Hoshi K, Ejiri S, Ozawa H
First Department of Oral Anatomy, Niigata University Faculty of Dentistry, Japan.
Ital J Anat Embryol. 2001;106(2 Suppl 1):141-50.
In order to elucidate the mechanisms of bone calcification, embryonic rat calvariae treated with chemical or cryo-fixation were observed using transmission electron microscopy by three techniques: fine structures, various cvtochemical localizations including nonspecific proteoglycan, decorin, chondroitin 4-sulfate, hyaluronan, alkaline phosphatase (ALP), and osteonectin, as well as the elemental mapping of calcium and phosphorus by energy-filtering electron microscopy. In the calvariae, the calcification sequence ran as follows crystallization within matrix vesicles, formation of calcified nodules, collagen calcification, and finally the establishment of an expansive calcified matrix. The osteoid contained an abundance of mesh-like fibers of proteoglycans, including decorin, chondroitin 4-sulfate, and hyaluronan, around collagen fibrils approximately 50 nm in diameter. Calcium tended to localize at the proteoglycan sites, while phosphorus was often mapped to the collagen fibril-structures in the osteoid. Calcium/phosphorus co-localization was found in and around the calcified nodules, where ALP and small sized proteoglycans were observed. During this stage, native proteoglycans surrounding the collagen fibrils disappeared, with the collagen fibrils fusing laterally, and attaining a diameter of more than 400nm. The calcified nodules expanded to occupy the entire space made available by the collagen fibril-fusion, following osteonectin accumulation in the calcified nodule/collagen fibril border. In conclusion, crystals present within the matrix vesicles became calcified nodules, in a process induced by the co-localization of calcium and phosphorus. ALP and proteoglycans may participate in the calcium/phosphorus co-localization. Decreases in the native proteoglycans, and the lateral fusion of collagen fibrils are thought to be involved in the expansion of calcified areas, followed by osteonectin-mediated collagen calcification.
为了阐明骨钙化的机制，采用三种技术，通过透射电子显微镜观察经化学或冷冻固定处理的胚胎大鼠颅骨：精细结构、包括非特异性蛋白聚糖、核心蛋白聚糖、硫酸软骨素4、透明质酸、碱性磷酸酶（ALP）和骨连接蛋白在内的各种细胞化学定位，以及通过能量过滤电子显微镜对钙和磷进行元素映射。在颅骨中，钙化序列如下：基质小泡内结晶、钙化结节形成、胶原钙化，最后形成扩张的钙化基质。类骨质在直径约50nm的胶原纤维周围含有大量蛋白聚糖的网状纤维，包括核心蛋白聚糖、硫酸软骨素4和透明质酸。钙倾向于定位于蛋白聚糖部位，而磷常映射到类骨质中的胶原纤维结构上。在钙化结节及其周围发现了钙/磷共定位，在那里观察到了ALP和小尺寸的蛋白聚糖。在此阶段，胶原纤维周围的天然蛋白聚糖消失，胶原纤维横向融合，直径达到400nm以上。随着骨连接蛋白在钙化结节/胶原纤维边界处积累，钙化结节扩展以占据胶原纤维融合所提供的整个空间。总之，基质小泡内的晶体在钙和磷共定位诱导的过程中变成钙化结节。ALP和蛋白聚糖可能参与钙/磷共定位。天然蛋白聚糖的减少以及胶原纤维的横向融合被认为与钙化区域的扩展有关，随后是骨连接蛋白介导的胶原钙化。
