Osteopontin in gingival crevicular fluid

Osteopontin (OPN) is a major glycosylated phosphoprotein in bone matrix and is produced by several cells including osteoblasts, osteoclasts and macrophages. OPN levels increase in active sites of bone metabolism. Recently, several bone-related proteins were identified in gingival crevicular fluid (GCF) to seek markers of alveolar bone resorption in periodontal disease. In this study, we investigated the existence of OPN in GCF and the correlation between OPN level in GCF and probing depth (PD) of sampling sites in 98 periodontitis patients and 35 healthy subjects. An immunoblotting analysis using 10% polyacrylamide gel showed that two forms of OPN with molecular masses of 54 and 66 kDa and several degraded fragments were detected in most GCF samples from diseased sites (PD > 4 mm). In GCF samples from healthy sites (PD < or = 3 mm), only one form (54 kDa) was observed, but any degraded fragments were not detected. When OPN amounts in GCF samples were determined by ELISA, a weak. but significant correlation was observed between OPN amount in GCF and PD (r=0.32, p=0.0013). These results demonstrate that OPN exists in GCF and that OPN level in GCF increases with the progression of periodontal disease.     

Interleukin-1 beta (IL-1 beta) levels in gingival crevicular fluid from adults with previous evidence of destructive periodontitis. A cross sectional study.

We have estimated the levels of Interleukin-1 beta (IL-1 beta) by ELISA in gingival crevicular fluid (GCF) at 58 sites from 37 patients with adult periodontitis. GCF was collected for 5 s on filter papers and a 2nd sample was collected for 30 s 1 min later. 68/116 strips yielded detectable levels of IL-1 beta. IL-1 beta was present in both the 1st and 2nd samples at 28 sites, in the 1st only at 4 sites and in the 2nd only at 8 sites; 18 sites were below the level of detection for the assay. When the concentrations of IL-1 beta were calculated in the original volume of GCF on each strip, the mean value for positive strips was 34.16 +/- 29.45 (SD) pg/microliters with a range from 1.75 to 97.13 pg/microliters. There were no statistically significant correlations with the plaque index, bleeding index or probable crevice depth (pocket depth). The results indicate that IL-1 is present in the GCF from a proportion of sites with evidence of previous periodontal destruction.     

Smoking cessation increases gingival blood flow and gingival crevicular fluid

OBJECTIVES: The purpose of the present study was to determine the effect of smoking cessation on gingival blood flow (GBF) and gingival crevicular fluid (GCF). MATERIAL AND METHODS: Sixteen male smokers (aged 22-39 (25.3+/-4.0) years), with no clinical signs of periodontal and systemic diseases, were recruited. The experiment was performed before (baseline) and at 1, 3 and 5 days, and at 1, 2, 4 and 8 weeks after smoking cessation. The status of smoking and smoking cessation was verified by exhaled carbon monoxide (CO) concentration, and by serum nicotine and cotinine concentrations. A laser Doppler flowmeter was used to record relative blood flow continuously, on three gingival sites of the left maxillary central incisor (mid-labial aspect of the gingival margin and bilateral interdental papillae). The GCF was collected at the mesio- and disto-labial aspects of the left maxillary central incisor and the volume was calculated by the Periotron 6000(R) system. The same measurements except for the GBF were performed on 11 non-smoking controls (four females and seven males), aged 23-27 (24.4+/-1.2) years. RESULTS: Eleven of 16 smokers successfully completed smoking cessation for 8 weeks. At 1 day after smoking cessation, there was a significantly lower CO concentration than at baseline (p<0.01). Also, nicotine and cotinine concentrations markedly decreased at the second measurement. The GBF rate of smokers was significantly higher at 3 days after smoking cessation compared to the baseline (p<0.01). While the GCF volume was significantly increased at 5 days after smoking cessation compared to the baseline (p<0.01), it was significantly lower than that of non-smokers until 2 weeks after smoking cessation (p<0.01). CONCLUSION: The results show that the gingival microcirculation recovers to normal in the early stages of smoking cessation, which could activate the gingival tissues metabolism/remodeling, and contribute to periodontal health.     

