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.     

Synthesis of proteoglycans in rat mucosal keratinocytes

The synthesis of hyaluronic acid and proteoglycans by rat mucosal keratinocytes of an established cell line (CCL-10) has been investigated. Proliferating cultures at or near confluency were grown in the presence of [35S] sulfate or D-[1-3H] glucosamine for 24 h, and the glycosaminoglycan composition of cells and medium was determined. Characterization of the 35S-labelled glycosaminoglycans showed that heparan sulfate was the major component (approximately 90%) and that small amounts (approximately 10%) of galactosaminoglycans had also been synthesized. Analysis of cultures labelled with D-[1-3H] glucosamine demonstrated that hyaluronic acid was also present, most prominently in the medium where approximately one third of the radioactivity in the glycosaminoglycan pool was found in the hyaluronic acid fraction. [35S]-labelled proteoglycans extracted from the cell layer in the presence of protease inhibitors showed substantial heterogeneity upon chromatography on Sepharose CL-6B. In contrast, the proteoglycans in the medium gave a major peak which was eluted at a Kav of 0.28. Gel chromatography of the glycosaminoglycan chains in the latter, isolated after proteolytic digestion, indicated a molecular weight of 17,000.     

Synthesis of proteoglycans in rat mucosal keratinocytes

The synthesis of hyaluronic acid and proteoglycans by rat mucosal keratinocytes of an established cell line (CCL-10) has been investigated. Proliferating cultures at or near confluency were grown in the presence of [³⁵S] sulfate or D-[1-³H] glucosamine for 24 h, and the glycosaminoglycan composition of cells and medium was determined. Characterization of the ³⁵S-laballed
glycosaminoglycans showed that heparan sulfate was the major component (∼90%) and that small amounts (∼10%) of galactosaminoglycans had also been synthesized. Analysis of cultures labelled with D-[1-³H] glucosamine demonstrated that hyaluronic acid was also present, most prominently in the medium where approximately one third of the radioactivity in the glycosaminoglycan pool was found in the hyaluronic acid fraction. [³⁵S]-labelled proteoglycans extracted from the cell layer in the presence of protease inhibitors showed substantial heterogeneity upon chromatography on Sepharose CL-6B. In contrast, the proteoglycans in the medium gave a major peak which was eluted at a K_av of 0.28. Gel chromatography of the glycosaminoglycan chains in the latter, isolated after proteolytic digestion, indicated a molecular weight of 17,000.     

Acid mucopolysaccharides in porcine gingiva

Acid mucopolysaccharides (AMPS) were extracted from porcine gingiva. The concentration of the uronic acid extracted was 0.23% of the ry-defatted issue. The AMPS were fractioned with a Dowex 1-X2 column.
The individual AMPS components of the various fractions obtained were determine on the basis of the contents of the hexosamine, unsaturated disaccharides, and chondroitin sulfate isomers. It was suggested from the results that the AMPS of porcine gingiva consisted of large amounts of chondrotin sulfates A and B, moderate amounts of hyaluronic acid and heparan sulfate, and a small amount of chondroitin sulfate C.     

Possible origin of sulfated glycosaminoglycans in human dental calculus.

The sulfated glycosaminoglycans present in human dental calculus have been shown to be dermatan sulfate and chondroitin-4-sulfate. The composition suggests that the glycosaminoglycans present in calculus, particularly subgingival material, could originate as a result of associated periodontal disease since closely similar compounds have previously been identified in normal and inflamed human gingiva.     

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%).     

Possible origin of sulfated glycosaminoglycans in human dental calculus

Abstract— The sulfated glycosaminoglycans present in human dental calculus have been shown to be dermatan sulfate and chondroitin-4-sulfate. The composition suggests that the glycosaminoglycans present in calculus, particularly subgingival material, could originate as a result of associated periodontal disease since closely similar compounds have previously been identified in normal and inflamed human gingiva.     

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.     

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.     

