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.     

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.     

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.     

Levels of glycosaminoglycans in peri‐implant sulcus fluid as a means of monitoring bone response to endosseous dental implants

The presence of certain glycosaminoglycans in peri‐implant sulcus fluid may be an effective means of monitoring changes in bone metabolic activity following initial loading of implant abutments. This study has investigated levels of chondroitin 4 sulphate and hyaluronan in peri‐implant sulcus fluid from titanium osseointegrated implants following initial abutment placement and exposure to masticatory stresses. Abutments were placed after a 3-month osseointegration period post‐initial surgical placement of the interosseous stage. 10 edentulous patients, each with 5 mandibular implants were reviewed at 2, 4, 6 and 8 days after abutment placement. Clinical details were assessed and recorded and sulcus fluid collected in microcapillary tubes for a 5‐min period for each abutment. Levels of glycosaminoglycans were assessed using cellulose acetate electrophoresis and densitometric scanning of alcian blue stained strips against known glycosaminoglycan standards. Maximum levels of sulcus fluid (0.3–1.25 /5 min) were evident at 4 days with a decrease towards 8 days. Levels of sulphated glycosaminoglycans were also maximal at 2–4 days (range 0.03–0.126 μg/5 min) and decreased at 6-8 days. Hyaluronan was detected within a similar range of values reaching maximal levels at 4 days and decreasing by 8 days. The results indicate that glycosaminoglycan levels of peri‐implant sulcus fluid is an effective means of measuring and maintaining changes in bone metabolism. The absence of proteodermatan sulphate precludes 1 soft tissues being a source of these markers.     

Altered collagen metabolism in nifedipine-induced gingival overgrowth

Fibroblasts from nifedipine-induced fibrotic gingiva (NFG) have been characterized with respect to several cellular functions which could contribute to the characteristic clinical overgrowth of the gingiva: collagen synthesis and breakdown, glycosaminoglycan production, fibronectin synthesis, and proliferation. Histologic examination of NFG tissue revealed a hyperplastic epithelium with elongated, branched rete pegs. The connective tissue consisted of densely-packed collagen fibers and numerous enlarged fibroblasts, as well as regions of thinner, disorganized collagen fibers in the vicinity of scattered inflammatory and mast cells. Results of in vitro experiments showed that the fibroblast strains from the fibrotic gingiva (NFG) produced significantly greater amounts of collagen and lower levels of collagenase activity when compared to age- and sex-matched normal human gingival fibroblast strains. The NFG fibroblasts did not produce significantly greater amounts of fibronectin, and their level of glycosaminoglycan production was less than that of the normal fibroblasts. The NFG fibroblasts did not proliferate significantly more rapidly than the normal fibroblast strains. These findings therefore show that there are defects in the regulation of collagen production by NFG fibroblasts in vitro , and suggest that these alterations in collagen metabolism contribute to the over-deposition of collagen in this tissue, rahter than hyperproliferation of the fibroblasts or through the production of increased amounts of fibronectin and glycosaminoglycans.     

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.     

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.     

Glycosaminoglycans in normal and osteoarthrotic human temporomandibular joint disks.

Glycosaminoglycans in normal and osteoarthrotic temporomandibular joint disks were studied by means of high-performance liquid chromatography methods. Normal disk tissue contains galactosaminoglycans (chondroitin sulfate and dermatan sulfate) as the main polysaccharides and with smaller amounts of hyaluronate and heparan sulfate. The galactosaminoglycans are mainly sulfated in 6-position, and some of the disaccharides contain iduronic acid. There was a slight general variation in glycosaminoglycan concentration with increasing age. In the severely arthrotic disks the content of glycosaminoglycans was considerably lower than in normal disk tissue. This decrease was far more extensive than that observed in relation to age in normal tissue. The 4/6-sulfate ratio of the galactosaminoglycans was increased, whereas the proportion of iduronic acid was markedly decreased.     

Extracellular glycosaminoglycan changes in healthy and overgrown gingiva fibroblasts after cyclosporin A and cytokine treatments

BACKGROUND: It has been demonstrated that cyclosporin A (CyA) blocks the immune system, acts on cytoskeleton and stimulates the production of extracellular matrix (ECM) and transforming growth factor-beta1 (TGF-beta1). This cytokine, such as transforming growth factor-alpha (TGF-alpha), induces deposition of glycosaminoglycans (GAG), proteoglycans and collagen fibres in the ECM. METHODS: In this work, we examined the effect induced by CyA, TGF-beta1 and TGF-alpha on cultures of healthy and overgrown human gingival fibroblasts in order to evaluate the glycosaminoglycan, cytoskeletal changes and the behaviour of fibroblasts after concanavalin A (Con A) treatment. Moreover, we examined gingival biopsies by Alcian blue histochemical staining and electron transmission microscopy. RESULTS: Total and extracellular sulphated GAG in overgrown gingiva specimens and in derived fibroblast cultures treated with CyA and cytokines were significantly higher than controls. The action of cytokines was increased (P < or = 0.01) compared with CyA with a greater effect of TGF-alpha in comparison with TGF-beta1; the electron microscopy showed ECM accumulation. The agglutinations showed the heterogeneity of fibroblast populations. CONCLUSIONS: Stimulation with Con A showed that the fibroblast population had cell surface heterogeneity, and could respond in a different way to both CyA and cytokine stimulus. Moreover, increased synthesis of GAG in overgrown gingiva compared with synthesis in normal fibroblasts before CyA treatment suggests a possible genetic origin of damage. As not all CyA-treated patients develop gingival overgrowth, a genetic predisposition may explain the different responses of gingival fibroblast populations.     

