Glycosaminoglycans in the lamina propria and submucosal layer of the monkey palatal mucosa.

Glycosaminoglycans (GAGs) in monkey palatal lamina propria plus both fatty and glandular zones of palatal submucosa were compared. Chemical analysis revealed that GAG contents of the lamina propria and glandular zone were higher than that of the fatty zone. The four GAGs identified by electrophoretic analysis were hyaluronic acid, dermatan sulfate, chondroitin sulfate and heparan sulfate. Each mucosal layer contained all four GAG components. The predominant GAG in both the lamina propria and glandular zone was dermatan sulfate followed by hyaluronic acid. The reverse situation (predominant hyaluronic acid, less prominent dermatan sulfate) was noted in the fatty zone of the submucosa. The three tissue regions showed different molar ratios of unsaturated chondroitin sulfate disaccharides. The ratio of delta Di-4S to delta Di-6S was lower in the lamina propria than in either the fatty or glandular submucosal zones.     

Qualitative and quantitative analysis of bovine, rabbit and human dental pulp glycosaminoglycans.

Bovine, rabbit and human dental pulp glycosaminoglycans were analyzed qualitatively and quantitatively using two-dimensional electrophoresis. The major components of bovine and rabbit dental pulp were chondroitin 4-sulphate and hyaluronic acid, while in the human dental pulp dermatan sulphate and chondroitin 4-sulphate were the major components.     

Isolation, identification, and quantitation of glycosaminoglycans synthesized by human gingival fibroblasts in vitro

The glycosaminoglycans synthesized by diploid fibroblasts obtained from healthy human gingivae of three donors were isolated, identified, and quantified. Degradation with specific enzymes identified the glycosaminoglycans as hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate; hyaluronic acid predominating. The distribution of the sulfated glycosaminoglycans in the cell layer and the medium was not the same. The cells contained mainly heparan sulfate (48.3%) and the medium mainly dermatan sulfate (47%).     

Sulfated glycosaminoglycan synthesis and its regulation by transforming growth factor-beta in rat clonal dental pulp cells

Dental pulps contain sulfated glycosaminoglycans (GAGs), such as chondroitin 4-sulfate (CSA/4CS), dermatan sulfate (CSB/DS), and chondroitin 6-sulfate (CSC/6CS). Sulfated GAGs play important roles in mineralization and collagen fibrillogenesis during primary, secondary, and reparative dentin formations. Transforming growth factor-beta (TGF-beta) is a potent regulator for several extracellular matrix (ECM) components and modulates the proliferation and differentiation. Using rat clonal dental pulp cells (RPC-C2A), we investigated the constituents of GAGs synthesized by the cells and the effect of TGF-beta on their synthesis by measuring the radioactivity of [35S]sulfate incorporated into GAG fractions. Cellulose acetate electrophoresis analysis revealed that RPC-C2A cells synthesized CSA and CSB but not CSC and that 10 ng/ml of TGF-beta increased the production of CSA and CSB in the cell/ECM fraction. Measurement of [35S]sulfate incorporation showed a significant increase in the amount of GAGs by TGF-beta, 1.3-fold CSA, and 1.2-fold CSB in the cell/ECM fraction. In the medium fraction the most secreted GAG was CSA, whereas CSB was stored in the cell/ECM fraction. Secreted CSA in the medium was markedly increased by 10 ng/ml of TGF-beta (1.7-fold). These findings indicate that CSA and CSB are major sulfated GAGs synthesized by RPC-C2A cells and that TGF-beta acts as a stimulator of sulfated GAG synthesis in dental pulp cells.     

Fibroblastic subpopulations in uninjured and wounded rabbit oral mucosa

Fibroblast cultures derived from uninjured and reparative rabbit buccal mucosa were compared in terms of extracellular glycosaminoglycan (GAG) content and cellular response to interleukin-1 (IL-1). Under identical growth conditions, proliferation of both cell lines was the same. Both lines incorporated [3H]-glucosamine into GAG in cellular, pericellular, and medium fractions, with the majority of incorporated label residing in the medium. Dermatan sulfate (DS) was the predominant GAG in the medium fraction of both normal and wound fibroblast cultures; however, the two cell lines differed in the identity of the medium fraction's secondary GAG: chondroitin sulfate (CS) for normal fibroblasts and hyaluronic acid (HA) for wound-derived cells. The GAG content of the pericellular matrix for all cultures was the same regardless of the tissue of origin: heparan sulfate (HS) accompanied by a very small amount of CS. Exposure to IL-1 produced limited but highly specific effects: It was not mitogenic for either cell line but did cause a quantitative change (increase) in overall incorporation into GAG for medium and pericellular fractions for both cell lines. Further, IL-1 induced a qualitative change in GAG composition for normal mucosal fibroblastic medium fractions by causing the synthesis/release of heparan sulfate (HS) and a variant form of DS. These data support the hypothesis that different fibroblastic substrains can populate a given oral site as a function of variables such as injury and/or healing status.     