Cystatin activity in gingival crevicular fluid from periodontal disease patients measured by a new quantitative analysis method

Cystatins are protein inhibitors of cysteine proteinases, which are believed to play an important role in the pathogenesis of periodontal disease. In this study, we report a new sensitive method for the quantitative analysis of cystatin activity in a small amount of crude sample such as gingival crevicular fluid. Cystatin activity in the crude sample was determined by using active site-titrated papain, which is a cysteine proteinase from the plant Carica papaya. Crude samples usually contain endogenous cysteine proteinases. These competed with the added papain for the active sites of the cystatins. The cystatin-cysteine proteinase complex was able to be dissociated by the addition of papain. This competition and dissociation could interfere with the determination of cystatin activity, since some of the cysteine proteinases, such as cathepsin B, hydrolyzed the specific substrate for papain during titration with the papain. In order to exclude this interference and measure total cystatin activity, the crude sample must be alkalinized (pH 11.0) for 5 min at 4 degrees C followed by 10 min at 40 degrees C before titration with papain. The minimum detectable amount of cystatins was 20 fmol/assay when it was calculated per mole of papain inhibitory sites. Using this method, significant levels of cystatin activity were detected in all the samples of gingival crevicular fluid taken from periodontal disease patients. These results suggest that cystatins could regulate the cysteine proteinases in gingival crevicular fluid and that this new method could be useful to clarify the role of cystatins in the pathogenesis of periodontal disease.     

Daytime variations of interleukin-1beta in gingival crevicular fluid

Interleukin-1 β (IL-1 β ) is an important parameter in periodontal research because of its role in inflammation and bone resorption. One measure used to assess local IL-1 β concentrations is analysis of its levels in gingival crevicular fluid (GCF). While studies on serum IL-1 β concentrations indicate a circadian rhythm of this parameter, nothing is known about daytime variations of IL-1 β in GCF. The present study thus aimed to analyse such variations. Daytime variations of GCF-IL-1 β between 08:00 and 22:00 h were assessed, with a time resolution of 2 h, in 28 periodontally healthy subjects.The data showed a significant variation throughout the day, with the lowest concentrations and total amounts in the morning and the highest in the evening. The effect sizes of comparisons between morning and evening samples were medium to high and corresponded in magnitude to those reported in other published research comparing healthy sites and those affected by periodontitis. The smallest daytime variations were found to occur between 12:00 h and 18:00 h. It is concluded that daytime variations in GCF-IL-1 β are large enough to be able to mimic or mask differences caused by clinical factors.     

Analysis of proteins in human gingival crevicular fluid by mass spectrometry

Kido J, Bando M, Hiroshima Y, Iwasaka H, Yamada K, Ohgami N, Nambu T, Kataoka M, Yamamoto T, Shinohara Y, Sagawa I, Nagata T. Analysis of proteins in human gingival crevicular fluid by mass spectrometry. J Periodont Res 2012; 47: 488–499. © 2012 John Wiley & Sons A/S Background and Objective: Gingival crevicular fluid is a bodily fluid transuded from periodontal tissues into the gingival crevice and periodontal pocket, and contains many species of components. Proteins in gingival crevicular fluid have been studied as markers for periodontal diseases. Mass spectrometric analysis is used for the analyses of proteins, lipids, saccharides and metals, and expected as an approach for disease diagnosis. For better analysis of the protein components in gingival crevicular fluid, we investigated proteins in gingival crevicular fluid samples from the healthy gingival crevice and periodontal pocket using mass spectrometry. Material and Methods: Gingival crevicular fluid samples were collected from subjects who gave their informed consent and were periodontally healthy or had diseased pockets. These samples were electrophoretically separated, and each fraction on the gels was analysed by nano liquid chromatography coupled with tandem mass spectrometry. Antimicrobial peptides detected in gingival crevicular fluid were confirmed by western blotting. Results: One hundred and four proteins were detected in gingival crevicular fluid samples from both healthy sites and sites of periodontitis; 64 proteins were contained only in gingival crevicular fluid from healthy sites and 63 proteins were observed only in gingival crevicular fluid from periodontitis sites. These proteins were blood‐, cytoskeleton‐, immunity‐, inflammation‐ and lipid‐related proteins and enzymes. Some proteins, including ceruloplasmin, glycogen phosphorylase, glutathione S‐transferase, phosphoglycerate mutase, psoriasin, S100A11 and resistin, were identified for the first time in gingival crevicular fluid. Antimicrobial peptides, such as lactoferrin, α1‐antitrypsin, lipocalin, S100A7, S100A8, S100A9 and cathelicidin, were observed by mass spectrometry and western blotting. Conclusion: Multiple protein components in gingival crevicular fluid were analysed at the same time using mass spectrometry, and this approach may be useful for the diagnosis of periodontal diseases.     

