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.     

Partial characterization of proteoglycans synthesized by human gingival epithelial cells in Culture

Proteoglycans (PGs) were extracted from the [³⁵S]-sulfate labelled medium and cell layer of proliferating human ginigival epithelial cells and anlyzed by ion exchanged and molecular sieve chromatorgraphy, and by SDS-PAGE. The majority of the incorporated radioactivity secreted into the medium eluted from a DEAE Sephacel ion exchange column as a single peak at 0.44 M NaCl with a small shoulder at 0.52 M NaCl. This material, when chromatographed on Sephartose CL-6B contained two spieces – a quantitatively major peak at K_av= 0.30 (M_t? 235 000 on SDS-PAGE) and a qantitatively major peak at K_av= 0.39. The major peak was sensitive to alkaline borohydride, shifting to K_av= 0.45, and nitrous acid degradtion, indicating the presence of heparan sulfate PG with glyscosaminoglycan chins with M_t? 26 000. The minor peak is chondroitin/dermatan sulfateP with glycpsminoglycan chains of M_t= 22 200 as indicted by sensitivity to alkaline borohydride (shifting to K_av= 0.48) and chondroitin ABC lyase digestion. The [³⁵S]-sulfate labelled material from the cell layer eluted in a broad peak between 0–0.50 M NaCL from DEAE Sephacel. Chromatography of this material on Sepharose CL-6B revealed the presence of three peaks at K_av=0.20, 0.31, and 0.75. The largest peak (K_av=0.20 and M_r? 245 000 on SDS-PAGE) shifted elution position to nitrous acid degradation. These results indicate that this peak contains heparan sulfate PG with glycosaminoglycan chains of M_t? 20000. Two peaks containing [³⁵S]-sulfate labelled glycosaminoglycan chains were detected by chromatography of the cell layer extract over Sepharose CL-6B with K_avs=0.42 (M_r? 30 500) and 0.75 (M_r? 5300). The larger peak was predominately chondroitin/dermatan glycosaminoglycan as indicated by susceptibility to chondroitin ABC lyase susceptibility to nitrous acid. These results indicate that cultured human gingival epithelial cells synthesize and secrete principally heparan sulfate PGs with small amounts of chondroitin/dermatan sulfate PGs. This work will serve as a basis for future studies designed to examine those factors involved in regulation of PG synthesis by these cells.     

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.     

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.     

Differential distribution of glycosaminoglycans in human cementifying fibroma and fibro-osseous lesions

OBJECTIVE: Differential diagnosis of cementifying fibroma, ossifying fibroma and fibrous dysplasia by histological evaluation is often difficult. The aim of this study was to examine the immunoreactivities for keratan sulfate (KS) and chondroitin-4-sulfate (C4S) glycosaminoglycans of the histological samples obtained from mandibles of patients with these diseases. MATERIALS AND METHODS: The samples were collected from three patients with cementifying fibroma, two with ossifying fibroma and three with fibrous dysplasia and were subjected to immunohistochemical analyses. RESULTS: The results demonstrated that a significant immunoreactivity for KS was found in lacunae housing cells in the cementum-particles of cementifying fibromas, while both ossifying fibromas and fibrous dysplasias showed no significant immunoreactivity for KS. For C4S, while the former showed little immunoreactivity, the latter two cases exhibited intensive immunostaining in the pre- and poorly mineralized matrices. CONCLUSIONS: These results suggest that cementifying fibromas could be distinguished from these fibro-osseous tumors by using immunohistochemical analysis for KS and C4S.     

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.     

Changes in amount of gingival crevicular fluid after a single episode of periodontal treatment

A total of 51 periodontal sites from 6 adults with no systemic diseases or medication were selected for the study. All sites showed radiologic bone loss and pockets of 4 mm or more. Crevicular fluid (CF) was collected by inserting filter paper strips into periodontal pockets for 5 s and was measured by Periotron. Samples were collected before and 2, 5, 10, 20, and 40 days after a single episode of periodontal treatment (scaling, root planing and curettage). Plaque Index (PII), Papilla Bleeding Index (PBI) and pocket depth (PD) were measured before and 40 days after treatment. The amount of bone loss was estimated from orthopantomograms taken immediately before the trial. Two days after treatment an increase in the amount of CF was seen. After this the amount of CF decreased, reaching the pretreatment level on day 5 after treatment and a level clearly below pretreatment level on day 10 after treatment. Forty days after treatment a slight increase in the amount of CF was seen. The difference between pretreatment values and values at days 2, 10, 20, and 40 was highly significant. In pretreatment samples, positive correlations were found between the amount of CF and PD, PBI and bone loss and, in samples collected 40 days after treatment between CF and PD. CF measurements made before treatment were of no value in predicting the changes in clinical parameters after treatment.     

