Ultrastructural and immunohistochemical studies of medullary bone calcification, with special reference to sulphated glycosaminoglycans

Histochemical, immunohistochemical and electron energy-loss spectroscopic studies were performed to examine the relationship between sulphated glycosaminoglycans and medullary bone calcification using oestrogen-injected male Japanese quail. Sulphated glycosaminoglycans, detected by high iron diamine (HID) or HID-thiocarbohydrazide-silver protein (HID-TCH-SP) methods, were distributed throughout the matrix of medullary bone, some periphery and extending tips of the trabeculae stained weakly, and the globular structures at osteoid areas were exclusively positive for HID-TCH-SP stain. Immunohistochemistry identified keratan sulphate located in the globular structures at osteoid areas and calcified matrix, but chondroitin-4 sulphate and chondroitin-6 sulphate were not detected in the matrix. Using electron spectroscopic imaging, sulphur was determined to be localized in the globular structures. These results demonstrate that medullary bone matrix accumulates keratan sulphate in the globular structures, which are the foci for calcification, and eventually in the calcified areas. This suggests that keratan sulphate containing sulphur is maintained in the calcified matrix. These results indicate a unique process of calcification exists in medullary bone.