Gingival Crevicular Fluid Elastase Research Articles

Methodological evaluation of gingival crevicular fluid volume and neutrophil elastase levels: sequential sampling, length of sampling time and two different sampling methods

Objectives: The mechanisms underlying the formation and composition of gingival crevicular fluid (GCF) and its flow into and from periodontal pockets are not understood very well. The aim of this study was to evaluate the length of sampling time and sequential sampling of GCF neutrophil elastase (NE) enzyme levels by using intracrevicular and orifice methods.Material and methods: Twenty adults (mean age of 41.8 years, ranged 31–60 years, 18 males and 2 females) with chronic periodontitis were enrolled and all completed the 3-d study. GCF was collected by both intracrevicular and intrasulcular methods, 720 samples of GCF were collected. In first, second and third day, the length of sampling time in seconds (s) and order were ‘5- 10-30-s’; ‘10- 30- 5-s’ and ‘30- 5- 10-s,’ respectively. GCF elastase levels were determined by hydrolysis of neutrophil specific substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide.Results: NE activity (µU) and NE activity/volume (µU/µl) were significantly different for order of sampling (p < .05), but not for the length of sampling time (p>.05).Conclusions: Within the limits of this study, the choice of sampling technique in GCF-profile studies seems to be a critical decision as it has the potential to affect the GCF volume and NE activity.

Neutrophil elastase levels in the gingival crevicular fluid following hyaluronan gel application in the treatment of chronic periodontitis: A randomized split-mouth study.

Neutrophils are the predominant leukocytes in the periodontium, which prevent infection from periodontal pathogens and subsequent tissue destruction. A potentially destructive role has been elucidated, especially due to elastase enzyme. Controlling its levels might be crucial in minimizing the tissue destruction. Hyaluronan, known to inhibit the release of this enzyme from neutrophils, might be a viable option to treat chronic periodontitis. The aim of this study is to evaluate and compare the effects of 0.2% hyaluronan gel adjunctive to scaling and root planing on the levels of elastase in gingival crevicular fluid (GCF). This split-mouth study included eighty (forty experimental and forty control) sites from twenty patients representing both sexes. GCF samples were collected from all the eighty sites; simultaneously, clinical periodontal parameters were recorded. Enzyme-linked immunosorbent assay was used to determine the levels of elastase at baseline and 6 weeks after therapy, following mechanical debridement and subsequent subgingival placement of the experimental drug. With the aid of statistical software (SPSS Version 13), Student's t-test and Pearson's correlation test were performed. There was a mean reduction in the elastase levels from baseline to 6 weeks after therapy in the experimental group. However, the difference between the groups was not statistically significant. Adjunctive use of hyaluronan following mechanical debridement resulted in comparable reduction in the elastase levels, suggesting that this substance has an inhibitory effect on elastase, and subsequent tissue destruction. Further long-term studies are mandatory to validate the results of this study.

The effects of stress on periodontal treatment: a longitudinal investigation using clinical and biological markers

To investigate the effects of psychosocial stress on the outcome of non-surgical periodontal treatment (NPT). Patients were categorized as stressed or unstressed, and the degree of stress was measured. One deep bleeding and one deep non-bleeding site ≥6 mm were selected in each patient for detailed investigation, and the clinical parameters were recorded before and at 6 months after NPT. Elastase and C-terminal teleopeptide of type I collagen (ICTP) were measured in gingival crevicular fluid (GCF) samples at both intervals. The baseline, clinical parameters and biological markers were similar in both stressed and unstressed groups, other than for GCF elastase levels, which were significantly higher in the stressed group of patients (p < 0.05). The effect of stress on the changes for clinical measurements and elastase levels in GCF was statistically significant for deep bleeding sites, with the response to treatment being poorer in the stressed group. The effects of smoking and the degree of stress were not statistically significant for any of the clinical or biological parameters (p > 0.05). Patients under psychosocial stress had a poorer outcome following NPT. The assessment of psychosocial stress may be valuable in the holistic management of periodontal disease.