Human gingival glycosaminoglycans in cyclosporin-induced overgrowth

Glycosaminoglycans in normal and cyclosporin‐induced gingival overgrowth were extracted by papain digestion and purified by Mono Q‐FPLC chromatography. The purified glycosaminoglycans were analyzed by agarose gel electrophoresis and by the pattern of degradation products formed by chondroitin lyases on HPLC chromatography. Our results on the glycosaminoglycan composition showed presence of chondroitin 4‐ and 6‐sulfate, dermatan sulfate, heparan sulfate and hyaluronic acid in both normal gingiva and cyclosporin‐induced gingival overgrowth. The total and relative amounts of glycosaminoglycans were similar between normal and overgrown gingiva. This suggests that the glycosaminoglycan composition is not changed in cyclosporin‐induced gingival overgrowth. Our present biochemical results conflict with histochemical and biosynthetic data previously reported by other groups. Those studies suggested that the affected tissues contained higher levels of glycosaminoglycans and that cyclosporin induced comparably high levels of these compounds in in vitro cultures of gingival fibroblasts. Therefore, these discrepant results suggest that a cyclosporin‐induced increase on gingival glycosaminoglycans still remains an open question. The implications of these conflicting results are discussed.     

Analysis of chondroitin sulfate isomers in the periodontium of the monkey using high-performance liquid chromatography

Glycosaminoglycan (GAG) was extracted from monkey periodontium, consisting of gingiva, periodontal ligament, alveolar bone and cementum, and from dental pulp and dentin by digestion with Pronase E. Unsaturated disaccharide isomers formed by chondroitinase AC digestion from chondroitin sulfate were labeled with dansylhydrazine and analyzed by high-performance liquid chromatography. These tissues showed different molar ratios of the unsaturated chondroitin sulfate disaccharides. The ratio of delta Di-4S to delta Di-6S was lowest in the dental pulp, followed by the gingiva, periodontal ligament, dentin, alveolar bone, and cementum, in that order. It was greater in the calcified than in the uncalcified tissues.     

Glycosaminoglycans of human cementum

The glycosaminoglycans in human cementum have been studied. Following proteolytic digestion of guanidine/EDTA and collagenase extracts of cementum, glycosaminoglycans were isolated and then separated by cellulose acetate membrane electrophoresis. After specific elimination by enzymatic and chemical treatments the glycosaminoglycans were identified as hyaluronic acid, chondroitin sulfate and dermatan sulfate. Neither heparan sulfate nor keratan sulfate were observed. Quantitation of the glycosaminoglycans in both extracts revealed chondroitin sulfate to represent the major species present. Hyaluronic acid was observed predominantly in the guanidine/EDTA extract while dermatan sulfate was a quantitative minor component of both extracts.     

Studies on matrix components relevant to structure and function of the temporomandibular joint]

Proteoglycans (PGs), or their component glycosaminoglycan (GAG) chains, have long been recognized as small in quantity but as significant components for many connective tissues. The temporomandibular joint (TMJ) disc and synovial fluid were rarely investigated in this regard. This research was aimed at the biochemical analysis of the composition of collagen, GAGs and PGs in the normal human and porcine TMJ discs, and of PGs in the synovial fluid. 1. The collagen type analysis by gel electrophoresis revealed that the human and porcine TMJ discs were composed only of type I collagen. 2. The human TMJ disc contained 69.9% chondroitin sulfate, 24.5% dermatan sulfate, 5.6% hyaluronic acid and a trace amount of keratan sulfate, and the porcine TMJ disc, 5.8%, 91.4%, 2.8% and a trace amount, respectively. Immunohistochemical staining of the functional parts of the TMJ disc showed a universal distribution of these GAGs. 3. PGs were extracted from the TMJ disc with 4 M guanidine chloride and separated by ion-exchange and gel-filtration chromatography. A high molecular weight PG resembling the cartilage PG and two low molecular weight PGs having a core protein of Mr 40 K were isolated. 4. PGs suggested to be derived from the matrix of other tissues were found in the abnormal human synovial fluid. From these results it was suggested that the TMJ disc was thought not to be fibrocartilage but a fibrous tissue adapted against to the compressive and tensile force.     

