Ultrastructural localisation of proteoglycans in the odontoblast-predentin region of rat incisor

Abstract

The localization of proteoglycans in the predentin of the rat incisor was investigated by ultrastructural histochemistry. Ruthenium red stained the cell coat of the odontoblasts as well as intracellular vesicles. There was also a staining of the extracellular matrix, but not of collagen fibers in the predentin. Treatment with the enzyme hyaluronidase prior to staining with ruthenium red abolished the staining of the vesicles and the extracellular matrix but not that of the cell coat. Bismuth nitrate and phosphotungstic acid gave similar staining of odontoblast vesicles and extracellular matrix. It is likely that the stained structures contain proteoglycans. The importance of these proteoglycans and their ultrastructural localization are discussed in relation to intracellular transport and the calcification process.

Structure and Ultrastructure of Dentine

“…Using rutheniumred staining, PG and GAG were apparent as fine granules under electron microscopy, either associated with or not associated with the collagen fibrils (NAGAI et al 1974;NYGREN et al 1976). The proteoglycans stain with cuprolinic blue, taking the form of ribbon-like structures and appearing as radiating filaments (GOLDBERG 1983;GOLDBERG and SEPTIER 1983).…”

Dentine proteoglycans: composition, ultrastructure and functions

“…In this context, cathepsin D, an acid proteinase, was identified chemically and by immunohistochemistry in the odontoblast-predentine layer. This enzyme may be involved in the degradation of PGs and GAGs (Linde & Persliden, 1977;Nygren et al, 1979). Nevertheless, some of the most essential data about dentine PGs, such as isolation and characterization of core proteins, are still lacking.…”

Ultrastructural visualization of complex carbohydrates in odontoblasts, predentin, and dentin matrix by the tannic acid-uranyl acetate method.

“…Of these, it is possible to omit osmium post-fixation with the HID-TCH-SP method [6], PA-TCH-SP method [6], and the silver methenamine method [3]. However, with the ruthenium red method [4], Alcian blue method [5], bismuth nitrate method [4,5], and the phosphotungstic acid method [3,4,5], post-fixation with lipophilic osmium tetroxide must be performed when observing the reaction products by electron microscopy. In addition, of the reagents used in these studies, ruthenium red cannot generally pass through the cell membrane because it is a macromolecular substance.…”

“…Therefore, it is impossible to detect intracellular substances that would be stained by ruthenium red[". Meanwhile, it is known that the secretory granules in odontoblasts are stained by the Alcian blue method [5], the bismuth nitrate method [4,5], and the phosphotungstic acid method [3,4,5]. However, because the secretory granules themselves have an essential affinity to osmium [6], it is impossible to differentiate them from the complex carbohydrates stained by these methods.…”

“…It has been shown previously by histochemical, biochemical, and autoradiographic methods that complex carbohydrates such as glycoproteins and proteoglycans are complex deeply involved in the formation of dentin [1,2]. The procedures used to detect these carbohydrates by electron-microscopic histochemical analysis are the phosphotungstic acid method [3,4,5], the silver methenamine method [3], the periodatethiocarbohydrazide-silver proteinate (PA-TCH-SP) method [6], the ruthenium red method [4,7], the Alcian blue method [5], the bismuth nitrate method [4,5], the colloidal thorium method [8], the high iron diamine (HID-TCH-SP) methodE6], and the cuprolinic blue method [9]. Most of these methods, however, have various drawbacks [6], so that there are in actuality few appropriate methods for the accurate detection of the complex carbohydrates involved in dentin formation.…”