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.     

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 sulfated glycosaminoglycans by human gingival fibroblasts from phenytoin-induced gingival overgrowth in vitro

The in vitro synthesis of sulfated glycosaminoglycans (GAGs) was studied in gingival fibroblasts from two patients exhibiting phenytoin(PHT)-induced gingival overgrowth, i.e. pseudopockets, which required surgical excision, from one patient on PHT medication not exhibiting pseudopockets and from two normal controls. The results showed that the newly synthesized GAGs were distributed to the culture medium, to a pericellular pool and to the cell fraction. Gingival fibroblasts from the PHT-induced gingival overgrowth showed a significantly increased incorporation of 35SO4(2-) into GAGs compared to the other strains, and this increase was mainly confined to the dermatan sulfate fraction. These results are in accordance with our previous biochemical studies where increased amounts of GAGs were found in gingival biopsies from the PHT-induced lesion.     

Synthesis of sulfated glycosaminoglycans by human gingival fibroblasts from phenytoin-induced gingival overgrowth in vitro

Abstract – The in vitro synthesis of sulfated glycosaminoglycans (GAGs) was studied in gingival fibroblasts from two patients exhibiting phenytoin(PHT)-induced gingival overgrowth, i.e. pseudopockets, which required surgical excision, from one patient on PHT medication not exhibiting pseudopockets and from two normal controls. The results showed that the newly synthesized GAGs were distributed to the culture medium, to a pericellular pool and to the cell fraction. Gingival fibroblasts from the PHT-induced gingival overgrowth showed a significantly increased incorporation of ³⁵SO₄^2- into GAGs compared to the other strains, and this, increase was mainly confined to the dermatan sulfate fraction. These results are in accordance with our previous biochemical studies where increased amounts of GAGs were found in gingival biopsies from the PHT-induced lesion.     

Stereologic study of cyclosporin A-induced gingival overgrowth in renal transplant patients

Gingival biopsies were taken from 13 renal transplant patients (mean age 26.5 yr), 11 of whom exhibited cyclosporin A (CsA)-inditced gingival overgrowth. Control material was obtained from seven volunteers (mean age 28 yr). Gingival tissue components were analyzed by quantitative microscopy (stereology) on 5-(μm-thick sections of interdental papillae. The volume density (Vv) of different tissue components and the surface density of epithelial ridges were calculated by conventional point and intersection counting. The study showed that the volume density of oral epithelium and the surface density of the epithelial ridges in the CsA-induced gingival overgrowth were significantly increased compared to normal gingival tissue. The connective tissue of the lesion exhibited a significant increase in volume density of cells, blood vessels and non-collagenous matrix with a corresponding decrease in the collagenous matrix. These results indicate that CsA-induced gingival overgrowth represents a tissue with an altered composition characterized by increased thickness of oral epithelium and relatively-increased amount of cells, vessels, non-collagenous matrix and decreased collagenous matrix in the connective tissue.     

Distribution of chondroitin sulfate and dermatan sulfate in normal and inflamed human gingivae.

The effect of inflammation on the distribution of chondroitin sulfate and dermatan sulfate proteoglycans was assessed after normal and inflamed human gingivae were stained with monoclonal antibodies against these extracellular matrix macromolecules. The tissues were obtained following periodontal surgery and reacted with specific antibodies after pre-treatment with chondroitinase ACII or chondroitinase ABC, and staining was visualized by the immunoperoxidase technique. The results indicated that these two proteoglycans were present in both the 4-sulfated and 6-sulfated isomeric forms. While chondroitin sulfate appeared to be uniformly distributed throughout the connective tissue, dermatan sulfate showed greater intensity of staining in the areas immediately subjacent to the epithelium. Positive staining for chondroitin sulfate was noted in the intercellular spaces of the epithelium. In inflamed tissues, there was significant staining associated with 4-sulfated dermatan sulfate and chondroitin sulfate, but this had lost the structured pattern of staining noted in normal sections. The 6-sulfated isomeric forms were greatly reduced in inflamed tissues and tended to show a predilection to be localized within the perivascular tissues. In the inflamed tissues, there was intense staining for chondroitin sulfate associated with the infiltrating inflammatory cells. These findings corroborate earlier biochemical studies on normal and inflamed gingival tissues. The specific tissue localization of dermatan sulfate and chondroitin sulfate in tissues damaged by inflammation indicates that, as opposed to the large loss of collagenous material noted during inflammation, there is not a corresponding large loss of proteoglycan. Indeed, at specific inflammatory foci, the intensity of staining for these macromolecules may intensify.     

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.     

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.     