Immunohistochemical study of chondroitin sulfate and dermatan sulfate proteoglycan in dental pulp and dentin

We investigated the localization of chondroitin sulfate and dermatan sulfate proteoglycans in the dental pulp and dentin of rats, using a combination of an immunohistochemical technique coupled with specific enzymatic digestion. Chondroitin 4-sulfate and dermatan sulfate were found to be widespread in pulpal connective tissue, predentin and dentinal tubules. The response to predentin was found to be particularly strong. Chondroitin 6-sulfate was stretched in pulpal connective tissue and predentin, but showed only a weak response.     

Production of colony-stimulating factor in human dental pulp fibroblasts

Class II major histocompatilibity complex (MHC)-expressing cells are usually distributed in dental pulp, and it was postulated that the colony-stimulating factor (CSF) derived from dental pulp fibroblasts contributes to the migration of class II MHC-expressing cells into pulp tissue. This study aimed to investigate the CSF production of human dental pulp fibroblasts. In pulp tissue sections, granulocyte (G)-CSF was detected from normal teeth, while G-CSF, macrophage (M)-CSF, and granulocyte-macrophage (GM)-CSF were detected from teeth with dentinal caries. In cultured dental pulp fibroblasts, G-CSF was detected by immunostaining, immunoprecipitation, and ELISA, and mRNAs of G-CSF, M-CSF, and GM-CSF were detected by RT-PCR. The dental pulp fibroblasts cultured with TNF-alpha were found to increase the G-CSF expression and to produce M-CSF and GM-CSF. These findings suggest that dental pulp fibroblasts usually produce G-CSF. In the presence of TNF-alpha, dental pulp fibroblast express M-CSF and GM-CSF.     

Glycosaminoglycan synthesis by adult rat submandibular salivary-gland secretory units

The synthesis of glycosaminoglycans (GAG) by a preparation of purified, functional submandibular-gland secretory units (acini and intercalated ducts) was examined. Such units were isolated from Sprague-Dawley rats by digestion of minced gland with hyaluronidase and collagenase followed by gentle sieving of the digest through a graded series of Teflon screens. They incorporated amino acids into exocrine proteins which could be released by stimulation with isoproterenol as in vivo, indicating their functional integrity. Secretory units, incubated for 2 h in medium containing [35S]-sodium sulphate alone or in combination with [3H]-glucosamine, were then washed, homogenized and digested in pronase. The resulting material was then sequentially digested by specific enzymic and chemical procedures and analysed by chromatography on Sephadex G-50 columns to identify the various GAG synthesized. Secretory units synthesized a GAG mixture which was 20-25 per cent hyaluronic acid, 70-75 per cent heparan sulphate, and only 3-5 per cent chondroitin or dermatan sulphates, similar to that synthesized in vivo. No GAG was present in the secretory material, suggesting that all the GAG synthesized was destined for the basement membrane or cell surface.     

Occurrence of multiple dentigerous cysts in a patient with the Maroteaux-Lamy syndrome (mucopolysaccharidosis, type VI)

The mucopolysaccharidoses (MPS) are a group of genetic lysosomal storage diseases. These diseases result from a defect in specific lysosomal enzymes required for the degradation of specific mucopolysaccharides. These incompletely degraded saccharides accumulate in tissues and are excreted in the urine. A general characteristic of these diseases is dysostosis multiplex. Dental complications can be severe and include unerupted dentition, dentigerous cystlike follicles, malocclusions, condylar defects, and gingival hyperplasia. This report examines multiple dentigerous cysts in a patient with a deficiency in N-acetylgalactosamine-4-sulfatase, Maroteaux-Lamy syndrome (MPS VI). The inability to hydrolyze the sulfate group from N-acetylgalactosamine-4-sulfate residue of dermatan sulfate due to a deficiency in this enzyme results in the accumulation of dermatan sulfate in tissues and its excretion in the urine. Examination of dentigerous cyst fluid revealed glycosaminoglycan content of 397 microgram per milliliter. Compositional analyses revealed 60% hyaluronic acid, 30% chondroitin 4- and -6-sulfate, and only 10% dermatan sulfate. This was consistent with dentigerous cyst fluid derived from persons without mucopolysaccharide-storage disorders but distinctly different from glycosaminoglycans assayed from other body fluids of this patient.     

