Matrix metalloproteinases and their inhibitors in gingival crevicular fluid and saliva of periodontitis patients

Abstract Matrix metalloproteinases (MMPs) and serine proteinases seem to be related to tissue destruction in periodontitis. The presence of MMPs in gingival crevicular fluid (GCF) and saliva, however, has not been studied comprehensively with the enzyme-linked immunosorbent assay (ELISA)-technique, We therefore examined the levels of MMP-1,-3.-8 and -9, and their endogenous inhibitor, tissue inhibitor of matrix metalloproteinases (TIMP-1). in GCF and saliva of patients with adult periodontitiss (AP) and localized juvenile periodontitis (LJP). Elevated levels of MMP-1 were detected in LJP GCF compared to AP and control GCF. Elevated levels of TIMP-1 were also detected in LJP GCF in comparison to AP and control GCF Higher MMP-8 levels were detected in AP GCF compared to LJP and control GCF. The relative low levels of MMP-3 were present in all studied GCF samples. Elevated levels of MMP-8 were further detected in saliva of AP compared to LJP and the controls. Both MMP-1 and TIMP-1 were detected in all studied saliva samples, but not significant differences were detected between the studied groups. Our ELISA-results confirm that (i) PMN MMP-8 and MMP-9 are the main collagenase and gelatinase in AP GCF, whereas GCF collagenase in LJP seems to be of the MMP-1-type; (ii) only low levels of TIMP-1. endogenous MMP-inhibitor. are present in AP GCF. which emphasises the importance of doxycycline as a possible adjunctive drug in the treatment of AP patients; (iii) tests based on specific antibodies against PMN MMPs. especially MMP-8, might serve as a reliable method of measuring and monitoring enzyme levels in GCF from different periodontitis patients.     

Effects of phase I periodontal treatment on gingival crevicular fluid levels of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1

OBJECTIVES: The aim of this study was to evaluate the effects of phase I periodontal treatment on gingival crevicular fluid (GCF) levels of matrix metalloproteinase (MMP)-3 and tissue inhibitors of metalloproteinase (TIMP)-1. METHODS: Plaque index, gingival index, pocket depth and clinical attachment loss were recorded and GCF samples were collected from 20 chronic periodontitis (CP) patients and 20 periodontally healthy controls (C) before treatment. CP patients received phase I periodontal treatment and all clinical parameters were recorded and GCF samples were collected once more after treatment. Assays were performed by an enzyme-linked immunosorbent assay. RESULTS: All of the clinical parameters improved significantly after the therapy (p<0.05). Baseline GCF levels of MMP-3 were significantly higher than C and that level was reduced significantly by treatment compared with baseline levels (p<0.05). Baseline GCF levels of TIMP-1 were lower than post-treatment levels and C (p<0.05). GCF levels of TIMP-1 increased significantly by treatment compared with baseline levels (p<0.05). CONCLUSION: This study shows that the clinical improvements after phase I periodontal therapy are accompanied by reduction in MMP-3 and increasing in TIMP-1 GCF levels.     

Correlation analysis for clinical and gingival crevicular fluid parameters at anatomically related gingival sites

This study was designed to evaluate the relationship of certain clinical and biochemical measures of periodontal pathology at anatomically related gingival sites. The maxillary first molar--second bicuspid region was studied in patients with gingivitis and periodontitis. The mesiobuccal site on the first molar was compared to the mesiopalatal and direct buccal sites on the molar and the distobuccal site on the second bicuspid. Probing depth, attachment level, gingival index, gingival crevicular fluid (GCF) volume, and GCF levels of the lysosomal enzyme B-glucuronidase (BG), the cytoplasmic enzyme lactate dehydrogenase, IgG and the protease-inhibitor alpha-2-macroglobulin were studied. For the 3 anatomical pairs that were analyzed, the correlation coefficients for the GCF constituents were generally higher than the correlations for the clinical parameters. The mean correlations for the GCF constituents were higher for the periodontitis patients as compared to the gingivitis patients. For the periodontitis patients, BG activity was correlated at adjacent proximal sites, approached significance at adjacent papillary sites, but was not significantly correlated at adjacent facial-proximal sites. This data suggests that sampling of BG activity from a mesiobuccal site provides information about the anterior papillary unit. In contrast, IgG in GCF collected from the mesiobuccal site on the first molar was significantly correlated with the total IgG in the 3 other sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigations into the cellular contribution to host tissue proteases and inhibitors in gingival crevicular fluid