Influence of Combinations of Bacteria on the Levels of Prostaglandin E2, Interleukin‐1β, and Granulocyte Elastase in Gingival Crevicular Fluid and on the Severity of Periodontal Disease

The aim of this study was to investigate the simultaneous presence of periodontal microbiota on inflammatory markers in gingival crevicular fluid from individuals with periodontal diseases. A total of 82 individuals with periodontal disease (mean age: 54.3 +/- 3.0 years) and 31 periodontally healthy individuals (mean age: 53.2 +/- 3.0 years) were randomly chosen and underwent clinical oral examinations in 2003 with the determination of the dental plaque index (PI), gingival index (GI), and periodontal probing depth (PD). The simultaneous presence of polymerase chain reaction (PCR)-assessed periodontal bacteria, levels of prostaglandin E(2) (PGE(2)), granulocyte elastase, interleukin 1-beta (IL-1beta), and total protein concentration were determined from the pockets. Marginal bone height percent was measured on x-rays. Analysis of variance and chi(2) tests were used to analyze the results. In sites with Tannerella forsythensis, levels of PGE(2) (pg/site), granulocyte elastase (monoclonal antibodies (mAbs)/site), and total protein (mg/ml) were significantly higher than in sites without T. forsythensis (P <0.05, P <0.01, and P <0.05, respectively). Those with periodontal disease with simultaneous presence of T. forsythensis and Porphyromonas gingivalis, or T. forsythensis and Prevotella nigrescens, showed significantly higher PI and GI, deeper PD, more loss of attachment, and more release of PGE(2) and granulocyte elastase than did periodontitis patients without these bacteria. The simultaneous presence of T. forsythensis and P. gingivalis, or T. forsythensis and P. nigrescens, seemed to promote the release of subgingival inflammatory mediators and seemed to be associated with more severe periodontal disease.

Granulocyte elastase in gingival crevicular fluid: improved monitoring of the site-specific response to treatment in patients with destructive periodontitis.

In 13 patients with severe destructive periodontitis, the response to periodontal therapy was estimated by granulocyte elastase level in gingival crevicular fluid (GCF). 62 sites were classified according to changes of probing depths (PD) and quantitative bone height (BH%) before and after 5-year regular maintenance treatment: (i) 17 consistently healthy sites with no changes of PD and BH%; (ii) 6 initially healthy sites with deterioration in PD and BH%; (iii) 14 diseased sites with improvement in PD and BH%; (iv) 25 diseased sites with no improvement in PD and BH%. GCF was collected by an intracrevicular washing system. The released elastase in the supernatants (EA-S) and the cell-bound elastase in the pellets (EA-P) were determined with a low molecular weight substrate specific for granulocyte elastase. The ratio of EA-S and EA-P (S/P-ratio) was used as a relative measure of elastase released by the granulocytes present. The sites classified as diseased with no improvement or initially healthy but deteriorating, had significantly higher EA-S, EA-P and S/P-ratios than the consistently healthy sites or diseased but improving sites (p < 0.01). Both EA-S and S/P-ratio showed strongly positive correlations with the current levels of gingival inflammation and periodontal destruction (p < 0.001). The present study suggests that increased elastase level is associated with disease progression, and may be used to monitor the response to longitudinal maintenance therapy.

Relationship between periodontal disease status and combination of biochemical assays of gingival crevicular fluid

Currently, no biochemical assay involving gingival crevicular fluid is utilized routinely as a screening test for periodontal disease. The objective of the present study was to evaluate the potential of gingival crevicular fluid assay as a screening methodology. The subject population was comprised of 27 volunteers. Nine participants were classified as 'subject with periodontal destruction' (SPD) exhibiting at least one site with pocket depth and attachment loss>3.5 mm, whereas the remaining individuals were categorized as 'subject with minimal periodontal destruction' (SMD). Gingival crevicular fluid was collected from fixed sites via a standardized method. Biochemical assays of 12 substances (hemoglobin, albumin, transferrin, alpha(1)-antitrypsin, fibronectin, IgA, IgG, IgM, lactoferrin, myeloperoxidase and neutrophil elastase) were conducted at a commercial laboratory. Power transformation of total quantities in gingival crevicular fluid was performed for statistical analysis. Relationships between total quantity of each substance and periodontal disease status were unclear. Logistic regression analysis yielded six predictive models, which consisted of substance pairs: neutrophil elastase/IgA, neutrophil elastase/hemoglobin, neutrophil elastase/alpha(1)-antitrypsin and neutrophil elastase/IgG, and IgA/albumin and IgA/transferrin (p<0.05). Regression lines for SPD and SMD on a scattergram of IgA and neutrophil elastase were nearly parallel within the range of amounts in gingival crevicular fluid. The predictive model derived from both substances afforded sensitivity and specificity of 88% and 94%, respectively. These results indicated that the combination of IgA and neutrophil elastase in gingival crevicular fluid may be crucial for prediction of periodontal disease status. Furthermore, these data suggested that biochemical assays employing both substances in gingival crevicular fluid may provide a satisfactory screening test for periodontal disease.