Collagen III aminoterminal propeptide in gingival crevicular fluid before and after periodontal treatment

The amount of procollagen III aminoterminal propeptide (PIIINP) in crevicular fluid (CF) was measured from three periodontitis patients before treatment (scaling, root planing, and curettage) and 2, 5, 10, 20, and 40 days after treatment. CF was collected by placing two paper strips in each pocket for 5 s, after which the amount of fluid was measured. Control samples were collected from three subjects with minimal gingival inflammation. PIIINP was extracted into saline solution and determined by a radioimmunological method. Plaque Index, Papilla Bleeding Index, and pocket depth were recorded before and 40 days after treatment. Forty days after treatment clinical parameters indicated healing. The CF PIIINP mean concentration was 162 μg/1 (range 0–430) in the pretreatment samples. Ten days after treatment, PIIINP mean concentration was 1400 μg/1 (range 1000–9000). After this, the concentration gradually decreased, reaching the pretreatment level 40 days after treatment. Most of the control samples showed undetectable amounts of PIIINP. It was suggested that elevated PIIINP concentrations in CF after periodontal treatment reflected increased type III collagen synthesis in gingiva. PIIINP concentrations before treatment reflected the rate of type III collagen turnover in inflamed periodontal tissues. It was further suggested that CF PIIINP has clinical value as an indicator of the healing process of inflamed gingiva.     

Calprotectin in gingival crevicular fluid correlates with clinical and biochemical markers of periodontal disease

Clinical and biochemical markers of periodontal disease have been used for precise objective diagnosis of periodontal inflammation. Interleukin 1beta (IL-1beta) and prostaglandin E2 (PGE2), inflammatory factors, levels in gingival crevicular fluid (GCF) of patients with periodontal disease are elevated and have been studied as biochemical markers. The levels of calprotectin, a leukocyte protein, in body fluids of patients with some inflammatory diseases are raised. Recently, we detected calprotectin in GCF and its concentrations in periodontal pockets were higher than those in healthy gingival crevices. In this study, we investigated the correlations between GCF calprotectin levels and clinical indicators (probing depth and bleeding on probing, BOP), and the IL-1beta or PGE2 levels in GCE Probing depth and BOP at 130 sites of 110 subjects with periodontal or other oral diseases were examined, then GCF samples were collected and their calprotectin, IL-1beta and PGE2 were determined by ELISA. The calprotectin level correlated positively with the probing depth and was significantly higher at BOP-positive than BOP-negative sites. There were significant, positive correlations between the calprotectin and IL-1beta or PGE2 concentrations. These results indicate that the calprotectin level in GCF correlates well with clinical and biochemical markers of periodontal disease and suggest that calprotectin may be useful for evaluating the extent of periodontal inflammation.     

Molecular forms and concentration of fibronectin and fibrin in human gingival crevicular fluid before and after periodontal treatment

A total of 23 periodontitis-affected sites from seven adults was selected for the study. Crevicular fluid (CF) samples were collected with paper strips before treatment (scaling, root planing, and curettage) and 2, 5, 10, 20, and 40 days after treatment. Each sample was eluted into sterile saline and two aliquots were drawn for gel electrophoresis: one for fibronectin and one for fibrin analysis. Peptides were transferred to nitrocellulose membranes, and molecules were detected by specific antibodies. The proportions of different molecular forms of fibronectin and fibrin were analyzed by laser densitometry. Plaque Index, Papilla Bleeding Index, and pocket depth were recorded before and 40 days after treatment. Radiologic bone loss was estimated from orthopantomograms. Two days after treatment, an increase was seen in the proportions of intact fibronectin, fibronectin fragments larger than 70 kDa, and fibrin-positive material with a greater molecular mass than intact fibrin. Between days 5 and 10, the proportions of these large fragments decreased. The highest fibronectin and fibrin concentrations were seen 10 days after treatment. These changes probably reflect degradation of the subgingival fibrin clot formed after treatment, and indicate resolution of the clot during the first 10 days of healing. This agrees well with previous observations of CF plasmin activity and concentration of collagen synthesis markers in CF after periodontal treatment, and with the histologic changes seen during periodontal healing. Results of the present study, together with earlier reported findings of collagen synthesis after periodontal treatment, also support the hypothesis of sequential appearance of fibronectin and collagens during the process of wound healing.     

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     

Enhanced expression of vascular endothelial growth factor by periodontal pathogens in gingival fibroblasts

Vascular endothelial growth factor (VEGF) has recently attracted attention as a potent inducer of vascular permeability and angiogenesis. Aberrant angiogenesis is often associated with lesion formation in chronic periodontitis. The aim of the present study was to investigate the properties of VEGF expression in human gingival fibroblasts (HGF) culture. HGF were stimulated with lipopolysaccharide (LPS), vesicle (Ve) and outer membrane protein (OMP) from Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. HGF constitutively produced VEGF and levels were significantly enhanced (P < 0.01) by stimulation with Ve and OMP from A. actinomycetemcomitans and P. gingivalis at concentrations of 10 microg/ml or higher. On the other hand, VEGF levels were not increased by LPS stimulation. VEGF mRNA expression was also observed in Ve- and OMP-stimulated HGF. A vascular permeability enhancement (VPE) assay was performed using guinea pigs to ascertain whether supernatant from cultures of Ve- and OMP-stimulated HGF enhance vascular permeability in vivo. Supernatant from cultures of Ve- and OMP-stimulated HGF strongly induced VPE. This was markedly suppressed upon simultaneous injection of anti-VEGF polyclonal antibodies with the supernatant. Heating and protease treatment of the stimulants reduced the VEGF enhancing levels in Ve and OMP in vitro. These results suggest that Ve and OMP may be crucial heat-labile and protease-sensitive components of periodontal pathogens that enhance VEGF expression. In addition, VEGF might be associated with the etiology of periodontitis in its early stages according to neovascularization stimulated by periodontal pathogens causing swelling and edema.     

