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

其基本目的是通过电子显微镜(EM)和细胞化学手段阐明生物钙化的机制。主要目的是揭示硬组织钙化初期的超微结构和元素分析特征。在液氦温度下采用冷冻取代法获得显影晶体的高分辨率电子显微照片，并用能谱定量分析元素。在基质囊泡(MV)钙化开始时，无论是大鼠颅骨还是胫骨生长板的MVS内的晶体都显示出分辨率较差的点阵图像(LIS)，约为0.25 nm周期，平均Ca/P摩尔比(MR)为1.3，表明为八钙磷酸钙(OCP)。从MVS延伸出来的晶体显示出明显的LIS相同时期的分解，并给出了平均Ca/P MR为1.5，表明磷酸三钙(TcP)。在钙化的较高级阶段，晶体的平均Ca/P MR为1.67，…更多的指示磷灰石(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。观察胎鼠顶骨ALPase活性的细胞化学定位，以阐明成骨细胞的细胞分化与钙化过程的关系。分化中的成骨细胞在质膜和MVS上表现出强烈的ALPase活性。离原始钙化区越近，MVS和成骨细胞的ALPase活性越强，而钙化范围较广的骨基质质膜上ALPase活性不明显。同时对鸡肌腱进行钙化检查。小腿肌腱中的纤维软骨细胞产生MVS，其中最初发生钙化，然后晶体在钙化的MVS附近的胶原纤维上生长。用铅作为钙沉积的示踪剂也得到了类似的结果。对大鼠皮下结缔组织注射醋酸铅后的异位钙化也进行了电子显微镜观察，结果表明，铅最初沉积在变性的胶原纤维内，而磷灰石晶体则在铅沉积的同一部位开始形成。较少

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。