Periodontal variables affecting nifedipine sequestration in gingival crevicular fluid

We previously demonstrated the sequestration of nifedipine in gingival crevicular fluid (GCF), especially in patients exhibiting significant gingival overgrowth. The aim of the present study is to determine the role of site specific periodontal factors in this phenomenon. 10 adult patients exhibiting nifedipine induced gingival overgrowth were studied. In each patient GCF was harvested from two sites that demonstrated inflammation and increased probing depth as well as from two clinically healthy sites. The concentration of nifedipine was determined using gas chromatography. Drug concentrations were significantly increased in the presence of inflammation (p=0.004) and plaque (p=0.029) whilst increased probing depths and gingival overgrowth were not significantly related to drug sequestration. We can conclude that inflammatory changes in gingival tissues appear to be a significant determinant for the sequestration of nifedpine in the GCF.     

Calcitonin gene-related peptide in gingival crevicular fluid in periodontal health and disease

The aims of the present study were to investigate whether calcitonin gene-related peptide (CGRP) was present in gingival crevicular fluid in both periodontal health and disease and to study the relationship with periodontal inflammation. Gingival crevicular fluid (GCF) was collected from a healthy, a gingivitis and a periodontitis site in 18 subjects with periodontitis and from a healthy site in 19 subjects without periodontitis. The volume of GCF was measured and each sample subsequently analysed for CGRP by radioimmunoassay. In subjects with periodontitis, CGRP immunoreactivity (CGRP-IR) was not detected in any periodontitis sites, nor in 67% of gingivitis and 28% of periodontally-healthy sites. The total amount of CGRP-IR was significantly elevated in periodontally healthy (p=0.0015) and gingivitis (p=0.027) compared with periodontitis sites. CGRP-IR was present in 89% of the healthy sites sampled in control subjects at comparable levels to those in healthy sites in periodontitis subjects. It is concluded that in periodontal inflammation, particularly in deep pockets, constituents of GCF process and degrade CGRP.     

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)     

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.     

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.     

