Relationships among gingival crevicular fluid biomarkers, clinical parameters of periodontal disease, and the subgingival microbiota

Abstract

Background: The objectives of this study were to measure levels of gingival crevicular fluid (GCF) biomarkers and subgingival bacterial species in periodontally healthy subjects and subjects with periodontitis to explore the relationships among these biomarkers, the subgingival microbiota, and the clinical parameters of periodontal disease.

Methods: Clinical periodontal parameters were measured at six sites per tooth in 20 subjects with periodontitis and 20 periodontally healthy subjects. GCF and subgingival plaque samples were obtained from the mesio-buccal aspect of every tooth. GCF levels of interleukin (IL)-1beta and IL-8 and matrix metalloproteinase 8 were measured using checkerboard immunoblotting, and the levels of 40 bacterial taxa were quantified using checkerboard DNA-DNA hybridization. A subset of "clinically healthy" sites from each group was analyzed separately. The significance of the differences between groups was determined using the unpaired t test or the Mann-Whitney test. Correlations among immunologic, microbiologic, and clinical data were determined using the Spearman rank correlation coefficient.

Results: There were positive correlations among mean clinical parameters, mean levels of the three biomarkers, and the proportions of orange and red complex species (P <0.05). Clinically healthy sites from subjects with periodontitis had higher levels of IL-1beta and IL-8 and higher proportions of orange and red complex species (P <0.05) than clinically healthy sites from periodontally healthy subjects. Red complex species were positively associated with the expression of all biomarkers (P <0.05), whereas purple and yellow complex species had negative correlations with IL-1beta and IL-8 (P <0.05).

Conclusions: Clinically healthy sites from subjects with periodontitis have higher levels of GCF biomarkers and periodontal pathogens than clinically healthy sites from periodontally healthy subjects. Different microbial complexes demonstrated distinct associations with specific GCF biomarkers.

Fig. 1

Mean counts (× 10⁵) of the 40 test species from up to 28 subgingival biofilm samples from each periodontally healthy and chronic periodontitis subject. Mean counts of each species were computed by averaging the data for each subject, and then averaging across subjects in each group separately. Significance of differences for each species between clinical groups was sought using the Mann-Whitney test; *p<0.05, ^†p<0.01, ^‡p<0.001 and adjusted for 40 comparisons. The species were ordered and grouped according to the complexes described by Socransky et al. (1998).

Fig. 2

Tukey box plots of the levels of IL-1β, IL-8, MMP-8 and GCF volume at “clinically healthy” sites (i.e. sites with pocket depth equal to 2 or 3 mm, no loss of attachment, no BOP, and no redness) in periodontally healthy and periodontitis subjects. The box indicates the lower and upper quartiles, the whiskers the 10^th and 90^th percentiles and the horizontal line in each box is the median. The circles indicate “outlier” values. The levels of each inflammatory mediator and the volume of GCF for this site category were averaged within a subject and then median determined across subjects in the 2 clinical groups separately. Significance of difference between the two groups was determined using the Mann-Whitney test. The numbers in parentheses represent the number of sites analyzed in each group.

Fig. 3

Bilateral bar chart of the mean percentage of DNA probe count for the 7 subgingival microbial complexes described by Socransky et al. (1998) in periodontally healthy and periodontitis subjects. Mean % DNA probe count of each complex was computed by averaging the data for each subject, and then averaging across subjects in each group separately. The bars indicate the means and the whiskers the SEM. Significance of differences between clinical groups (*p<0.05, ^†p<0.01, ^‡p<0.001) was sought using the unpaired t-test or the Mann-Whitney test depending on the result of the D’Agostino-Pearson normality test. Insert: percentile plot of mean % DNA probe count of the Red complex species for periodontally healthy (white circles) and periodontitis (red circles) subjects. The y-axis represents the mean % DNA probe count and the x-axis represents the percentiles. Each circle depicts the mean value for an individual subject and the horizontal lines indicate the median values.

Fig. 4

Grid-plot of the Spearman rank correlation coefficients among the mean proportions of the subgingival microbial complexes and the mean levels of GCF biomarkers in periodontally healthy and periodontitis subjects (*p<0.05, ^†p<0.01, ^‡p<0.001).