Abstract Gingival crevicular fluid (GCF) was collected from chronic periodontitis patients using plastic micropipettes and coverslip smears stained with antibodies for leukocyte markers and Toluidine Blue for mast cells. The smears consisted of 70–80% granulocytes, 10–20% monocytes/macrophages. 5% mast cells and 5% T lymphocytes: no B lymphocytes were found. Proteases and inhibitors in GCF cells were investigated by enzyme cytochemistry using 2-methoxy-4-naph-thylamine-linked peptide substrates and simultaneous coupling to Fast Blue B and immunocytochemistry using biotinylated secondary antibodies and an alkaline phosphatase/new fuchsin detecting system. Elastase was detected in granulocytes, cathepsin B in macrophages. dipeptidyl peptidases II and IV in a small proportion of macrophages, dipeptidyl peptidase IV in a few T lymphocytes, tryptase in mast cells and α-1-proteinase inhibitor and α-2-macroglobulin in some macrophages. GCF was also collected on filter paper strips and eluted into buffer for biochemical enzyme assays. Lysis of cells by addition of detergent to the elution buffer increased activities to 140–240% of control values. Removal of cells by centrifugation reduced measured activities to 1–30% of original figures; this effect was less if samples were pre-treated with detergent. Proteases from inflammatory cells therefore appear to make up most of the measured enzyme activity in GCF, and this association may explain recent correlations with periodontal disease progression.     

慢性牙周炎患者龈沟液中白细胞介素-4的检测和意义

目的检测慢性牙周炎患者牙周基础治疗前后龈沟液中白细胞介素-4(IL-4)的质量浓度,探讨IL-4与牙周炎的关系及其在牙周炎发病机制、病情进展等方面所起的作用。方法用滤纸条浸润法采集成年健康者和牙周炎患者治疗前后的龈沟液样本,用酶联免疫吸附测定检测样本中IL-4的质量浓度。结果慢性牙周炎患者龈沟液中IL-4的质量浓度低于健康对照组(P<0.05)。经牙周基础治疗1个月后,IL-4的质量浓度无明显变化,治疗前后的差异无统计意义(P>0.05);IL-4的质量浓度与探诊深度呈显著负相关,与牙龈指数和附着丧失无明显相关性。结论IL-4缺乏可能会导致牙周病的发生,IL-4可作为早期诊断牙周病和检测易患人群的敏感性指标。     

The gingival crevicular fluid ciprofloxacin level in subjects with gingivitis and periodontitis, and its effects on clinical parameters

