Characteristics and localization of rat submandibular gland proteoglycans.

The submandibular gland proteoglycans were investigated biochemically and immunohistochemically in male Sprague-Dawley rats. Proteoglycans were extracted with 4 M guanidine-HCl, followed by ultracentrifugation in a CsCl density gradient, and fractionated by ion-exchange chromatography and gel filtration. The molecular weight of PGs was estimated by SDS-PAGE and immunoblot analysis with monoclonal antibodies (HepSS-1 or 6-B-6). The glycosaminoglycan side-chains in the proteoglycan fractions were identified by electrophoresis on cellulose acetate membrane. Three proteoglycan fractions were obtained. One was a heparan sulphate proteoglycan that migrated as a diffuse band of about 210 kDa. The other two fractions contained at least two dermatan sulphate proteoglycans of 70-85 kDa and 40-50 kDa. Digestion of these two proteoglycans with chondroitinase ABC, but not heparitinase, produced two bands of 50 and 21 kDa, which were core proteins. The smaller dermatan sulphate proteoglycan may be a portion of the other, as the core protein of both bound to 6-B-6 antibody, and sugar chains of both were the same (20-30 kDa). Heparan sulphates recognized by antibody HepSS-1 were observed widely in the basement membrane, fibrous connective tissue, and striated and excretory ductal cells, while dermatan sulphate proteoglycans recognized by antibody 6-B-6 were located in the connective tissue surrounding striated and excretory ducts.

Disaccharide analysis of chondroitin sulphate in human gingival crevicular fluid using high-performance liquid chromatography.

Gingival crevicular fluid (GCF) was collected into capillary tubes from healthy gingiva and sites of advanced periodontitis. Following digestion with Pronase E, the glycosaminoglycans were isolated by successive precipitation into 5% cetylpyridinium chloride and 95% ethanol. Unsaturated disaccharide isomers of chondroitin sulphate, obtained following chondroitinase ACII digestion, were analysed by high-performance liquid chromatography. Chondroitin sulphate was found in all GCF samples, with greater amounts in patients with periodontal disease than at control sites with a relatively healthy periodontium. The predominant isomer in the periodontal diseased group was delta Di-4S, while that in the control group and serum samples was delta Di-0S. Comparison of the relative proportions of the unsaturated disaccharides in GCF with previously reported values for alveolar bone, cementum, gingiva and periodontal ligament, as well as for serum, indicates that the chondroitin sulphate present in GCF of patients with periodontal disease originated from the mineralized connective tissues of the periodontium, notably alveolar bone, possibly with some contributions from soft connective tissues of gingiva and periodontal ligament and from serum.

Biochemical analysis of extracellular matrix in bovine periodontal ligament.

The molecular relationships of extracellular matrix (ECM) from bovine periodontal ligaments to supporting mechanisms of the ligament were investigated. Glycosaminoglycan (GAG) and collagen components of the ECM were compared quantitatively and qualitatively for periodontal tissue adjacent to mandibular anterior (APL) and maxillary posterior (PPL) teeth. There was no significant quantitative difference GAG between the APL and PPL. GAGs in both APL and PPL consisted primarily of dermatan sulfate (DS), chondroitin sulfate (CS) and hyaluronic acid (HA). Heparan sulfate was present as a minor component. Analysis of unsaturated disaccharides obtained from the GAG revealed that delta Di-0S from CS in the PPL was significantly higher than that in the APL, and delta Di-4S from DS in the APL was significantly higher than within the PPL. Furthermore, there was significantly more collagen (as determined by Hyp content) in the APL than in the PPL although the chemical composition of collagen was the same for both. These results suggest that ECM molecules in the APL exhibit both quantitative and qualitative differences to those in the PPL, possibly owing to contrasting mechanisms of tooth support in response to different masticatory functions.