Nitrendipine-induced gingival overgrowth in dogs

This study assessed the development of gingival overgrowth in dogs given nitrendipine, a new antihypertensive dihydropyridine. Nine male Beagle dogs with established plaque and gingivitis were used. Following a baseline examination, which involved assessment of plaque, gingivitis, and gingival enlargement, six dogs (test) received nitrendipine twice daily in a high dose, while 3 dogs (control) received placebo. Clinical scorings were repeated after 10 and 20 weeks. At termination of the study gingival biopsies were excised and examined morphometrically. Already at the 10-week examination definite changes in gingival size had occurred, and following 20 weeks of nitrendipine treatment the gingival enlargement had markedly increased. In none of the control dogs were there any signs of gingival size changes. The histopathological examination showed that the only principal changes in histopathological morphology were that areas of non-infiltrated connective tissue in test specimens showed an increase in vascularity and appeared more loose compared with the dense tissue in control specimens. The morphometric analysis demonstrated minor differences between test and control specimens in regard to all tissue fractions observed; however, such differences were numerically very small, and were not considered biologically significant. Thus, the results demonstrated that nitrendipine administered to Beagle dogs during a 20-week period causes marked overgrowth of gingival tissue of apparently normal composition. The fact that cases of gingival overgrowth have been reported among patients receiving other dihydropyridines suggest that the observed gingival overgrowth is an effect of this class of drugs.     

Ⅱ°根分叉病变患者引导组织再生治疗术前术后龈沟液中糖氨多糖水平的变化

目的　观察Ⅱ°根分叉病变患者引导组织再生治疗术 (guidedtissueregeneration ,GTR)前后龈沟液(gingivalcrevicularfluid ,GCF)中糖氨多糖 (glycosaminoglycans ,GAG)水平变化的同时 ,探讨GCF中GAG能否作为判断GTR术后组织成熟的指标。方法　对 6例Ⅱ°根分叉病变的患牙采用GTR治疗 ,并于手术前 ,手术后 1、2、3、4、5、6周收集GCF。用 0 .1%阿尔辛兰 (Alcianblue)染色 ,分光光度法测定GCF中总的硫酸化GAG及硫酸软骨素 (chondroitinsulfate ,CS)的水平。结果　GTR术后 1～ 2周 ,GCF中CS明显降低 (P <0 .0 5 ) ,然后逐渐升高 ,第 6周恢复到基线水平。而GCF中总的硫酸化GAG则在术后 1周明显升高 (P <0 .0 5 ) ,然后下降 ,到第 6周升高并超过基线水平。结论　GCF中总的硫酸化GAG ,尤其是CS可用作监测牙周伤口愈合和组织再生的一个潜在指标 ,但是否可以用作GTR术后组织成熟的指标 ,还需加大样本并结合病理进行进一步的纵向观察     

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.     

Morphological evaluation of combined effects of cyclosporin and nifedipine on gingival overgrowth in rats

It has previously been shown that, while cyclosporin A (CsA) and nifedipine both cause gingival overgrowth in the rat, the combined use of these drugs increases the severity of overgrowth. The aim of this study was to describe the histometry and densities of fibroblasts, collagen fibers and vessels in the gingival tissue of rats that were treated with CsA and nifedipine, either alone or in combination. Rats were treated for 60 days with a daily subcutaneous injection of 10 mg/kg body weight of CsA and/or with 50 mg/kg body weight of nifedipine added to the chow. The results confirmed that CsA causes a more severe overgrowth than nifedipine, and that the combined use of these drugs increases the overgrowth severity. All the rat groups that were studied showed that, as the severity of overgrowth increased, there was a parallel increase in fibroblasts and collagen, and a decrease in vessel content. Therefore, independently of whether the gingival overgrowth was caused by CsA alone, nifedipine alone, or both treatments in combination, the fibroblast and collagen density increased in parallel with the severity of the overgrowth.     

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.     

The effects of up to 240 days of tacrolimus therapy on the gingival tissues of rats--a morphological evaluation

BACKGROUND: Tacrolimus, an immunosuppressive drug used in organ transplantation, has been reported not to induce gingival overgrowth. However, prevalence studies are limited, and the methods used for assessing gingival overgrowth varies among studies. OBJECTIVE: The purpose of this study was to evaluate the effects of up to 240 days of tacrolimus therapy on gingival tissues of rats. MATERIALS AND METHODS: Rats were treated for 60, 120, 180 and 240 days with daily subcutaneous injections of 1 mg/kg body weight of tacrolimus. After histological processing, the oral and connective tissue, volume densities of fibroblasts (Vf), collagen fibers (Vcf) and other structures (Vo) were assessed in the region of the lower first molar. RESULTS: After 60 and 120 days of treatment with tacrolimus, gingival overgrowth was not observed. The gingival epithelium, connective tissue, as well as the values for Vf, Vcf, and Vo were similar to those of the control rats (P>0.05). After 180 and 240 days of the treatment, gingival overgrowth was associated with a significant increase in the gingival epithelium and connective tissue as well as an increase in the Vf and Vcf (P<0.05). CONCLUSIONS: Within the limits of the experimental study, it may be concluded that the deleterious side effects of tacrolimus on the gingival tissues of rats may be time-related.     

