑誌. 39. 36- (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Shimizu Y. et al: "Suppression of osteoclast-like cell formation by periodontal ligament cells" J. Bone Miner Metab. 14. 65-72 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Kagayama M. et al.: "Localization of glycosaminoglycans in periodontal ligament during physiological and experimental tooth movement" J. Periodont Res.31. 229-234 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Kagayama M. et al.: "An improved mounting method for observation of thick specimens using confocal microscopy" Biotechnic and Histochemistry. 71(5). 231-233 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 清水 義信ら: "In vitro培養したヒト末梢血リンパ球/単球から形成された破骨細胞様細胞と骨吸収窩" Jpn. J. Oral Biol.39(2). 11-20 (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Shimizu Y. et al.: "Resorbable bioactive glass and resorbable hydroxyapatite in bone remodeling" Implant Dentistry. 6(3). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] 清水 義信ら: "骨改造に対する生体活性ガラス、ハイドロキシアパタイトおよび脱灰凍結乾燥人骨粉" 生体材料. 15(2). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Kagayama M. et al.: "Confocal microscopy of cementcytes and their lacunae and canaliculi in rat molars." Anat Embryol. (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Kagayama M. et al.: "Expression of osteocalcin in cementoblasts forming acellular cementum" J. Periodont Res.(1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Yoko Iwamatsu: "On the devdoping periodontal ligament of rats,using a new specimen preparation methed for SEM in comparison with histo chenistory" Acta Aratomica Nipponica. 70. 322-329 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Yoshinobu Shimizu: "Suppressing activity of Periodontal Ligament cells on formation of osteoclast like cells" Journal of Bone and Mineral Metabolism. 14. (1995)

    • Related Report
      1995 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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