The acidic glycosaminoglycans of gingiva of diabetic rats

Rats made diabetic by administration of alloxan monohydrate showed in gingiva a 35.9 % reduction in total glycosaminoglycans concentration. The concentration of hyaluronic acid was 48·4 % and that of chondroitin-4-sulfate 45·2 % decreased. Heparin concentration was 23·8 % increased. The other sulfate fractions did not appear to be affected. The administration of insulin almost entirely restored the level of the affected glycosaminoglycans to normal values with the only exception of heparin.     

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.     

Biochemical changes in periodontal ligament ground substance associated with short-term intrusive loadings in adult monkeys (Macaca Fascicularis)

Proteoglycan-like fractions (PG) were isolated from the ligaments of teeth undergoing various degrees of intrusive loadings. The PG were characterized by their molecular-size profiles on Sepharose 4B, the presence of uronic acid in the separated fractions and by the electrophoretic detection of constituent glycosaminoglycans including heparan sulphate, hyaluronic acid, dermatan sulphate and chondroitin-4-sulphate. The high molecular-weight fraction, peak i (estimated minimum size, 2 X 10(6) daltons) of the normal-functioning (stressed) ligament was reduced approx. 70 per cent, compared with ligament undisturbed for 3 h. There was a decrease in peak-i size between 0.25 and 1 N loadings of approx. 72 per cent. The 4 N loadings produced a further decrease followed by an increase during a 3 h undisturbed recovery phase. Thus changes in the chemistry and properties of the ground-substance components of the periodontal ligament could partly explain changes in tooth mobility.     

Analysis of the relationship between the severity of periodontal destruction and proteoglycan metabolism of gingiva and gingival crevicular flu

BACKGROUND: Although it is well-described that proteoglycans (PGs) are among the major non-collagenous components of the matrix which are degraded during periodontal diseases, the relationship between PG metabolism and seventy of periodontal breakdown, the extent of degradation of PGs together with the resulting end-products, and the elimination pathways of these catabolic end-products is likely to need further clarification. OBJECTIVE: The main aim of the present study was to analyze the possible impact of severity of periodontal destruction on PG metabolism of gingiva and gingival crevicular fluid (GCF). MATERIAL AND METHODS: For this purpose, gingiva and GCF samples obtained from patients (n = 45) exhibiting sites (n = 57) with moderate periodontal breakdown (MP) or severe periodontal breakdown (SP) were analyzed for PG metabolism via spectrophotometric determination of uronic acid levels. Gingiva and GCF samples were obtained from the same sites in every patient to analyze the possible relationship between uronic acid content of gingival tissue and GCF. RESULTS: No significant differences were found in uronic acid levels between sites with MP and SP (p > 0.05). The uronic acid content of GCF and gingiva showed significant overlaps between MP and SP sites and uronic acid levels did not present any constant correlation with the clinical parameters (p > 0.05). In a similar manner, uronic acid content of GCF and gingival tissue was not correlated (p > 0.05). CONCLUSION: The lack of a significant correlation between the uronic acid content of gingival tissue and GCF may suggest that the passage of PG metabolites from gingiva to GCF is likely to be under the influence of multifactorial interactions rather than being linear. As a general measure of PG metabolism, uronic acid levels do not seem to be related with the severity of periodontal destruction and tend to act as different measures when compared to traditional clinical parameters.     

Proteoglycans in human gingiva: Molecular size distribution in epithelium and in connective tissue

The major extracellular, non-fibrous macromolecules in human gingiva are proteoglycans. These sulphated, uronic acid-containing macromolecules are synthesized in vitro by both gingival epithelium and its underlying connective tissue. Biochemical analyses were carried out on cultured human predominantly gingival epithelium and predominantly connective tissue in media containing appropriate radioactive precursor molecules. The proteoglycans were extracted with salt solutions of varying concentration and characterized by means of column chromatography and liquid scintillation counting. The major differences noted between the macromolecules extracted from these two closely apposed regions were molecular size, and the ability or not to form large self-aggregatable macromolecules. In general, the epithelial proteoglycans are larger than connective tissue proteoglycans. Proteoglycans from gingival epithelium are able to self-aggregate to form large macromolecules with mol. wt in excess of 2 × 10⁶ which are essential for the maintenance of tissue integrity.     