OBJECTIVES: The purpose of this study, conducted on patients with gingivitis and periodontitis, was twofold: to find out the serum and gingival crevicular fluid concentration of ciprofloxacin, which is a common drug used effectively against Actinobacillus actinomycetemcomitans and to determine the effects of ciprofloxacin administration on clinical parameters. METHOD: A total of 32 adult patients, consisting of 16 subjects with gingivitis and 16 subjects with untreated chronic periodontitis, were included in the study. The subjects were divided into four groups: group I included eight subjects with chronic gingivitis who had not previously received any ciprofloxacin; group II included eight subjects with chronic gingivitis to whom three doses of ciprofloxacin were administered (Siprosan 500 mg) to establish adequate gingival crevicular fluid and serum concentrations of the agent; group III consisted of eight subjects with chronic periodontitis who had not received any ciprofloxacin; group IV included eight subjects with chronic periodontitis to whom three doses of ciprofloxacin were administered to establish adequate gingival crevicular fluid and serum concentrations of the agent. All patients were systemically healthy, free of pain and reported no current medication usage. Each patient was treated with scaling and/or root planing using specific hand instruments under local anesthesia. Gingival index, plaque index and clinical attachment levels of the teeth were used to determine the clinical condition of the subjects and findings were recorded at the beginning, seventh day, 21st day and third month of the study. Serum ciprofloxacin level was measured in venous blood. Approximately 5 ml of venous blood was drawn from subjects in groups II and IV using a standard venipuncture technique. Gingival crevicular fluid samples were sampled from six interproximal sites with six paper strips in the posterior region of upper jaw (excluding third molar) and all gingival crevicular fluid and serum samples were evaluated by high-performance liquid chromatography. RESULTS: The serum concentrations of ciprofloxacin at the first and 72nd hour were not significantly different in subjects with periodontitis compared to subjects with gingivitis. But the gingival crevicular fluid concentrations of ciprofloxacin at the same hours were significantly high in subjects with periodontitis compared to subjects with gingivitis. Both subjects with gingivitis and periodontitis had significantly higher ciprofloxacin levels in the gingival crevicular fluid than in serum. The application of ciprofloxacin did not have any positive or statistically significant effect upon the clinical parameters of the subjects with gingivitis. On the other hand, a significant decrease in the clinical attachment level scores of the subjects with periodontitis (group IV) was observed compared to group III in the 21st day and third month. CONCLUSION: According to these results, the use of ciprofloxacin as an alternative drug in subjects with periodontitis but not gingivitis can be recommended. However, long-term studies are also needed to assess the effects of ciprofloxacin on clinical parameters.     

Levels of interleukin-17 in gingival crevicular fluid and in supernatants of cellular cultures of gingival tissue from patients with chronic periodontitis

BACKGROUND AND AIMS: Interleukin-17 (IL-17) is a T-cell-derived cytokine that may play an important role in the initiation or maintenance of the pro-inflammatory response and has recently been found to stimulate osteoclastic resorption. The purpose of the present study was to determine the presence of IL-17 in gingival crevicular fluid (GCF) samples and in the culture supernatants of gingival cells from patients with chronic periodontitis. METHOD: GCF samples were collected during 30 s from two sites in 16 patients from periodontally affected sites (probing depth > or =5 mm, attachment loss > or =3 mm). The comparison with healthy controls was carried out by collecting GCF samples from eight healthy volunteers. GCF was collected using a paper strip and ELISA was performed to determine the total amount of IL-17. Supernatant cellular cultures of gingival cells were obtained from periodontal biopsies taken from 12 periodontitis patients and from eight healthy control subjects during the surgical removal of wisdom teeth. Spontaneous and phytohaemagglutinin (PHA)-stimulated levels of IL-17 were determined by ELISA. RESULTS: The total amount of cytokine IL-17 was significantly higher in the periodontitis group than the control group (45.9 versus 35.6 pg, p=0.005). Significantly higher GCF volume and amount of total proteins were obtained from periodontitis patients as compared with control subjects (0.98 versus 0.36 microl, p=0.0005; 0.12 versus 0.05 microg, p=0.0005, respectively). A higher concentration of IL-17 was detected in culture supernatants from periodontitis patients compared with healthy subjects, either without stimulation (36.28+/-8.39 versus 28.81+/-1.50 microg/ml, p=0.011) or with PHA stimulation (52.12+/-14.56 versus 39.00+/-4.90 microg/ml, p=0.012). Treatment with PHA induced a significant increase in the production of IL-17 in healthy subjects and periodontitis patients (p=0.001 and 0.003). CONCLUSIONS: The total amount of cytokine IL-17 in GCF samples and in the culture supernatants of gingival cells are significantly increased in periodontal disease.     

Intracytoplasmic enzymes in gingival crevicular fluid of patients with aggressive periodontitis

