Ultrastructural and cytochemical studies on the hard tissue calcification mechanism.

硬组织钙化机制的超微结构和细胞化学研究。

• 批准号: 59440076
• 负责人: OZAWA Hidehiro
• 金额: $ 14.72万
• 依托单位: Niigata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (A)
• 财政年份: 1984
• 资助国家: 日本
• 起止时间: 1984 至 1986
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-59440076/
• 关键词: Freeze-substitution; matrix vesicles; calcification; elemental analysis; ALPase; lead acetate; ectopic calcification; 異所性石灰化; 膠原線維; 基質小胞性石灰化; コラーゲン性石灰化; 急速凍結置換法; 鉛トレーサー実験; アルカリホスファターゼ活性

The basic objective is to elucidate the biological calcification mechanism by means of electron microscopy(EM) and cytochemistry. The major approach was designed to reveal the ultrastructural and elemental analytical characteristics of the initial stage of the hard tissue calcification. Freeze-substitution methods at the liquid helium temperature were employed to obtain high resolution electron micrograaphs of developing crystals, and to detect elements by the quantitative EDX. At the beginning of matrix vesicle(MV) calcification, crystals within the MVs of either rat calvaria or the tibial growth plate showed the lattice images (LIs) poorly resolved as about 0.25nm period, and gave a mean Ca/P molar ratio(MR) of 1.3, indicating octacalcium phosphate(OCP). Crystals extended from MVs showed the distinct LIs resolved as the same period, and gave a mean Ca/P MR of 1.5, indicating tricalcium phosphate(TCP). At the more advanced stage of calcification, crystals gave a mean Ca/P MR of 1.67, … More indicating apatite(HA). This suggests that calcification at the early stage may occur from ACP or OCP within the MVs, and transform into HA through TCP. The cytochemical location of ALPase activity of fetal rat parietal bones was observed to elucidate the relationship between cyto-differentiation of osteoblasts and the calcification process. Differentiating osteoblasts showed intense ALPase activity on the plasma membranes as well as MVs. The closer to the initially calcified area, the stronger was the ALPase activity in both MVs and osteoblasts, though the activity was not shown in the plasma membrane surfacing on the bone matrix where the calcification was more extensive. The chicken tendon calcification was also examined. Fibrocartilage cells in the leg tendon produced MVs in which the initial calcification occured, and in turn crystals extended on the collagen fibrils adjacent to the calcified MVs. The similar result was obtained by using lead as a tracer for calcium deposition. The ectopic calcification induced by the administration of lead acetate to the rat subcutaneous connective tissue was also studied by EM, resulting that the initial lead deposition occured within the degenerated collagen fibrils, and in turn apatite crystal formation began at the same sites of lead deposition. Less

基本目的是通过电子显微镜（EM）和细胞化学方法阐明生物钙化机制。主要的方法旨在揭示硬组织钙化初期的超微结构和元素分析特征。采用液氦温度下的冷冻置换法，获得了高分辨的显影晶体电子显微图，并用能谱仪进行了元素的定量分析。在基质囊泡（MV）钙化开始时，大鼠颅骨或胫骨生长板MV内的晶体显示晶格图像（LI）分辨率低，约为0.25nm周期，平均Ca/P摩尔比（MR）为1.3，表明磷酸八钙（OCP）。从MV延伸的晶体显示出相同时期分辨的不同LI，并给出1.5的平均Ca/P MR，表明磷酸三钙（TCP）。在钙化的更晚期，晶体的平均Ca/P MR为1.67， ...更多信息 指示磷灰石（HA）。这表明早期钙化可能发生在MVs内的ACP或OCP，并通过TCP转化为HA。本实验观察了胎鼠顶骨成骨细胞分化与钙化过程的关系。分化的成骨细胞在质膜和MV上显示出强烈的ALcase 3活性。越靠近最初钙化区域，MV和成骨细胞中的ALcase 3活性越强，尽管在钙化更广泛的骨基质上的质膜表面没有显示出活性。还检查了鸡肌腱钙化。肌腱中的纤维软骨细胞产生了最初钙化发生的MV，而晶体又在钙化MV附近的胶原纤维上延伸。用铅作钙沉积的示踪剂也得到了类似的结果。用电镜观察了醋酸铅对大鼠皮下结缔组织的异位钙化，结果表明，铅最初沉积在变性的胶原纤维内，而磷灰石晶体的形成则是在铅沉积的同一部位开始的。少

项目成果

T.Uchida: Proc.VIIIth Intl.Cong.on Calcium Regulating Hormones.8A. 441-443 (1984)

T.Uchida：Proc.VIIIth Intl.Cong.on 钙调节激素.8A。

H.Ozawa: Proc.IVth Intl.Symposium on the Composition,Properties and Fundermental Structure of Tooth Enamel.146-150 (1984)

H.Ozawa：Proc.IVth Intl.Symposium on the Composition,Properties and Fundamental Structure of Tooth Enamel.146-150 (1984)

T.Yamamoto: Histochemistry. 83. 221-226 (1985)

T.Yamamoto：组织化学。

小澤英浩: 歯科基礎医誌. 27. 751-774 (1985)

小泽秀宏：基础牙科医学杂志 27. 751-774 (1985)

Y.Takano: Cell Tissue Res.,. 243. 91-99 (1986)

Y.Takano：细胞组织研究。