Immunoelectron microscopic studies of glycosaminoglycans in the metaphyseal bone trabeculae of growing rats

Abstract

The types and distribution of glycosaminoglycans (GAGs) were studied immunocytochemically in osteoid, mineralized bone matrix, and cartilage matrix of growing rat metaphyseal bone after aldehyde fixation and EDTA demineralization, using four monoclonal antibodies (mAbs 1-B-5, 2-B-6, 3-B-3 and 5-D-4). These mAbs specifically recognize epitopes in non-sulphated chondroitin (C0-S); chondroitin 4-sulphate (C4-S) and dermatan sulphate (DS); chondroitin 6-sulphate (C6-S) and C0-S; and keratan sulphate (KS) respectively. In osteoid, all mAbs except 1-B-5 weakly stained matrix material on and between collagen fibrils, and moderately stained organic material corresponding to bone nodules, which are known sites of mineralization. However, the staining of osteoid abruptly decreased at the mineralization front; weak staining was confined mostly to the organic material of bone nodules in mineralized bone matrix, with very weak or no staining of the rest of the bone matrix. This staining progressively decreased toward the mineralized cartilage matrix and became negative. The mineralized cartilage matrix and lamina limitans reacted strongly with all mAbs except 5-D-4. These results indicate that osteoid contains sulphated proteoglycans containing C4-S and/or DS, C6-S and KS, and subsequent bone matrix mineralization appears to require accumulation of these macromolecules within bone nodules and eventual loss of these substances for complete mineralization, whereas proteoglycans containing C0-S, C4-S and/or DS, and C6-S still exist in mineralized cartilage matrix and lamina limitans.

Dentine proteoglycans: composition, ultrastructure and functions

“…The intense immunostaining in these sites accounts for the findings of the light microscopic and ultrastructural cytochemical studies which demonstrate an increased affinity of the wall of dentinal tubules for cationic reagents (Wislocki & Sognnaes, 1950;Fulimer & Alpher, 1958;Symons, 1961;Takuma & Eda, 1966;Symons, 1968;Takagi et al, 1981), and is also comparable to the staining of the walls of bone lacuna and canaliculus in rat bone (Takagi et al, 1991). Similarly, the decreased immunostaining in the dentine resembles that seen in the mineralization front of rat bone (Kazama et al, 1992). These results indicate that similar processes are occurring in those two tissues.…”

“…This was not the case with aqueous EDTA (Goldberg et al, 1980). This conclusion is also supported by the biochemical and immuno-and lectin-histochemical investigation carried out on bone matrix proteins by Takagi et al (1992). From all these data, it can be concluded that it is necessary to study PG distribution on material fixed either with cryotechniques (although there are many problems inherent to such methods, for example: ice crystal damage unbound, radiosulphate also visible in the autoradiograms) or in the presence of cationic dyes or cationic detergents.…”

“…In this context, many studies conclude that definite differences exist between PGs in predentine and dentine (Takagi et al, 1990). This was also shown in bone (Takagi et al, 1991;Kazama et al, 1992). However, there are still some possibilities that, in predentine, a few minor PGs, as, for example, PG-II or decorin-although this component has not yet been characterized in predentine -implicated in other tissues as a controller for collagen fibrillogenesis, may be eventually incorporated in dentine together with the collagen fibres when these later are processed into the mineralized compartment.…”

“…If EDTA demineralization appears to be necessary for highly mineralized tissues, ethanolic triethylammonium EDTA (Scott & Kyffin, 1978) can be utilized, rather than standard aqueous EDTA. The application of the ethanolic triethylammonium EDTA method to ultrastructural cytochemistry and immunohistochemistry offers a valuable approach to localizing glycoconjugates in bone matrix with further increase in staining intensity, compared to that of conventionally demineralized specimens (Takagi et al, 1991(Takagi et al, , 1992.…”

Localizational Alterations of Calcium, Phosphorus, and Calcification-Related Organics Such as Proteoglycans and Alkaline Phosphatase During Bone Calcification

“…( 6,15,16 ) At the electron microscopic level, ruthenium red,( 17 ) colloidal thorium dioxide,( 17 ) high‐iron diamine,( 18 ) or cuprolinic blue (CB)( 19,20 )‐based cytochemical studies revealed PGs in osteoid and calcified nodules. These results were confirmed by immunoelectron microscopic detection of chondroitin sulfates by pre‐embedding methods( 21 ) as well as hyaluronan‐binding PG by postembedding methods. ( 22 )…”