Castro CE, Koss MA, López ME. Intracytoplasmic enzymes in gingival crevicular fluid of patients with aggressive periodontitis. J Periodont Res 2011; 46: 522–527. © 2011 John Wiley & Sons A/S Background and Objective: Biochemical parameters of crevicular fluid could provide evidence of periodontal tissue disease. The aim of this study was to analyze enzymes in crevicular fluid in aggressive localized and generalized periodontitis. Material and Methods: One hundred and twenty‐four subjects were classified as having localized (n = 36) or generalized aggressive periodontitis (n = 38) and subclassified into moderate and severe groups. Controls were 50 periodontitis‐free subjects. Activities of the enzymes lactate dehydrogenase, neutrophil elastase, alkaline phosphatase and aspartate aminotransferase were determined. Data were analyzed using one‐way ANOVA and Tukey’s test. Results: Among the subjects with localized aggressive periodontitis, values of lactate dehydrogenase and alkaline phosphatase increased notably in moderate and severe periodontitis compared with control subjects. Values for aspartate aminotransferase increased with the severity of the disease, and neutrophil elastase was increased in the moderate and severe states. In generalized aggressive periodontitis, lactate dehydrogenase showed higher values than in control subjects in both periodontal subgroups. Alkaline phosphatase and neutrophil elastase showed higher significant differences between moderate and severe periodontitis compared with the control group. Aspartate aminotransferase showed differences between the severe and moderate periodontitis groups compared with the control group. Of all the enzymes analyzed, only lactate dehydrogenase showed higher values in localized than in generalized aggressive periodontitis. Conclusion: Lactate dehydrogenase may distinguish localized and generalized aggressive periodontitis. Alkaline phosphatase increases from moderate to severe states in both types of periodontitis. Aspartate aminotransferase and neutrophil elastase only increase with strong evidence of periodontal destruction.     

Phylloquinone in gingival crevicular fluid in adult periodontitis

Abstract. Phylloquinone is a lipid soluble vitamin which is an absolute growth requirement for black-pigmented anaerobes, many of which are implicated in the aetiology of periodontal diseases. This cross-sectional study aimed to detect the levels of phylloquinone in GCF from healthy and diseased sites in subjects with adult periodontitis, in order to investigate further its potential role in the disease process. The sample consisted of eighteen patients with adult periodontitis. Periodontal probing depths, attachment levels and gingival indices were recorded from one healthy and one diseased site in each subject. GCF was sampled and the amount of phylloquinone in each sample was determined using reverse-phase high performance liquid chromatography coupled to electrochemical detection. The mean amount of phylloquinone in accumulated GCF from diseased sites was 406 pg/site and 80 pg/site from healthy sites ( p =0.013). When the amounts of phylloquinone in GCF were expressed as concentrations the values were 228 ng/ml and 3350 ng/ml for diseased and healthy sites respectively ( p =0.084). These findings suggest the levels of phylloquinone in GCF differs in periodontal health and disease in subjects with adult periodontitis. The total phylloquinone at diseased sites may provide the nutritional requirements favouring the growth of black-pigmented anaerobes.     

Chemokine RANTES in gingival crevicular fluid of adult patients with periodontitis

Background, aims: This study presents the first evidence on the presence of the chemokine RANTES in the gingival fluid crevicular (GCF) of patients with periodontitis. RANTES is a chemokine that selectively attracts and activates macrophages and lymphocytes. Leucocytes play a critical rôle in the host response to the subgingival microflora. Method: In this study, the presence de RANTES in GCF was determined in samples obtained from adult patients with periodontitis and from control subjects with clinically healthy gingiva. GCF was collected from different probing depths (<3 mm, 4–6 mm, >6 mm) (n=72); and active (n=12) and inactive sites (n=12). An active site was defined as attachment loss >2 mm, as determined by sequential probing and the tolerance method. GFC was collected for 30 s using Periopaper^® strips, and RANTES was quantified by ELISA. Results: The presence of RANTES was detected exclusively in the group of patients with periodontitis, presenting a total amount of 40.43±16 pg and a concentration 67.80±41 pg/μl. RANTES concentration was significantly higher in probing depth <3 mm than in probing depth >6 mm (87.24 versus 51.87, p=0.014). Total amount and concentration in the GCF samples from active sites were higher that in inactive sites (p>0.05). Conclusions: The finding that RANTES is found only in patients with periodontitis, may represent a general feature of chronic inflammatory in periodontal diseases. Finally, RANTES may be implicated in the biological mechanisms underlying the pathogenesis and progression of periodontal disease.     