Crevicular fluid elastase levels in relation to periodontitis and metabolic control of diabetes.

The purpose of this study was to investigate the associations between gingival crevicular fluid (GCF) elastase levels, clinical measures of periodontal status, and metabolic control of diabetes in insulin dependent (type 1) diabetes (IDDM) and non-insulin dependent (type 2) diabetes (NIDDM) patients. Sixty patients were recruited from the Diabetes Center at the University of California in San Francisco. Thirty subjects were type 1 diabetics and 30 subjects were type 2 diabetics. Metabolic control was evaluated by glycosylatted hemoglobin (HbA1c) levels. Demographic information was obtained using a structured interview with the subjects. Clinical measurements and GCF samples were taken from the mesio-buccal surfaces of 2 premolars and 2 molars from the most diseased sextant. GCF elastase was determined by measurement of p-Nitroanalide resulting from hydrolysis of elastase specific peptide. Crevicular fluid elastase levels were significantly correlated with gingival index, bleeding index, probing depth and attachment level in both type 1 and type 2 diabetes groups (0.01 <p < 0.001). HbA1c levels were not correlated with clinical measurements and GCF elastase. The results suggest that GCF elastase. age and smoking are risk indicators for periodontitis in patients with diabetes mellitus, and periodontal status is not associated with the duration and metabolic control of diabetes.

A 2-year longitudinal study of elastase in human gingival crevicular fluid and periodontal attachment loss.

The purpose of this study is to determine whether gingival crevicular fluid (GCF) elastase total activity (TA) and concentration (EC) correlate with and predict progressive attachment loss (AL). 75 previously untreated patients with moderate periodontitis were recruited. GCF was collected from 16 molar and premolar mesiobuccal sites and probing attachment loss (PAL), probing depth (PPD), gingival index (GI), gingival bleeding index (GBI) and plaque index (Pl.I) were measured. PAL and PPD were measured with an electronic, constant pressure probe. Patients were given basic periodontal treatment prior to baseline when the above procedures were repeated. In addition, carefully localised radiographs were taken of the test teeth and repeated annually. Patients were seen at 3 months intervals to 2 years and the procedures were repeated. 119 AL sites were detected and 89 of these were rapid AL sites (RAL) and 30 were gradual AL sites (GAL). Elastase levels (TA & EC) at RAL sites were significantly higher (p < or = 0.0001) than paired control sites in the same patient at both the attachment loss time (ALT) and the prediction time (PT). The mean levels (TA & EC) over the study period at GAL sites were significantly higher (p < or = 0.0001) than paired control sites in the same patient. Using a critical value (CV) of 125 micronsU/30 s (TA) and 400 micronsU/micronsL (EC) in 2 x 2 contingency tables showed a sensitivity of 100% and specificity of 99.95% (TA) and a sensitivity of 100% and specificity of 99.91% (EC) at the PT with very similar values at the ALT. Patient level comparisons showed that the mean elastase levels (TA & EC) were significantly higher (p < or = 0.0001) at RAL and GAL sites than non-attachment loss (NAL) sites in AL patients and that the mean levels were significantly higher (p < or = 0.0001) in AL patients than NAL patients. All these results indicate that these CVs for GCF elastase activity may serve as a predictors of future attachment loss.

Variations in crevicular fluid elastase levels in periodontitis patients on long-term maintenance.

Granulocyte elastase was determined in the gingival crevicular fluid (GCF) of 18 periodontitis patients. They initially had similar severity of disease but had responded differently to 5-yr maintenance, 13 responders and 5 non-responders. A total of 102 sites were investigated and categorized as: i) consistently healthy, ii) healthy after treatment, iii) gingivitis, and iv) periodontitis, according to clinical criteria. GCF elastase activity was determined with a granulocyte-specific substrate. The sites from non-responders had consistently higher elastase levels than the corresponding category of sites from responders, despite similar gingival inflammation and periodontal destruction, with the exception of consistently healthy sites. Within the non-responders, the periodontitis sites had higher elastase levels than the gingivitis sites commensurate with probing depth, while no difference existed between gingivitis sites and sites healthy after treatment, despite a difference in probing depth. In contrast, in the responders similar elastase levels were found at the periodontitis sites and gingivitis sites despite difference in probing depth, while both diseased sites had higher elastase levels than the sites healthy after treatment, commensurate with probing depth. This study suggests that increased granulocyte-specific elastase levels in GCF may serve as a diagnostic marker for refractory periodontitis patients.

