A histochemical study of the cementum mineralization in unfixed, undemineralized sections

固定的脱矿切片通常用于牙骨质的组织学和组织化学研究。然而，固定和脱矿可能导致结构变化和/或基质损失。钙在脱矿组织中尤其不能被检测到。为了克服这一困难，我们采用胶膜法制备了大鼠磨牙的非固定、非矿化冰冻切片，用偶氮染料染色法检测了碱性磷酸酶（ALP）的定位，用乙二醇双（2-羟基苯胺）（GBHA）染色法检测了钙离子的定位，用免疫组化法检测了骨唾液蛋白（BSP）和骨桥蛋白（OPN）的定位。我们发现以下内容：1）随着牙本质矿化的开始，矿化牙本质上出现初始的脱细胞牙骨质; 2）初始的脱细胞牙骨质缺乏纤维，富含BSP和OPN ; 3）脱细胞牙骨质形成初期，成牙骨质细胞显示强烈的ALP反应，并出现小的GBHA反应颗粒（=cal 关于我们 4）随着主纤维附着在牙骨质上，碱性磷酸酶反应减弱，GBHA反应颗粒减少，无细胞牙骨质形成初期，根面出现基质囊泡和含钙小球，但主纤维附着后，两者均消失。结果表明：1）在脱细胞牙骨质形成初期，成牙骨质细胞释放基质囊泡，形成含钙小球; 2）含钙小球诱导脱细胞牙骨质矿化; 3）主纤维附着后，矿化沿着主纤维进行，基质囊泡消失。此外，本研究表明，组织化学和免疫组织化学反应在未固定的，未矿化的部分比在固定的，脱矿的部分保存得更好。少

Fixed, demineralized sections have been generally used for histological and histcochemical studies of the cementum. The fixation and demineralization, however, may cause the structural change and/or loss of matrices. Calcium especially can not be detected in demineralized sections. To overcome the difficulty, unfixed,undemineralized sections are necessary.We prepared unfixed, undemineralized frozen sections of rat molars by an adhesive film method, and examined the localization of alkaline phosphatase (ALP) by Azo-dye staining, calcium by glycol bis(2-hydroxyanil)(GBHA) staining, and bone sialoprotein(BSP) and osteopontin(OPN) by immunohistochemistry. We found the following : 1) with the onset of dentin mineralization, the initial acellular cementum appears on the mineralized dentin ; 2) the initial acellular cementum is fibril-poor, and rich in BSP and OPN ; 3) at the initial acellular cementogenesis, cementoblasts show the intense ALP reaction, and small GBHA reactive particles (=cal … More cium-GBHA comprex, about 1μm in diameter) appear on the root surface; 4) as principal fibers attach to the initial cementum, the ALP reaction became low and GBHA reactive particles decrease in number.It has been shown that matrix vesicles and calciferous spherules appear on the root surface at the initial acellular cementogenesis, but after principal fiber attachment, either of the two structures is not seen. From the above findings, we concluded that: 1) at the initial acellular cementogenesis, cementoblasts release matrix vesicles, which result in calciferous spherules corresponding to the GBHA reactive particles; 2) calciferous spherules induce the mineralization of the initial acellular cementum; 3) after principal fiber attachment, the mineralization advances along principal fibers without matrix vesicles. In addition, this study showed that the histochemical and immunohistochemical reactions are preserved better in unfixed, undemineralized sections than in fixed, demineralized sections. Less