Evaluation of a novel point-of-care test for active matrix metalloproteinase-8: agreement between qualitative and quantitative measurements and relation to periodontal inflammation.

BACKGROUND AND OBJECTIVES: Severe periodontitis affects about 10% of the world population. In addition, associations between periodontitis and systemic diseases exist. Therefore, the diagnosis should be made quickly and at an early stage. Matrix metalloproteinase-8 (MMP-8) is the most prominent collagenase found in inflamed periodontal tissues. Its active form (aMMP-8) is increasingly used as a diagnostic biomarker. Aim of the present study is to evaluate the diagnostic accuracy of a novel aMMP-8 point-of-care (POC) test in comparison to the standard laboratory test to diagnose the disease rapidly and reliably. MATERIAL AND METHODS: In a prospective, mono-center, double-blinded, case-control study, participants with healthy gums (n = 35), gingivitis (n = 60) and periodontitis (n = 35) were investigated before and after therapy. Beside clinical variables for plaque and inflammation, aMMP-8 concentrations were determined in oral rinsing specimens by the enzyme-linked immunosorbent assay (ELISA) and by POC. Positive and negative percent agreements with their exact one-sided lower 95% confidence limits were calculated. RESULTS: Of 130 participants, 111 finished the study. Overall, positive percent agreements were 75.8% (57.7-88.9) before treatment and 73.7% (56.9-86.6) after treatment. Negative percent agreements were 92.8% (85.7-97.0) before and 93.3% (85.1-97.8) after treatment. Positive test results (POC and ELISA) ranged from 5.7% to 8.6% in healthy patients, 25.0-29.8% in patients with gingivitis and 40.0-48.1% in patients with periodontitis. Patients who had positive aMMP-8 test results (POC) showed higher scores for plaque and inflammation. CONCLUSIONS: The novel POC test to detect aMMP-8 has proved to agree strongly with the standard method, ELISA. The test can be recommended to screen patients at risk for periodontitis in dental offices, at the general practitioner and at specialists for associated diseases.

Full-mouth profile of active MMP-8 in periodontitis patients.

BACKGROUND AND OBJECTIVE: MMP-8 in gingival crevicular fluid is considered as a protease with high destructive potential because of its ability to degrade collagen in periodontitis-affected patients. The aim of this study was to investigate whether there was a relationship between clinical diagnostic parameters and the concentration of active MMP-8 (aMMP-8) in gingival crevicular fluid in a site-level full-mouth analysis. Based on these data, the prognostic value of aMMP-8 levels in relation to pocket depth may be evaluated. MATERIAL AND METHODS: Clinical measurements of pocket depth, bleeding on probing (BOP), plaque index (PlI) and gingival index (GI), as well as samples of gingival crevicular fluid, were obtained from four sites of each tooth of nine healthy female patients with chronic generalized periodontitis. The aMMP-8 concentration in gingival crevicular fluid was quantified by ELISA using specific monoclonal antibodies. Multiple linear regression models for the single measures of aMMP-8 and pocket depth were calculated with GI and BOP as additional variables. RESULTS: Between 92 and 112 recordings were obtained for each parameter in each patient. Mean values of between 31.5 and 88.8% were calculated for pocket depths of ≥ 4 mm. Mean pocket depths ranged from 3.11 to 4.73 mm, the mean BOP values ranged from 34.0 to 96.7% and the mean full-mouth gingival crevicular fluid aMMP-8 concentration ranged from 3.2 to 23.7 ng/mL. CONCLUSION: In this sample of female periodontitis patients, a broad range of intra-individual and interindividual aMMP-8 values was found. Although the explained variance was rather weak, a statistically significant relationship between aMMP-8 and pocket depth was proven.

Oral rinse MMP-8 point-of-care immuno test identifies patients with strong periodontal inflammatory burden.

OBJECTIVE: To determine whether oral rinse matrix metalloproteinase (MMP)-8 levels, measured by three different methods, tissue inhibitor of matrix metalloprotease-1 (TIMP-1) levels and elastase activity differentiate subjects with different periodontal condition; and second, to find out if MMP-8 levels were comparable among the methods used. METHODS: MMP-8 levels were analysed with an immunofluorometric method (IFMA), dentoELISA and commercial ELISA. Also TIMP-1 levels and elastase activity were measured. For statistical analysis 214 study subjects were categorized into four groups, specified by the presence and number of moderate (4-5mm) and deep (≥6mm) periodontal pockets, and bleeding on probing percentage. RESULTS: MMP-8 levels especially measured by dentoELISA and adjusted to the number of teeth per subject differentiated the study group with strong periodontal inflammatory burden from groups with lower levels. This was also verified with receiver operating characteristic (ROC) analysis. Elastase activity associated with higher IFMA and dentoELISA MMP-8 levels. IFMA MMP-8/TIMP and dentoELISA MMP-8/TIMP-1 tended to be higher with the increasing level of periodontal inflammatory burden. TIMP-1 levels decreased with increasing age. CONCLUSIONS: Oral rinse MMP-8 together with TIMP-1 analysis may have potential in complementary periodontal diagnostics. dentoELISA can be applied in quantitative oral rinse chair side biomarker diagnostics.