龈沟液中与牙周病有关的细胞因子的研究进展

牙周病时龈沟液内含有多种可作为诊断指标的细胞因子,由于取样简单无创,且能重复取样,易为患者所接受。因此,近年来学者们对龈沟液内的细胞因子在牙周炎活动期的诊断﹑治疗及疗效评价中作用的研究很多。本文对龈沟液中与牙周病有关的细胞因子的研究进展作一综述。     

Collagen I carboxyterminal propeptide in human gingival crevicular fluid before and after periodontal treatment

The amount of procollagen I carboxyterminal propeptide (PICP) in crevicular fluid (CF) was measured in three periodontitis patients. Samples were collected from 29 sites before treatment (scaling, root planing, and curettage) and 2, 5, 10, 20, and 40 days after treatment, by placing two paper strips in periodontal pockets for 5 s. The amount of fluid in strips was measured by the Periotron device. Control samples were collected from subjects with minimal gingival inflammation. PICP was extracted into saline solution and determined by a radioimmunologic method. Plaque index, papilla bleeding index, and pocket depth were recorded before and 40 days after treatment. The CF PICP mean concentration was 4.2 mg/1 in the pretreatment samples. Five days after treatment a statistically significant increase in PICP concentration was seen in all subjects. The peak appeared on days 5 or 10 in 27 sites. The mean peak PICP concentrations of the subjects were 5-10 times higher than the pretreatment values. Twenty days after treatment, mean PICP concentration decreased to pretreatment level. PICP concentrations did not correlate with the clinical parameters. In control samples PICP amounts were below the detection limit. CF PICP is a new marker of type 1 collagen metabolism in periodontal tissues. It was concluded that elevated PICP concentrations in CF after periodontal treatment reflected increased type I collagen synthesis in periodontal tissues and that the peak in type I collagen synthesis takes place 5-10 days after treatment.     

Elastase in gingival crevicular fluid from smokers and non-smokers with chronic inflammatory periodontal disease

OBJECTIVE: To compare elastase concentrations in gingival crevicular fluid (GCF) from individual sites of smokers and non-smokers.
MATERIALS AND METHODS: Twelve pairs of smokers and non-smokers with untreated, moderate to advanced chronic inflammatory periodontal disease were matched for gender, age, ethnicity and the clinical and radio-graphic extent of disease. Durapore filter strip samples were collected over 30 s from two mesiopalatal sites on upper left posterior teeth. Samples were analysed for: I) polymorphonuclear neutrophil leucocyte (PMNL) cell counts; 2) PMNL elastase-αI-antitrypsin complex in the GCF supernatant by ELISA; and 3) functional elastase, free or bound to α2-macroglobulin, estimated from activity against N-tert-butoxycarbonyI-alanyl-prolyl-nor-valylg-chlorothiobenzyl ester in supernatant and lysates of GCF PMNLs.
RESULTS: There were no differences in disease parameters between groups except that bleeding on probing was less extensive in smokers (P< 0.001). Cell counts and elastase content of crevicular PMNLs showed no differences between groups. Lower concentrations of elastase were found in GCF supernatants from smokers than non-smokers. This difference was observed for functional elastase (mean [s.d.] = 30.21 [17.60] against 73.77 [75.26] ng μI^-1, P <0.05) and elastase complexed with αl-antitrypsin (8.97 [6.54] ng μl^-1 against 25.71 [22.07] ng μI^-1, P < 0.001).
CONCLUSIONS: Smokers have lower elastase concentrations in GCF than non-smokers. Further investigation is required to elucidate the underlying cause and its relationship with periodontal disease.     

Relationship between gingival inflammation and painfulness of periodontal probing

The relationship of gingival inflammation to the pain associated with periodontal probing was assessed at baseline (B), and 1 month (1M) and 3 months (3M) later in 46 subjects. At each of the 3 sessions, clinical measures of gingival inflammation included an observational gingival index and bleeding score. In addition, periodontal probings of all existing teeth was performed with a constant force probe (25 g). Following completion of the probing at each session, subjects rated the global painfulness of the probing using a visual analog scale for pain. Results of this study showed that judged painfulness of probing was related to clinical inflammation (bleeding score) at baseline and 1 month and suggest that the degree of periodontal inflammation is related to the pain and discomfort associated with periodontal probing.