The effect of interleukin-1 β on hyaluronic acid synthesized by adult human gingival fibroblasts in vitro

The effect of recombinant interleukin-1β (IL-lβ) on hyaluronic acid synthesis by human gingival fibroblasts was studied. IL-1bT caused a dose-dependent increase in the incorporation of (³lucosamine into hyaluronic acid. The ³⁵S/35H ratios of labeled macromolecules did not change regardless of the presence or absence of TL-lβ and indicates stimulation of hyaluronic acid synthesis. Inhibition of cell proliferation by hydroxyurea caused an increase in hyaluronic acid synthesis. The effect of IL-1β on hyaluronic acid synthesis in the presence of hydroxyurea was increased over untreated and IL-lβ-treated controls, but equivalent to the hydroxyurea-treated controls. Thus the effect of IL-1β on hyaluronic acid synthesis may be independent of cell proliferation. Furthermore, inhibition of prostaglandin E2 synthesis by indomethacin abolished the effect of IL-1β on hyaluronic acid synthesis. Inhibition of new protein synthesis by cycloheximide negated the effect of IL-β on hyaluronic acid synthesis. This may be related to inhibition of new hyaluronate synthetase synthesis, since IL-1β stimulated the level of hyaluronate synthetase activity. Sepharose CL-2B chromatography revealed that most of the newly synthesized hyaluronic acid was of large molecular size. The cells exposed to IL-1β retained more large molecular size hyaluronic acid in their cell layer environment than did the control cells. These responses by fibroblasts to IL-1β may be indicative of early tissue repair.     

Chemical and immunochemical characteristics of proteoglycans in bovine gingiva and dental pulp

Proteoglycans were extracted with 4 M guanidinium chloride at 6 degrees C and purified by ion-exchange chromatography and precipitation with cetyl-pyridinium chloride. Chromatography on Sepharose CL-4B under dissociating conditions separated larger (PG1) and smaller (PG2) proteoglycans. Gingival PG2, by virtue of its amino-acid composition and the exclusive presence of L-iduronate-rich dermatan sulphate, was a proteodermatan sulphate (PDS) with a similar molecular weight to periodontal-ligament PDS. Reaction with four monoclonal antibodies to bovine skin PDS confirmed the relationship between these small proteoglycans and that of skin. Their glycoprotein cores, liberated by digestion with chondroitinase ABC, were similar in size (mol. wt = 55,000 by SDS-gel electrophoresis). Pulp PG2 had a small amount of PDS but the main component contained D-glucuronate-rich sulphated galactosaminoglycans. Similar galactosaminoglycans, which included chondroitin sulphate, characterized the larger proteoglycans of gingiva and pulp; significant amounts of L-iduronic acid-rich dermatan sulphate or heparan sulphate were not present.     

A biochemical and immunohistochemical study of the proteoglycans of alveolar bone

The purpose of this investigation was to study the proteoglycans in alveolar bone of three animal species. Alveolar bone was obtained from humans, pigs, and rabbits. Portions were fixed, sectioned, and stained with monoclonal antibodies against keratan sulfate and chondroitin sulfate. In other samples, biochemical analyses were performed. After removal of the organic matrix by 4 mol/L guanidinium HCl extraction in the presence of proteinase inhibitors, proteoglycans in the mineralized matrix were extracted with 4 mol/L guanidinium HCl/0.5 mol/L EDTA/proteinase inhibitors, and characterized on the basis of their glycosaminoglycan content (cellulose acetate membrane electrophoresis), charge (DEAE-Sephacel and hydroxylapatite chromatography), size (Sepharose CL-6B chromatography and agarose/polyacrylamide gel electrophoresis), and amino acid content. The results indicated that keratan sulfate could be detected immunohistochemically and biochemically in rabbit bone only. The predominant glycosaminoglycan in pig and human alveolar bone was chondroitin sulfate, although some hyaluronate, dermatan sulfate, and heparan sulfate were also detected. The proteoglycans were found to be slightly smaller than gingival proteoglycans, but similar to those in cementum, dentin, and other bones. In addition to intact proteoglycans, some free glycosaminoglycan chains were also extracted from the mineralized matrix. Amino acid analyses showed some subtle differences between alveolar bone proteoglycan and those of the soft tissues of the periodontium.     