Longitudinal Evaluation of Elastase as a Marker for the Progression of Periodontitis

To determine whether elastase levels in gingival crevicular fluid (GCF) could serve as a marker for the progression of periodontitis, we monitored GCF elastase and periodontal status in selected sites in 32 periodontally healthy volunteers and 31 periodontitis patients at intervals over a 6-month period. Clinical measurements included plaque index, gingival index, bleeding on probing, suppuration, probing depth, clinical attachment level, and relative attachment level measured with an automated disk probe. GCF elastase, detected by reaction with a fluorescent substrate, was assessed visually against fluorescence standards and quantitatively with a fluorometer. Bone loss was detected by subtraction radiography of standardized vertical bite-wing radiographs at baseline and 6 months. Mean visual elastase scores (VES) and quantitative elastase measurements were significantly higher (P < 0.001) in sites from periodontitis patients than in sites from healthy volunteers. When bone loss was used as the criterion for disease progression, significantly higher (P < 0.001) visual and quantitative GCF elastase levels were found at progressing sites than in nonprogressing sites in the periodontitis patients. The odds ratios (OR) for the event of developing bone loss with positive 4-minute and 8-minute VES tests were 4.2 (P < 0.001) and 7.4 (P < 0.001), respectively. When corrected for the tendency of progressing sites to be clustered within a subpopulation of patients, the OR for developing bone loss with the 4-minute and 8-minute VES tests were 3.1 (P < 0.007) and 4.9 (P < 0.001), respectively. These data indicate that sites with high levels of elastase are at significantly greater risk for progressive bone loss as assessed by digital subtraction radiography.

Granulocyte elastase in gingival crevicular fluid. A possible discriminator between gingivitis and periodontitis.

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.

Influence of oral hygiene on elastase concentration of gingival crevicular fluid.

PMN elastase concentration of gingival crevicular fluid (GCF) was investigated in 11 healthy volunteers before professional tooth cleaning and after a 5-week period of intensive oral hygiene. GCF was collected using filter paper strips and the enzyme concentration was evaluated by the ELISA-technique. Intensive daily oral hygiene led to a considerable improvement in the clinical indices and to a reduction in the concentration of elastase in GCF. Despite the changes in the oral hygiene status, functional elastase was still present in the samples at the end of the experiment. This could mean that even at clinically healthy sites there is a lack of alpha-1-proteinase inhibitor, the major serum protein inactivating PMN elastase.

Elastase as an indicator of periodontal disease progression.

THIS STUDY SOUGHT TO EVALUATE the ability of gingival crevicular fluid (GCF) elastase to predict attachment and bone loss in human periodontitis. Thirty subjects who were medically healthy and had a history of progressive periodontitis were studied with an automated probe. Five sites in each patient were monitored bi-monthly for a 6-month period for attachment loss. Subtraction radiography was utilized at the beginning and end of the study to monitor bone loss. GCF elastase was measured at 0 month and then bi-monthly by collecting GCF on paper strips impregnated with PMN leukocyte elastase substrate inserted into the gingival crevice for 15 seconds. After 8 minutes of reaction time, the strips were scored relative to fluorescent standards in an ultraviolet view box. Strips were then eluted in methanol and total elastase measured by spectrofluorometry. Total elastase was significantly higher in sites demonstrating progressive attachment loss than in inactive sites (2.81 +/- .29 versus 2.03 +/- .07, P less than 0.0005) and sites demonstrating bone loss (2.32 +/- .17 versus 2.01 +/- .08 P less than 0.05). When considering the joint presence of bone loss and attachment loss of 1.0 mm or greater in the 6-month period following a visual elastase kit score of 2 or greater, the test kit shows a sensitivity and specificity of 82% and 66%, respectively. This study demonstrated that GCF elastase levels are significantly higher in sites demonstrating progressive periodontal attachment and bone loss assessed 6 months later and may serve as a predictor of future bone and attachment loss.