Matrix metalloproteinase-8 levels and elastase activities in gingival crevicular fluid from chronic adult periodontitis patients

AIM: To make an initial assessment of the periodontal diagnostic potential of immunoreactive matrix metalloproteinase-8 (MMP-8) in gingival crevicular fluid (GCF) by comparison with elastase activity which has previously been associated with disease severity and progression. METHODS: GCF was collected from molar and premolar sites of 16 chronic adult periodontitis patients before treatment and 13 of this group 2 weeks after scaling and root planing. Samples were analysed for MMP-8 by immunofluorometric assay and for elastase activity with a fluorogenic substrate. RESULTS: Mean patient clinical parameters and GCF enzyme totals both decreased significantly after treatment. Total MMP-8 levels and elastase activities generally correlated significantly with gingival and bleeding indices. For GCF concentrations, only MMP-8 showed a significant fall after treatment, and some significant correlations with clinical parameters. Amounts of the 2 enzymes correlated significantly with each other. CONCLUSIONS: Similarities between MMP-8 and elastase probably reflect the fact that both enzymes are associated mainly with neutrophils: MMP-8 levels may have fallen more after treatment because the assay, unlike that for elastase, would most likely not have detected much enzyme bound to alpha-macroglobulin. The immunoassay for MMP-8 is more specific and convenient than functional collagenase assays, and might be suitable for monitoring the periodontal condition.     

Interleukin-8 and anti-interleukin-8 autoantibodies in gingival crevicular fluid from patients with periodontitis

Interleukin-8 (IL-8) is believed to play an important role in the pathogenesis of various forms of periodontitis. In addition, the anti-IL-8 autoantibody has been recently recognized as a potent modulator of IL-8 function. In the current study, the concentrations of IL-8 and its autoantibody in gingival crevicular fluid from patients with chronic generalized periodontitis were compared to those in gingival crevicular fluid from patients with refractory chronic periodontitis. Gingival crevicular fluids were collected from patients treated in a private periodontal clinic. Nine patients who were identified as having chronic generalized periodontitis and four with refractory chronic periodontitis were selected for the study. Patients included in the latter group had undergone supportive periodontal therapy for more than 10 years, and during that time had experienced many episodes of periodontal destruction. The gingival crevicular fluid concentrations of total protein, IL-8, free anti-IL-8 autoantibody and IL-8 bound to the autoantibody (anti-IL-8:IL-8 complexes) were examined. There were no differences in concentration of total protein, but significantly higher levels of IL-8 were detected in patients with chronic generalized periodontitis in comparison to patients with refractory chronic periodontitis (P < 0.05). In addition, anti-IL-8:IL-8 complexes were present in 90% of patients with chronic generalized periodontitis, but in only 50% of patients with refractory chronic periodontitis. The results suggest that elevated concentrations of free and complexed IL-8 can differentiate patients with chronic generalized periodontitis from patients with refractory chronic periodontitis.     

Granulocyte elastase in gingival crevicular fluid

The granulocyte elastase activity and the immuno-reactive (antigenic) granulocyte elastase of gingival crevicular fluid (GCF) were studied in 16 periodontitis patients and in 10 gingivitis patients. The elastase activity was measured with a low molecular weight substrate specific for granulocyte elastase. The antigenic elastase was determined with specific antibodies against granulocyte elastase. Intracrevicular sampling of GCF with paper strips for 30 s seemed to provide representative values of elastase. The elastase activity correlated with probing depth and attachment loss and appeared to be a measure of the degree of tissue destruction. Antigenic elastase represents the number of granulocytes in GCF and should thus be related to the degree of inflammation. The periodontitis patients and the gingivitis patients both had a similar degree of inflammation as measured by antigenic elastase per microliter GCF and gingival index. The elastase activity per microliter GCF, however, was higher in the periodontitis group. Elevated granulocyte elastase activity in GCF seems to be independent of inflammation and could thus be an indicator of patients at risk for periodontitis.     

