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，固定和脱矿可能会导致基质的结构变化和/或丢失。尤其是在脱矿部分检测不到钙。为了克服这一困难，必须进行无固定、无脱钙的切片。我们用粘贴膜法制备了无固定、无脱矿的大鼠磨牙冰冻切片，用偶氮染料染色检测碱性磷酸酶(ALP)的定位，用乙二醇双(2-羟基苯胺)(GBHA)染色检测钙，用免疫组织化学方法检测骨涎蛋白(BSP)和骨桥蛋白(OPN)。我们发现：1)随着牙本质矿化的开始，在矿化的牙本质上出现了最初的脱细胞牙骨质；2)最初的脱细胞牙骨质缺乏纤维，富含骨钙素和骨桥蛋白；3)在脱细胞牙骨质形成的初始阶段，成牙骨质细胞表现出强烈的碱性磷酸酶反应，并有较小的GBHA活性颗粒(=Cal…4)随着主纤维附着在牙骨质上，碱性磷酸酶反应减弱，GBHA活性颗粒减少。研究表明，在脱细胞牙骨质形成初期，牙根表面出现基质小泡和钙化球体，但主纤维附着后，两种结构均不见。根据以上发现，我们得出结论：1)在脱细胞牙骨质形成初期，成牙骨质细胞释放基质小泡，形成与GBHA活性颗粒相对应的含钙球体；2)含钙球体诱导初始脱细胞牙骨质矿化；3)在主纤维附着后，矿化沿着没有基质小泡的主纤维推进。此外，这项研究表明，在未固定的、未脱钙的切片中，组织化学和免疫组织化学反应比固定的、脱钙的切片保存得更好。较少

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