Ultrastructure of the organic matrix of embryonic avian bone after en bloc reaction with various electron-dense 'stains'.

The morphological distribution of noncollagenous proteins in bone matrix is poorly known, chiefly because their amount changes with the type of bone, and they are not easily recognizable under the electron microscope. The aim of this study was to obtain further ultrastructural data on these proteins and on their relationships with collagen fibrils and the calcification process. Specimens of woven bone from the upper metaphysis of the tibia of chick embryos were examined after en bloc treatment with acetone solution of KMnO4, or aqueous or alcoholic solutions of phosphotungstic acid or uranyl acetate. Untreated specimens were used as controls. Untreated, decalcified, and decalcified and stained sections were examined. As expected, the results showed that the woven bone matrix of the chick embryo consists of irregularly oriented collagen fibrils which outline wide interfibrillary spaces. These contain calcification nodules at the calcification front and are full of irregularly oriented crystals in completely calcified matrix. These spaces contain greater than expected amounts of filamentous or granular organic structures (probably corresponding to noncollagenous proteins), which are in a close relationship with needle- and filament-like crystals. The collagen fibrils are calcified to a lower degree than the interfibrillary spaces, and often appear uncalcified, or decalcified, when the interfibrillary spaces do not. This suggests that either the former calcify after the latter, or the respective mineral phases differ in their degree of solubility, so that the decalcification of collagen fibrils precedes that of interfibrillary spaces. The results confirm that woven bone contains an amount of interfibrillar noncollagenous material greater than lamellar bone, and show that woven bone calcification occurs not only in and on collagen fibrils, but chiefly between them, in relationship with noncollagenous filamentous material.