Langerhans cells express matrix metalloproteinases 9 and 2 and tissue inhibitors of metalloproteinases 1 and 2 in healthy human gingival tissue and in periodontitis

BACKGROUND: As antigen-presenting cells, Langerhans cells may play an important role in the initiation and maintenance of periodontal disease. This study is the first report that extends our knowledge of the expression of matrix metalloproteinases and their endogenous tissue inhibitors by Langerhans cells in healthy and diseased gingival tissues. METHODS: Single and double immunolabeling procedures were carried out using monoclonal antibodies against CD1a, matrix metalloproteinases 2 and 9, and tissue inhibitors of matrix metalloproteinases 1 and 2, and analyzed by conventional and confocal microscopes. RESULTS: Langerhans cells expressed matrix metalloproteinases 2 and 9, and tissue inhibitors of matrix metalloproteinases 1 and 2 in healthy and diseased gingival tissues. The tissue inhibitors of matrix metalloproteinase-positive Langerhans cells were mainly observed in the upper epithelial layers. Matrix metalloproteinase 9-positive Langerhans cells were observed especially during periodontitis and in the basal epithelial layer or crossing the basement membrane. CONCLUSION: During periodontal disease, changes in the expression of matrix metalloproteinases and their tissue inhibitors by gingival Langerhans cells could be implicated in the migration of the cells towards the connective tissue.     

Levels of tissue inhibitor of metalloproteinases-1 and matrix metalloproteinases-1 and -8 in gingival crevicular fluid following treatment with enamel matrix derivative (EMDOGAIN)

The mechanism of enamel matrix derivative (EM D) action on the periodontal wound healing process is not well understood. However, earlier in vitro studies from our laboratory demonstrated that EMD stimulated the proliferation of both periodontal ligament and gingival fibroblast cells. Therefore, the purpose of this study was to further evaluate the effect of EMD on the early wound healing process by assessing the protein levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in gingival crevicular fluid (GCF). Sixteen patients, each of whom had one or two pairs of infrabony defects located contralaterally in the same arch, were included in this clinical trial. Thirty-six infrabony defects were randomly assigned treatment with flap surgery plus EMD or flap surgery plus placebo. At baseline and at 2, 4 and 12 week follow-up evaluation visits, GCF was sampled with paper strips. After determination of GCF volume, TIMP-1, MMP-1 and MMP-8 GCF levels were measured by an enzyme-linked immunosorbent assay. Intragroup analysis: At week 2 following surgery, when compared to baseline all parameters in each study group, except MMP-1, significantly increased (p<0.05). There were no significant differences between 4 or 12 weeks and baseline in either study group. Intergroup analysis: At 4 weeks after surgery, GCF volume and TIMP-1 levels showed a significant decrease (p<0.05) in the EMD group, when compared to the placebo group. MMP-1 levels at weeks 2, 4 and 12, and MMP-8 levels at weeks 4 and 12 were significantly lower (p < 0.05) in the EMD group compared to the placebo group. EMD compared to placebo treated sites demonstrated a more rapid return to baseline levels of TIMP-1, MMP-1 and MMP-8. These findings suggest that treatment with flap surgery and EMD, compared to flap surgery with placebo, accelerated healing at an earlier stage of wound healing following surgery.     

广泛型侵袭性牙周炎患者龈沟液中白介素-17的检测

目的 比较广泛型侵袭性牙周炎患者与健康人龈沟液中白介素-17(IL-17)的表达水平及与临床指标的关系。方法 选择广泛型侵袭性牙周炎患者18例和健康人16例,共68颗牙,记录临床牙周检查指标,用滤纸条法收集龈沟液。采用抗体夹心ELISA法测定广泛型侵袭性牙周炎患者治疗前、治疗后3个月及健康对照者龈沟液中IL-17总量和浓度。结果 广泛型侵袭性牙周炎患牙治疗前龈沟液中IL-17的总量和浓度高于牙周健康牙,基础治疗3个月后广泛型侵袭性牙周炎患牙龈沟液中IL-17的总量和浓度较治疗前下降。广泛型侵袭性牙周炎患牙龈沟液中IL-17浓度与探诊深度、附着丧失和出血指数呈正相关关系,而IL-17总量仅与探诊深度呈正相关关系。结论 龈沟液中IL-17浓度可能与广泛型侵袭性牙周炎牙周破坏及炎症的严重程度相关。?