Molecular size distribution of proteoglycans in human inflamed gingival tissue

Proteoglycans were extracted from human gingiva with 2 M CaCl2. The extracts were examined by gel filtration on Sephacryl S-400 in 2 M CaCl2 under dissociative conditions. The 280 nm absorbance profiles of clinically uninflamed, inflamed and severely-inflamed tissues showed that material was present with molecular weights of between 2 X 10(6) or greater, and 16,000. Proteoglycans were examined by cellulose-acetate electrophoresis with subsequent identification of the constituent glycosaminoglycans after protease digestion, and finally by chondroitinase AC digestion of the liberated glycosaminoglycans. The relative proportion of each glycosaminoglycan was calculated by scanning each cellulose-acetate sheet on an integrating densitometer. Heparan sulphate was found only in fraction I (mol. wt 2 X 10(6) or greater), together with hyaluronic acid and chondroitin-4-sulphate, these being present in all of the glycosaminoglycan-containing fractions (I-IV). Dermatan sulphate was absent from fraction I, but present in II-IV, apparently existing on the same protein core as chondroitin-4-sulphate. The relative proportions of these two glycosaminoglycans was related to molecular size, and with the degree of inflammation for a given molecular species.     

Proteoglycans and glycosaminoglycans in phenytion-induced gingival overgrowth

Proteoglycans and glycosaminoglycans in normal gingival and phenytoin-induced gingival overgrowth were studied by gel electrophoresis and HPLC methods after extration with guanidinium hydrochloride and subsequent cesium chloride gradient centrifugation. The results showed that normal gingival. The relative collagen content was decreased in the phenytoin lesion. These results are in agreement with our revious stereological study, which reported an accumulation of the non-collagenous matrix chondroitin sulfates Containing non-sulfated, 4-sulfated and 6-sulfated disaccharide units, dermatan sulfate, hyaluronic acid and presumably also heparan sulfate in both normal gingival. and phenyton-induced gingival overgrowth. The increased amounts of PGs seen in the PHT lesion were associated with an increase mainly in chondroitinase sensitive glycosaminoglycans of high molecular weight and with a relative increase in iduroinc acid content. This study is consistent with the view that the PHT-lesion represents a tissue with an altered composition of the connective tissue.     

In vivo and in vitro glycosaminoglycans from human dental pulp.

A qualitative assessment was made of the type of glycosaminoglycans (GAG) present in normal human dental pulp using electrophoresis on cellulose-acetate plates. A comparison was also made between the GAG derived directly from the dental pulp (in vivo) and those derived from cultured pulp fibroblasts from the same individual (in vitro). The results of this study showed four main types of GAG in normal human dental pulp tissue, which were dermatan sulfate, heparan sulfate, hyaluronic acid, and chondroitin sulfate. GAG synthesis from cultured pulp fibroblasts in vitro was different from the GAG present in the dental pulp (in vivo). Extracellular GAG, as well as pericellular GAG consisted of dermatan sulfate, hyaluronic acid, chondroitin sulfate, and heparin. Cellular GAG, however, contained only dermatan sulfate, hyaluronic acid, and chondroitin sulfate. There was no difference in type of GAG from the second and fourth passaged pulp fibroblasts.