Effect of p-nitrophenyl-xyloside on the biosynthesis of proteoglycan in rat ovarian granulosa cells--analyses of glycosaminoglycan synthesis in the Golgi apparatus

Biosynthesis of proteoglycans and glycosaminoglycans in the presence of p-nitrophenyl-xyloside was studied using a primary rat ovarian granulosa cell culture system. Addition of p-nitrophenyl-xyloside into cell culture medium caused about a 700% increase of [35S]sulfate incorporation (ED50 at 0.03 mM) into macromolecules, which included free chondroitin sulfate chains initiated on xyloside and native proteoglycans. Free chondroitin sulfate chains initiated on xyloside were almost exclusively secreted into the medium. The molecular size of chondroitin sulfate chains decreased from 40,000 to 21,000 as the total [35S]sulfate incorporation was enhanced, suggesting that enhanced synthesis of chondroitin sulfate perturbed the normal mechanism of glycosaminoglycan chain termination. Biosynthesis of heparan sulfate proteoglycans was reduced by approximately 50%, likely due to competition at the level of UDP-sugar precursors. [35S]Sulfate incorporation was shut down by the addition of cycloheximide with an initial half time of approximately 2 hr in the presence of xyloside, while that in the absence of xyloside was about 20 min. The difference likely reflects the turnover rate of glycosaminoglycan synthesizing capacity as a whole. The turnover rate of glycosaminoglycan synthesizing capacity observed in ovarian granulosa cells was much shorter than that observed in chondrocytes, reflecting the relative dominance of proteoglycan biosynthetic activity in the total metabolic activity of the cells.     

ELISA detection of glycosaminoglycan (GAG)-linked proteoglycans in gingival crevicular fluid

The purpose of this study was to develop an ELISA method to detect chondroitin sulfate isomer-linked proteoglycans in gingival crevicular fluid (GCF), and to lelucidate the role played by the glycosaminoglycans (GAGs) in GCF during experimentally-induced periodontitis in dogs. Experimental periodontitis was induced by placement of a silk ligature below the gingival margin of the molar teeth in 3 mongrel dogs. GCF was collected using microcapillary tubes at 0. 7.21 and 60 days after ligature placement. Too compare with GAG in GCF, bovinenasal cartilage proteoglycan monomer, dog's serum and supernatant of homogenized gingival tissuw were prepared. Combination of monoclonal antibodies, 3B3 and 9A2, and specific enzymatic digestion made possible the indentification of chondroitin 4 sulfate (C4S), chondroitin 6 sulfate (C6S) and dermatan sulfate (DS). The ELISA method detected very small amounts of chondroitin sulfate (CS) isomers (15-1000 ng/ml of bovine nasal cartilage proteoglycan). The ELISA value of CS isomers in GCF was lower than that of homogenized gingival tissue but higher than that of the serum. The ELISA value of C4S, C6S and DS, although fluctuating, increased in proportion to the severity of the inflammation.     

Glycosaminoglycan patterns in gingival proteoglycans of rat with age.

Among the potential biochemical indices that are closely associated with craniofacial development are the proteoglycans. Gingival segments from the palate of 4-, 6-, 8-, 12- and 18-week-old rats were incubated for 4 h in medium containing [3H]-glucosamine and [35S]-Na2SO4, and subjected to proteoglycan isolation and glycosaminoglycan analysis. Two distinct proteoglycan fractions differing in the degree of sulphation were obtained by ion-exchange chromatography. The incorporation of both labels in the undersulphated fraction increased with age; there was a pronounced decrease with age in the sulphated proteoglycan fraction. The undersulphated proteoglycans showed an age-dependent decrease in hyaluronic acid, and increase in dermatan sulphate and chondroitin 4- and 6-sulphates. Gel filtration of the sulphated proteoglycan fraction yielded high and low molecular-weight proteoglycans, the glycosaminoglycans of which were particularly rich (61-76%) in dermatan sulphate. Smaller quantities of chondroitin 4- and 6-sulphates, and heparan sulphate were also present. All glycosaminoglycans showed a decrease in content with age. The findings suggest a possible correlation between gingival proteoglycan/glycosaminoglycan patterns and development.     

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.     

Incidence of verapamil-induced gingival hyperplasia in a dental population.