Interleukin-1 and IL-1 receptor antagonist in gingival crevicular fluid

BACKGROUND/AIMS: This study aimed to investigate the cytokine IL-1beta and its receptor antagonist IL-1ra in gingival crevicular fluid (GCF), in patients with adult periodontitis. METHOD: A total of 40 GCF samples were harvested from 10 subjects with moderate to severe adult periodontitis and 10 healthy controls. Subjects were selected from both genders, with all the upper anterior teeth present, and with no relevant systemic illness, pregnancy or recent medication. All subjects were non-smokers and had not received any periodontal therapy within the preceding 3 months. Deep bleeding sites, deep non-bleeding sites and healthy sites were investigated in relation to upper anterior teeth. Clinical measurements were recorded for each site, after obtaining a GCF sample. IL-1beta and IL-1ra were quantified using new commercially available ELISA kits (Quantikine), and could be detected in all samples. RESULTS: The mean concentration for IL-1beta was 0.11 (SD 0.14) pg/microl for bleeding periodontitis sites, 0.04 (0.05) pg/microl for non-bleeding periodontitis sites, and 0.01 (0.03) pg/microl for healthy sites (p<0.001). In contrast, the mean concentration for IL-1ra was 6.99 (9.78) pg/microl for healthy sites, 0.59 (0.44) pg/microl for non-bleeding periodontitis sites, and 0.44 (0.36) pg/microl for bleeding periodontitis sites (p<0.001, except for comparisons between bleeding and non-bleeding periodontitis sites, p>0.05). For healthy sites, a strong inverse relationship was found between IL-1beta and IL-1ra levels in GCE. CONCLUSIONS: The results suggest a strong relationship between the severity of adult periodontitis and the increasing GCF levels of IL-1beta and decreasing levels of IL-1ra.     

Cytokines in gingival crevicular fluid of adolescents and young adults

Background/aim:  The purpose of this study was to compare the levels of the cytokines interleukin-1β (IL-1β), IL-4, and IL-8 in the gingival crevicular fluid (GCF) of adolescents and young adults.
Methods:  Twenty-five adolescents aged between 14 and 16 years (Group A) and 20 periodontally healthy young adults aged between 25 and 35 years (Group B) were selected from two private dental clinics limited to pedodontics and periodontics respectively in Piraeus Greece. All subjects were systemically healthy. Clinical examination included probing pocket depth (PPD), presence or absence of plaque, and bleeding on probing (BOP). GCF was collected from four sites per subject. IL-1β, IL-4, and IL-8, measured as total amounts (pg/30 s), were evaluated in 180 samples using a commercially available sandwich enzyme-linked immunosorbent assay.
Results:  IL-1β mean levels of Groups A and B were adjusted for BOP and PPD. Differences of IL-1β mean levels between the two age groups were statistically significant ( F  = 50.245, P  < 0.001) in favour of Group A. Adolescents showed statistically significantly lower mean levels of IL-4 than young adults in the presence of BOP ( F  = 10.690, P  = 0.001). There was no statistically significant difference between adolescents and adults for the means of IL-8 adjusted for BOP and plaque presence ( F  = 2.032, P  = 0.161).
Conclusions:  Within the limits of this study the differences reported in mean levels of IL-1β and IL-4 may be attributed to the different age status.     

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 pharmacokinetics of phenytoin in gingival crevicular fluid and plasma in relation to gingival overgrowth

Abstract The aim of this study was to determine whether phenytoin (PHT) could be detected in gingival crevicular fluid (GCF), and to relate its concentration to both plasma level and degree of gingival overgrowth. 23 patients medicated with phenytoin for at least 6 months were clinically examined for signs of periodontal disease and gingival overgrowth. 12 patients out of these demonstrated clinically significant overgrowth and their plaque scores and gingival inflammation were greater than for the non-overgrowth group (p<0.001). Phenytoin concentrations were determined by high performance liquid chromatography, and was detected in GCF. There was a significant correlation between the GCF and plasma phenytoin concentrations (p<0.05), but it was not related to the extent of gingival overgrowth. Inflammation increased the GCF volume, but was not a determinant of GCF phenytoin concentration. It is concluded that effusion of phenytoin into GCF is regulated by the plasma levels of the drug, but its concentration in GCF is not related to the incidence of gingival overgrowth.     