Quantitative histopathologic assessment of developing phenytoin-induced gingival overgrowth in the cat

Abstract Phenytoin (Dilantin®) induces gingival overgrowth characterized by an accumulation of connective tissue. The cell-to-matrix ratio in the mature lesion is normal, yet there must be more fibroblasts per oral cavity if there is excessive tissue mass. Using a mongrel cat model system, we studied the early, developing phenytoin-induced lesion by quantitating fibroblasts per unit of tissue in papilla biopsies collected over a 3-month period of daily drug administration. At 6 and 8 weeks, the number of fibroblasts per unit of tissue increased dramatically. By 3 months, as the lesions matured, the fibroblast-to-matrix ratio returned to normal. We suggest that the drug interacts with resident gingival fibroblasts, causes them to proliferate and thus induces a true, but transient, hypercellularity. Cell division then appears to slow or cease, and rapid production of connective tissue matrix ensues, returning the cell-to-matrix ratio to normal.     

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.     

The role of nickel accumulation and epithelial cell proliferation in orthodontic treatment-induced gingival overgrowth

The aim of this study was to investigate the role of nickel in orthodontic treatment-induced gingival hyperplasia. The nickel concentration in gingival tissues with and without overgrowth, histopathology of gingival overgrowth, and epithelial cell proliferation response to different nickel concentrations were analysed. Ten patients receiving orthodontic therapy (eight females and two males, mean age 15.4 years) were included in the study. Hyperplastic and healthy gingiva samples were collected from the same patients. The amount of nickel in the gingival tissue samples was analysed using the atomic absorption spectrometry technique. The tissues removed from hyperplastic areas during gingivectomy were also used for histological analysis. To analyse the effect of nickel on epithelial cell proliferation, four different nickel concentrations (0.5, 2, 5, and 10 microg) were incubated with keratinocyte cells for 11 days. Mann-Whitney U-test, analysis of variance, and Tukey's test were used in the statistical analyses. The results did not show any difference in nickel concentration between the study and control gingiva tissue samples, but histological analysis demonstrated an increase in epithelial thickness and a significant increase (P = 0.031, 0.02, 0.02) in epithelial cell proliferation in response to low-dose nickel concentrations, with a toxic response to a higher dose. In the limitations of this study, it is plausible that the effect of a continuing low-dose nickel release to epithelium is the initiating factor of gingival overgrowth induced by orthodontic treatment.     

Connective tissue growth factor in drug-induced gingival overgrowth

BACKGROUND: Drug-induced gingival overgrowth is a known side effect of certain chemotherapeutic agents used for the treatment of systemic disorders. The pathogenesis and mechanisms responsible for this condition are not fully understood. This study assesses for the presence and localization of connective tissue growth factor (CTGF) in drug-induced gingival overgrowth tissues. CTGF immunostaining was compared with sections stained with transforming growth factor (TGF)-beta1 and CD31 antibodies in order to investigate possible pathogenic mechanisms. METHODS: Gingival overgrowth samples were obtained from patients undergoing therapy with phenytoin (n = 9), nifedipine (n = 4), cyclosporin A (n = 5), and control tissues from systemically healthy donors (n = 9). Tissue sections were subjected to peroxidase immunohistochemistry and were stained with CTGF and TGF-beta1 polyclonal primary antibodies. Possible relationships between CTGF staining and angiogenesis were also studied using an anti-CD31 antibody as a marker for endothelial cells. Staining was analyzed by computer-assisted quantitative and semiquantitative methodology at 5 defined sites in all samples based on the location of specific landmarks including epithelium and underlying connective tissues. RESULTS: Cellular and extracellular CTGF content in phenytoin gingival overgrowth tissues was significantly (P<0.05) higher compared to the other gingival overgrowth tissues and the controls. Higher CTGF staining in phenytoin gingival overgrowth tissues was accompanied by an increased abundance of fibroblasts and connective tissue fibers. No strong association of CTGF staining with TGF-beta1 or CD31 staining was found. CONCLUSIONS: The data from the present study show significantly higher CTGF staining in phenytoin-induced gingival overgrowth tissues compared to controls, cyclosporin A-, or nifedipine-induced gingival overgrowth. Moreover, semiquantitative analyses of histologic samples support the concept that the phenytoin overgrowth tissues are fibrotic. These associations suggest a possible role for CTGF in promoting development of fibrotic lesions in phenytoin-induced gingival overgrowth.