Interaction of acid glycosaminoglycans (mucopolysaccharides) with hydroxyapatite

Abstract— Selected acidic glycosaminoglycans (GAG) have been used as a model series to study some binding properties of hydroxyapatite. The order of binding was heparin, heparin sulfate, dermatan sulfate/chondroitin-4-sulfate/chondroitin-6-sulfate (all similar) and hyaluronic acid, indicating that the negative charge on the molecules is a major determinant in the binding process. Both calcium and saliva pretreatrnent led to an increase in uptake of selected GAG whereas fluoride even at 1 and 3 parts/10⁶ levels led to a graded reduction in uptake. The clinical significance of these findings may be related to the presence of certain GAG in the dental integuments and to the interaction of a chemically defined group of compounds at a specific solid surface. Such findings may also have an application in studies on endogenous mineralization. The results are in accord with the generally held view that polyanions may interact electrostatically with calcium sites present in the crystal lattice of the hydroxyapatite.     

Interaction of acid glycosaminoglycans (mucopolysaccharides) with hydroxyapatite.

Selected acidic glycosaminoglycans (GAG) have been used as a model series to study some binding properties of hydroxyapatite. The order of binding was heparin, heparin sulfate, dermatan sulfage/chondroitin-4-sulfate/chondroitin-6-sulfate (all similar) and hyaluronic acid, indicating that the negative charge on the molecules is a major determinant in the binding process. Both calcium and saliva pretreatment led to an increase in uptake of selected GAG whereas fluoride even at 1 and 3 parts/106 levels led to a graded reduction in uptake. The clinical significance of these findings may be related to the presence of certain GAG in the dental integuments and to the interaction of a chemically defined group of compounds at a specific solid surface. Such findings may also have an application in studies on endogenous mineralization. The results are in accord with the generally held view that polyanions may interact electrostatically with calcium sites present in the crystal lattice of the hydroxyapatite.     

Glycosaminoglycans of gingival epithelium and connective tissue during experimental periodontitis in dogs

The purpose of this study was to investigate the changes in concentration of glycosaminoglycans (CAGs) and to investigate the incorporation of 3H-glucosamine into GAGs in vitro in the epithelium and sub-epithelium connective tissue separated from the gingiva during a period of experimental periodontitis. Periodontitis was induced by placement of a silk ligature below the gingival margin in dog molars. The GAGs extracted from gingival samples obtained 0, 7, 21, 60 and 90 days before and after the ligature placement were separated by cellulose acetate membrane electrophoresis for both qualitative and quantitative analysis. Hyaluronic acid content of the epithelium was decreased significantly at the acute phase of inflammation. In the connective tissue, the amounts of dermatan sulfate and hyaluronic acid were higher, but chondroitin sulfate and heparan sulfate levels lower than in the control. The incorporation of 3H-glucosamine into GAGs in the epithelium was greater than that in connective tissue at the acute phase. The greatest incorporation of 3H-glucosamine was found in chondroitin sulfate at the acute phase, and did not return to the basal level at the chronic phase. These findings suggest that the biochemical response of GAGs in the epithelium to inflammation might be different from that in connective tissue.     

The effect of chronic inflammation on gingival connective tissue proteoglycans and hyaluronic acid