The records of 5,000 dental patients were reviewed for history of verapamil use between 1987 and 1990. Twenty-four dentate patients who received verapamil for more than 1 year were identified. Of these, gingival hyperplasia occurred in 1 patient (4.1%) that was limited to the mandibular attached gingiva. Onset of gingival overgrowth was associated with drug dosage, bacterial accumulation, and gingival inflammation. Histologically, the findings resembled that seen in hyperplasia induced by phenytoin, cyclosporin, and other calcium channel blockers. Our data suggest that gingival hyperplasia caused by verapamil occurs less frequently than nifedipine-induced gingival hyperplasia.     

Disaccharide analysis of chondroitin sulfate in peri-implant sulcus fluid from dental implants

We collected peri-implant sulcus fluid by capillary tubes from sites around titanium osseointegrated implants and determined the chondroitin sulfate released into the peri-implant sulcus fluid by high-performance liquid chromatography. Chondroitin sulfate was found in all peri-implant sulcus fluid samples, and its content was similar to that in gingival crevicular fluid obtained around natural teeth. The predominant unsaturated disaccharide isomer was ΔDi-OS, followed by ΔDi-4S. ΔDi-6S was present in trace amounts. The amount of ΔDi-OS was greater in peri-implant sulcus fluid than in gingival crevicular fluid. Assaying chondroitin sulfate disaccharides in peri-implant sulcus fluid may be an effective method of monitoring the peri-implant condition of dental implants.     

Gingival tissue proteoglycan and chondroitin-4-sulphate levels in cyclosporin A-induced gingival overgrowth and the effects of initial periodontal treatment

OBJECTIVES: Cyclosporin A (CsA) is a potent immunosuppressive drug used in organ transplant patients to prevent graft rejection. CsA-induced gingival overgrowth is one of the side effects of this drug and its pathogenesis is still unclear. The present study was planned to comparatively analyse total proteoglycan (PG) and chondroitin-4-sulphate (C4S) levels in CsA-induced overgrown gingival tissue samples obtained before and after initial periodontal treatment and to compare these findings with the situation in healthy gingiva. MATERIAL AND METHODS: Gingival tissue samples were obtained from nine patients with CsA-induced gingival overgrowth before and 4 weeks after initial periodontal treatment including oral hygiene instruction and scaling and also from 10 healthy control subjects. Total PG and C4S levels were determined by biochemical techniques. PG levels were analysed using modified Bitter and Muir method. C4S assay was carried out using chondroitin sulphate lyase AC and chondroitin-6 sulphate sulphohydrolase enzymes. The results were tested statistically using non-parametric tests. RESULTS: All clinical measurements in the CsA-induced gingival overgrowth group demonstrated significant reductions 4 weeks after initial periodontal treatment (p<0.05). There was no significant difference between the levels of baseline total PG in CsA-induced gingival overgrowth and healthy control groups (p>0.05). The gingival tissue levels of PG in CsA-induced gingival overgrowth group decreased significantly 4 weeks after treatment (p=0.043). Gingival tissue C4S levels in the overgrowth group were significantly higher than the healthy control group at baseline (p=0.000). C4S levels of the overgrowth group were significantly reduced after treatment (p=0.033), but these levels were still significantly higher than the healthy control group (p=0.000). CONCLUSION: The observed prominent increase in gingival tissue C4S levels may be interpreted as a sign of an increase in C4S synthesis in CsA-induced gingival overgrowth. Furthermore, remission of clinical inflammation by means of initial periodontal treatment had a positive effect on tissue levels of these extracellular matrix molecules.     

Cyclosporin A inhibits production and activity of matrix metalloproteinases by gingival fibroblasts

Cyclosporin A (CyA) is a potent immunosuppressor used in organ transplantation and in the management of various autoimmune diseases. Gingival overgrowth is one of the side-effects of the CyA-treatment, affecting the attached gingiva of 25-81% of treated patients. To investigate the production and activity of matrix metalloproteinases (MMPs) in the CyA-induced gingival overgrowth, 2 well-documented models were utilized: the in vivo CyA-induced rat gingival overgrowth and primary cultures of human gingival fibroblasts treated with CyA. Our results obtained from the Western blot assays demonstrated clearly that the production of MMP-1 and MMP-3 was significantly inhibited by CyA at similar concentrations found in the serum of patients undergoing CyA-treatment. Moreover, the gelatinolytic activity of MMP-2 was also reduced both in cultured fibroblasts and in the rat CyA-induced gingival overgrowth. Taken together, the data presented here suggest that these inhibitory effects may contribute to the extracellular matrix (ECM) components accumulation in the CyA-induced gingival overgrowth.     

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.     

Histologic evidence for impaired growth control in diphenylhydantoin gingival overgrowth in man.

Fibroblast density and collagen content in non-inflamed gingiva from patients with gingival overgrowth due to sodium 5,5-diphenylhydantoinate therapy were measured and compared to normal tissue. Direct counting of fibroblast nuclei as well as stereologic point-counting precedures were performed using stained microtome sections. In no case were fibroblasts more numerous or collagen fibres more ubiquitous in DPH-enlarged gingiva. The mature, fibrous-type DPH gingival lesion represents neither hypertrophy, hyperplasia nor fibrosis, but is an example of uncontrolled growth of a connective tissue of apparently normal cell and fibre composition.