Detection of gingival crevicular fluid MMP-8 levels with different laboratory and chair-side methods.

OBJECTIVE: The aim of the study was to compare four methods for gingival crevicular fluid (GCF) matrix metalloproteinase (MMP)-8 detection. METHODS: Matrix metalloproteinase-8 levels from 20 GCF samples from two periodontally healthy subjects, 18 samples from two patients with gingivitis and 45 samples from six patients with moderate to severe periodontitis, altogether 83 samples, were analysed using (1) a time-resolved immunofluorometric assay (IFMA), (2) an MMP-8 specific chair-side dip-stick test, (3) a dentoAnalyzer device and (4) the Amersham ELISA kit. Western immunoblot using same monoclonal anti-MMP-8 as in IFMA and dentoAnalyzer was used to identify molecular forms of MMP-8 in GCFs. RESULTS: Correlation between IFMA and dentoAnalyzer results calculated with Spearman's correlation coefficient was 0.95 (P = 0.01). The chair-side dip-stick test results were well in line with these assays. Periodontitis sites with unstable characteristics were differentiated with these methods. The Amersham ELISA results were not in line with the findings by other methods. CONCLUSIONS: Immunofluorometric assay and dentoAnalyzer can detect MMP-8 from GCF samples and these methods are comparable. Using Western immunoblot, it was confirmed that IFMA and dentoAnalyzer can detect activated 55 kDa MMP-8 species especially in periodontitis-affected GCF. dentoAnalyzer is among the first quantitative MMP-8 chair-side testing devices in periodontal and peri-implant diagnostics and research.

Effect of N-chlorotaurine mouth rinses on plaque regrowth and plaque vitality.

The purpose of this 4-day plaque regrowth study was to assess the effect of N-chlorotaurine (NCT) mouth rinses on plaque inhibition and plaque vitality. Eighty volunteers participated in this investigator-blind, randomized, clinical controlled study in parallel groups. No oral hygiene was permitted except rinsing with a 2% or 3% NCT mouth rinse, a positive or a negative control. Primary parameters were the plaque index (Silness and Löe, Acta Odontol Scand, 22:121-135, 1964) and plaque vitality (Netuschil et al., J Clin Periodontol, 16:484-488, 1989) after the final rinse. In addition, another plaque index (Turesky et al., J Periodontol, 41:41-43, 1970), plaque area, and bleeding on probing were recorded. All parameters were taken at baseline and day 5. U test was applied on a 5% error level. No differences in plaque inhibition were found between the two NCT formulations and the negative control. However, a statistically significant reduction of plaque vitality compared to the negative and positive control was observed. Discoloration of the tongue and unpleasant taste were recorded in participants in the NCT groups. NCT mouth rinses did not inhibit plaque regrowth, but they did reduce the vitality of plaque bacteria. Methods of prolonging the substantivity of the NCT mouth rinses should be investigated to enhance the antibacterial properties of these formulations.

Evaluation of immunoassay-based MMP-8 detection in gingival crevicular fluid on a point-of-care platform.

A novel immunology-based point-of-care test has been designed to assess the activated form of matrix metalloproteinase-8 (aMMP-8) for diagnosis and monitoring of periodontal diseases. The test has been automated using an analyzer, which quantitatively measures aMMP-8 in 18 min in gingival crevicular fluid (GCF). Fluid samples were collected from healthy, gingivitis-, and periodontitis-affected teeth. The test results from the analyzer were compared with quantitative aMMP-8 immunofluorometric assay (IFMA) and in-house enzyme-linked immunosorbent assay (ELISA) as well as with the periodontal state. Preliminary results of analyzer measurements of these 34 clinical samples showed a good agreement with the results from IFMA and in-house ELISA and with the clinical picture.

Rapid quantitative chairside test for active MMP-8 in gingival crevicular fluid: first clinical data.

In a first pilot field study 64 gingival crevicular fluid (GCF) samples were collected from patients of dental practitioners. The dentists (one orthodontist one periodontist, and one general practitioner) were asked to monitor the respective clinical status of the sites of sampling and to collect, if possible, sulcus fluid samples from healthy as well as affected sites from the same patient. The concentration of activated matrix metalloproteinase-8 (aMMP-8) in the GCF was recorded using a set of monoclonal antibodies and a novel DentoAnalyzer. From all three dental offices the distribution of the aMMP-8 values in GCF showed a congruent pattern, where healthy and periodontitis-affected inflamed sites were clearly disparate.