Proteoglycans have been isolated and analysed from extracts of normal and chronically inflamed human gingiva in order to determine the effects of chronic inflammation on these important soft connective tissue extracellular macromolecules. The uronic acid content of glycosaminoglycans isolated by papain digestion of normal and inflamed gingiva did not differ significantly. Likewise, electrophoretic analysis revealed that the content of hyaluronic acid, heparan sulfate, dermatan sulfate and chondroitin sulfute was similar. The sulfated glycosaminoglycans from both sources eluted from a Sepharose C1-6B column with a Kav of 0.45 (approximate M_r 25,000). However, hyaluronic acid from normal gingiva was predominantly of a large size eluting in the void volume of a Sepharose. CL-6B column, while that isolated from inflamed tissue was mostly a small molecular weight species which elutccl in the included volume of a Sepharose CL-6B column. Using dissociative conditions, intact proteoglycans could be more readily extracted from inflamed tissues (90% of the total tissue uronic acid) than from normal tissues where only 80% of the total tissue uronic acid was extractable. Even though DEAE-Sephacel ion-exchange chromatography revealed no differences in charge between normal and inflamed gingival proteoglycans, Sepharose CL-4B chromatography revealed more molecular size polydispersity in samples from inflamed tissue than from normal tissue. Taken together, these results indicate that while hyaluronic acid is depolymerized in inflamed tissue, no evidence of sulfated glycosaminoglycan degradation was found. Therefore, the most likely cause for disruption to the molecular integrity of the proteoglycans is via proteolytic alteration to the proteoglycan core protein.     

Qualitative and quantitative analyses of bovine gingival glycosaminoglaycans.

Bovine gingival glycosaminoglycans have been analysed qualitatively and quantitatively by two-dimensional electrophoresis on a cellulose acetate strip. The four spots observed were identified as chondroitin 4-sulphate, dermatan sulphate, hyaluronic acid and heparan sulphate. Neither chondroitin 6-sulphate nor heparin and keratan sulphate were observed.The major components of bovine gingival glycosaminoglycans were chondroitin 4-sulphate, 32–40 per cent; dermatan sulphate, 33–37 per cent; hyaluronic acid, 17–27 per cent. Heparan sulphate was present only in a limited amount. The total uronic acid content of bovine gingiva, however, decreased with age, especially during the first three years of life, possibly due to the marked decrease of both chondroitin 4-sulphate and dermatan sulphate. After 3 years of age, the decrease of these glycosaminoglycans slowed down considerably. Hyaluronic acid decreased rather slowly from the time of birth to 10 years of age, and heparan sulphate decreased initially but increased later.     

Rapid determination of dry weight in human dental pulp by a colorimetric reaction

This study was designed to assess total carbohydrate (TC) concentration, as well as the noncollagenous protein content, in human dental pulp. Pulps were obtained from eight premolars (13.10 +/- 4.33 mg weight, mean +/- SD) and homogenized in saline solution. TC content was as follows: 16.68 +/- 9.49 micrograms/mg of tissue (mean +/- SD); 3.22 +/- 1.69 ng of TC/mg protein (mean +/- SD); and 16.23 +/- 6.80 micrograms of TC/mg total organic material (mean +/- SD). The high concentration of carbohydrates observed in the pulp is a result of the presence of glycoproteins, glycosaminoglycans (chondroitin-6-sulfate, heparan sulfate, hyaluronic acid, and dermatan sulfate), and proteoglycans, whose structural, biochemical, and physiological functions have been well documented. Dry weight determination using dichromate solution was used because it shows less dispersion than when the data on carbohydrates and proteins are expressed as wet weight.     

Molecular size distribution analysis of human gingival glycosaminoglycans in cyclosporin- and nifedipine-induced overgrowths

Glycosaminoglycans are thought to accumulate in formative lesions like drug-induced gingival overgrowth. Recent evidences, however, suggest that the amounts of glycosaminoglycans are comparable in overgrown and healthy gingiva. Besides, alterations in the size distribution of glycosaminoglycan molecules isolated from phenytoin-induced overgrown samples have also been suggested. Therefore, we sought to determine possible differences in molecular size distribution of gingival glycosaminoglycans in other types of drug-induced overgrowths. Purified gingival glycosaminoglycans from healthy and cyclosporin- and nifedipine-induced overgrown gingival tissues were analyzed by agarose gel electrophoresis and their molecular-size distribution was evaluated by both gel filtration chromatography and polyacrylamide gel electrophoresis. Our results on the gingival glycosaminoglycan composition showed presence of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronic acid in all types of gingival tissues examined. In addition, hyaluronic acid was predominantly of a large size eluting near to the void volume of a Superose-6 column, while the sulfated glycosaminoglycans were mainly composed of low molecular size glycosaminoglycans. Our results show no differences in the molecular-size distribution of hyaluronic acid and sulfated glycosaminoglycans among healthy and drug-induced overgrown gingival tissues.