Methodological considerations in the assessment of gingival crevicular fluid volume

In 4 studies on gingival crevicular fluid volume (GCFV) determination, the reliability of measurements, influences of plaque, circadian rhythms and stability over 24 h were examined. Samples were taken at 2 sites with a modified intracrevicular method. Reliability (n=40): repeated GCFV determinations within 5 min revealed good reliability coefficients (r(tt)>0.80). Influences of supragingival plaque (n=80): repeated GCFV determinations within 5 min with plaque removal between measurements in fourty subjects, the other subjects serving as control, revealed no group differences with respect to the differences between measurements. Circadian rhythms (n=20): GCFV was assessed 6x throughout the day. Repeated measures ANOVA revealed no significant time effect. Stability over 24 h under constant clinical conditions (n=20): measures were taken at 16:00 h on 2 consecutive days, several disturbing variables were kept constant. Retest correlations revealed a low stability (i.e., high variability) of GCFV measures under constant clinical conditions (r(tt)=0.38 for tooth 11 and r(tt)= -0.25 for tooth 26). It is concluded that GCFV determination can be done with high reliability, the validity of measurements neither being affected by supragingival plaque nor by diurnal rhythms. The low stability of measurements questions the validity of GCFV for diagnostic purposes.     

龈沟液弹性蛋白酶洗提和保存方法的初步研究

目的 :观察不同保存温度、时间和不同缓冲液以及不同洗提方法对龈沟液弹性蛋白酶 (GCF-EA)活性的影响。方法 :采用底物反应法分别于取样即刻和 1、2、3、4周时检测 - 2 0℃和 - 70℃保存的PBS组和Tris-HCl组以震荡法和震荡 +离心法洗提的GCF样本中EA的活性。结果 :不同洗提方法间洗提效果的差异具有显著性 (0 .0 1

0 .5 )。在相同时间内 ,PBS组或Tris -HCl组 - 2 0℃保存的GCF样本EA活性与- 70℃者间的差异无显著性 (P >0 .0 5 ) ,但 - 2 0℃保存者有略高于 - 70℃者的倾向 ,且 - 2 0℃比 - 70℃条件下保持EA活性的时间略长。结论 :将滤纸条上的GCF样本进行洗提时 ,采用“震荡 +离心法”优于“震荡法”。GCF样本的保存过程中 ,缓冲液的种类以及保存温度可影响GCF -EA的活性。以PBS作为缓冲液、- 2 0℃保存可能更利于其中EA活性的稳定。     

Subantimicrobial-dose doxycycline and cytokine-chemokine levels in gingival crevicular fluid

Background: The present randomized, double‐masked, placebo‐controlled, parallel‐arm study examines the impact of adjunctive subantimicrobial‐dose doxycycline (SDD) on the local inflammatory response through cytokine and chemokine levels in gingival crevicular fluid (GCF) samples from patients with chronic periodontitis. Methods: Forty‐six patients with chronic periodontitis received scaling and root planing with or without adjunctive SDD. GCF samples were collected and clinical parameters including probing depth, clinical attachment level, gingival index, and plaque index were recorded every 3 months for 12 months. GCF tumor necrosis factor‐α, interleukin (IL)‐6, IL‐4, IL‐10, IL‐13, IL‐17, macrophage inhibitory protein 1α, macrophage inhibitory protein 1β, monocyte chemoattractant protein 1, and regulated on activated normal T‐cell expressed and secreted protein levels were determined by xMAP multiplex immunoassay. Results: Significant improvements were observed in all clinical parameters in both groups over 12 months (P <0.0125), whereas the SDD group showed significantly better reduction in gingival index, probing depth, and gain in clinical attachment compared to the placebo group (P <0.05). Decrease in IL‐6 in the SDD group was significantly higher compared to the placebo group at 6 and 9 months in deep pockets (P <0.05), whereas tumor necrosis factor‐α was significantly reduced in moderately deep pockets (P <0.05). SDD resulted in a stable IL‐4 and IL‐10 response while reducing the monocyte chemoattractant protein 1 levels at 3 months (P <0.05). Conclusions: These results show that SDD, as an adjunct to non‐surgical periodontal therapy, stabilizes the inflammatory response by promoting the suppression of proinflammatory cytokines and increasing the anti‐inflammatory cytokines. The chemokine activity would account for the regulation of the inflammatory response to SDD therapy.