Antibody-mediated polysome precipitation as a method for the size determination of viral mRNA species: viral envelope glycoprotein mRNA of avian sarcoma viruses.

A method was developed that allows the in situ isolation of viral mRNA, i.e. from polysomes of infected cells. This was achieved by precipitating intact polysomes via their nascent virus polypeptides using virus-specific antibodies. As a model, antibodies to the major envelope glycoprotein (gp85) of avian sarcoma viruses were employed to precipitate those polysomes from infected cells which synthesized the corresponding p70 virus glycoprotein precursor. Normal immunoglobulin and polysomes from uninfected chicken embryo fibroblasts served as controls. The radioactivity labelled mRNA from antibody-precipitated polysomes could subsequently be extracted and characterized for size. It was found that avian sarcoma virus gp85 envelope glycoprotein is predominantly synthesized by a 22--28 s viral mRNA. In addition, minor amounts of gp85-specific mRNAs of 16--21 s and 28--35 s could be demonstrated. The data indicate the presence in polysomes of viral mRNA species coding for i) gp85 only (16--21 s RNA), ii) gp85 and pp60src protein from the adjacent src-gene (22--28 s RNA), and iii) a large viral precursor protein (28--35 s RNA).

A pilot study of confocal laser scanning microscopy for the assessment of undisturbed dental plaque vitality and topography.

Confocal microscopy and vital fluorescence techniques were combined for the first time to investigate ex vivo human dental plaque. The vital fluorescence technique used discriminates vital from dead cells, while confocal laser scanning microscopy allows the optical sectioning of undisturbed biofilms leaving the samples intact during analysis. The concomitant use of both methods made an examination of the three-dimensional architecture of dental plaque possible. The topography of plaque biofilms that were allowed to accumulate in situ on glass and enamel was recorded. The distribution of plaque microflora vitality as well as its accumulation varied according to plaque age. A plaque thickness of up to 8, 35 and 45 microm was estimated ex vivo on enamel after 1, 2 and 3 days, respectively. Young and sparse plaque biofilms consisted mainly of dead material. Vital bacteria were observed on top of this dead layers.

An approach to differentiate between antibacterial and antiadhesive effects of mouthrinses in vivo.

An experimental set-up allowing differentiation in vivo between antibacterial and antiadhesive properties of mouthrinses is described. The percentage of vital bacteria (= microbial vitality) and the bacterial counts were microscopically evaluated in saliva and in supragingival dental plaque both collected simultaneously at various times during de novo plaque formation. In a cross-over design, 12 healthy participants refrained from all oral hygiene for four separate periods of 2 x 4 h and 2 x 72 h after having rinsed with either an amine fluoride/stannous fluoride solution (Meridol) or 0.9% NaCl (placebo). Stimulated whole saliva was collected before and after the rinse. Together with whole-saliva samples, representative 4, 24 and 72-h-old plaque samples were separately taken from defined vestibular tooth surfaces that had been either exposed to the mouthrinse (unprotected sites) or temporarily covered with inert plastic films (protected sites) during rinsing. The pooled plaque and saliva were stained with fluorescent dyes to differentiate vital from dead micro-organisms which permitted the estimation of the percentages of vital bacteria. The total bacterial counts were quantified under the darkfield microscope. The Wilcoxon test was used for selected pairwise comparisons (alpha = 0.05). The percentage of vital bacteria in saliva fell significantly from 80-95% to about 50-60% as a result of the antibacterial activity of the test solution. These baseline values and those found in the presence of 4 and 24-h-old plaque were frequently lower than those recorded after the placebo rinse. In comparison to the placebo, microbial vitality was significantly reduced in early supragingival plaque formed on unprotected sites after applying the test solution. The similar total bacterial counts in 4-h-old plaque recorded after the use of the test solution on the unprotected and the protected areas did not point to an antiadhesive effect of the agent. It is concluded that this new experimental set-up allows decoding of the mode of action of a mouthrinse.

Spatial distribution of vital and dead microorganisms in dental biofilms.

To examine the spatial structure of dental biofilms a vital fluorescence technique was combined with optical analysis of sections in a confocal laser scanning microscope (CLSM). Enamel slaps were worn in intraoral splints by three volunteers for five days to accumulate smooth-surface plaque. After vital staining with fluorescein diacetate and ethidium bromide the specimens were processed for CLSM examination. Optical sections 1 microm apart were analysed in the z-axis of these dental biofilms. One of the films was 15 microm high, sparse and showed low vitality, i.e. <16%, while the others were taller (25 and 31 microm) and more vital, i.e. up to 30 and 69%, respectively. In all instances the bacterial vitality increased from the enamel surface to the central part of the plaque and decreased again in the outer parts of the biofilm. The spatial arrangement of the microorganisms in the biofilm showed voids outlined by layers of vital bacteria, which themselves were packed in layers of dead material.

