Ultrastructural analysis of bone calcification by using energy-filtering transmission electron microscopy.

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.