Ultrastructural and cytochemical studies on the hard tissue calcification mechanism.

基本目的是通过电镜和细胞化学手段阐明生物钙化机制。主要方法旨在揭示硬组织钙化初始阶段的超微结构和元素分析特征。采用液氦温度下的冷冻取代方法获得了显影晶体的高分辨率电子显微图，并通过定量EDX检测元素。在基质囊泡钙化开始时，大鼠颅骨和胫骨生长板基质囊泡内的晶体在0.25nm周期内显示晶格图像（LIs）分辨率较差，平均Ca/P摩尔比（MR）为1.3，表明为磷酸八钙（OCP）。从mv延伸出的晶体显示出相同周期的明显的li，并且平均Ca/P MR为1.5，表明磷酸三钙（TCP）。在钙化较晚期，晶体的平均Ca/P MR为1.67，更多表明磷灰石（HA）。这表明早期钙化可能发生在心肌内的ACP或OCP，并通过TCP转化为HA。通过观察胎鼠顶骨ALPase活性的细胞化学定位，阐明成骨细胞分化与钙化过程的关系。分化成骨细胞在质膜和细胞膜上表现出强烈的ALPase活性。越接近初始钙化区域，MVs和成骨细胞中的ALPase活性越强，尽管在骨基质表面的质膜上没有显示出活性，那里钙化更广泛。同时对鸡肌腱进行钙化检查。腿肌腱中的纤维软骨细胞产生初始钙化发生的MVs，然后晶体延伸到钙化MVs附近的胶原原纤维上。用铅作为钙沉积的示踪剂，也得到了类似的结果。电镜研究了醋酸铅对大鼠皮下结缔组织的异位钙化作用，发现最初的铅沉积发生在退化的胶原原纤维内，而在铅沉积的相同部位开始形成磷灰石晶体。少

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