Microbial accumulation and vitality on different restorative materials.

A new technique using standardized test facings was designed to evaluate interdental plaque accumulation on different restoration materials. In 10 volunteers, a total of 40 samples, including enamel, two different ceramics and a bonding composite, were inserted one by one into a precision attachment in an experimental inlay bordering on either the lower second premolar or the lower first molar. Bacterial accumulation on each approximal specimen was allowed to mature for 3 d. Following microbiological processing of the plaque samples, total bacterial counts, colony forming units and the bacterial vitality were determined. The results revealed different accumulation rate patterns. Both ceramics accumulated less plaque with a reduced vitality compared to enamel, while the bonding composite showed no significant differences compared to the natural tooth substance. No significant differences were detected when comparing the two examined ceramics.

The influence of a 0.2% chlorhexidine mouthrinse on plaque regrowth in orthodontic patients. A randomized prospective study. Part I: clinical parameters.

In a prospective plaque regrowth study focusing on oral hygiene during fixed appliance therapy 12 adolescent patients (mean age 14.1 +/- 1.5 years) were evaluated twice over 2-day test periods. In the randomized, double-blind study the influence of a 0.2% chlorhexidine (CHX) mouthrinse (Corsodyl) and a commercially available dentifrice supplementing fluoride (Odol-med-3) were compared intra- and interindividually in a crossover design with regard to plaque and gingivitis. Before starting the first test phase there was a 14-day preliminary phase for upgrading the oral hygiene. Between the 2 test phases was a 5-day "washout". On the last day of the second test phase the patients were asked to fill in a questionnaire concerning their experiences during the study. The 0.2% Corsodyl reduced the plaque index scores significantly (p < 0.001). The gingival index revealed a similar reduction (2nd day of test: p = 0.03). Until the 5th day of washout a clear-cut carryover effect of the chlorhexidine rinse on the gingival index was observed. Both the lower mean values of the 2 clinical parameters at the beginning of the test phases as compared with those at the beginning of the preliminary phase and the evaluation of the questionnaires indicated a possible Hawthorne effect. 0.2% Corsodyl may be employed as an adjunct to other preventive measures such as professional care and patient-oriented instruction on an intermittent basis in order to reduce the plaque-induced iatrogenic side effects and to enhance the efficacy of oral hygiene measures in connection with orthodontic therapy with fixed appliances.

The influence of a 0.2% chlorhexidine mouthrinse on plaque regrowth in orthodontic patients. A randomized prospective study. Part II: Bacteriological parameters.

In a prospective plaque regrowth study focusing on oral hygiene during fixed appliance therapy 12 adolescent patients (mean age 14.1 +/- 1.5 years) were evaluated twice over 2-day test periods. In the randomized, double-blind study the influences of a 0.2% chlorhexidine mouthrinse (Corsodyl, CHX) and a commercially available dentifrice supplementing fluoride (Odol-med-3) were compared intra- and interindividually in a crossover design with regard to the bacteriological parameters. The bacteriological parameters of vital fluorescence, bacteriological counts (BC), colony forming units (CFU), plating efficiency (PE) and mutans streptococci (MS) were related to the clinically assessed indices of plaque and gingivitis. All parameters analyzed demonstrated significant differences between the control and the test (chlorhexidine) group. Where-as the values of BC, CFU, and PE progressively increased in the control group from T0 to T2, these parameters distinctly decreased in the chlorhexidine group. All values of vital flora (VF) scored around 75% in the control group compared to values of 30% in the test group. BC, CFU und PE correlated significantly. The score of mutans streptococci persisted or increased in the controls whereas mutans streptococci approached 0 in the chlorhexidine group. Until the 5th day of washout a clear-cut carry over of the chlorhexidine rinse on mutans streptococci as well as on the gingival index was evident. Since dead microorganisms remain on the tooth surface and in order to maintain oral health, chlorhexidine application might advisedly be supplemented by mechanical plaque control.

Human dental plaque formation on plastic films. A quantitative SEM study.

The initial colonization of bacteria in previously clean teeth or artificial surfaces inserted in mouth has been reported to occur at various periods of time. Rönström et al., using light microscopy, saw that bacteria were present 10 seconds after prophylaxis. Bacterial culture studies have shown that microorganisms associated with the surface coating on tooth surfaces appeared within minutes after prophylaxis. Furthermore, Rönström et al. noted that the number of bacteria increased over a period of four hours. In contrast, ultrastructural investigations of early plaque have demonstrated bacterial colonization only within the first two hours of plaque development in a few samples obtained on a smooth surface and in most samples on a rough surface. However, microorganisms have been found regularly after four hours of plaque accumulation in subjects with healthy gingiva. The aim of the present investigation was to determine the earliest occurrence of bacterial colonization in situ and to observe the pattern of the initial formation of human dental plaque.