Assessment of craniofacial skeletal maturity is of great importance in orthodontic diagnosis and treatment planning. Traditional radiographic methods suffer from clinician subjectivity and low ...reproducibility. Recent biochemical methods, such as the use of gingival crevicular fluid (GCF) protein biomarkers involved in bone metabolism, have provided new opportunities to assess skeletal maturity. However, mass spectrometry (MS)-based GCF proteomic analysis still faces significant challenges, including the interference of high abundance proteins, laborious sample prefractionation and relatively limited coverage of GCF proteome. To improve GCF sample processing and further discover novel biomarkers, we herein developed a single-pot, solid-phase-enhanced sample-preparation (SP3)-based high-field asymmetric waveform ion mobility spectrometry (FAIMS)-MS protocol for deep quantitative analysis of the GCF proteome for skeletal maturity indicators. SP3 combined with FAIMS could minimize sample loss and eliminate tedious and time-consuming offline fractionation, thereby simplifying GCF sample preparation and improving analytical coverage and reproducibility of the GCF proteome. A total of 5407 proteins were identified in GCF samples from prepubertal and circumpubertal groups, representing the largest dataset of human GCF proteome to date. Compared to the prepubertal group, 61 proteins were differentially expressed (31 up-regulated, 30 down-regulated) in the circumpubertal group. The six-protein marker panel, including ATP5D, CLTA, CLTB, DNM2, HSPA8 and NCK1, showed great potential to predict the circumpubertal stage (ROC-AUC 0.937), which provided new insights into skeletal maturity assessment.
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•SP3 combined with FAIMS simplified GCF sample processing, eliminating the common but laborious sample fractionation.•The SP3-based FAIMS-MS protocol improved the coverage of GCF proteome and showed good quantitative performance (R2 > 0.98).•We generated the largest dataset of human GCF proteome (5407 proteins) to date.•A novel six-protein marker panel (AUC = 0.937) was provided for skeletal maturity assessment.
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)‐1β 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‐1β 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‐1β 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.
Periodontitis is a prevalent chronic inflammatory disease associated with dysbiosis. Although complement inhibition has been successfully used to treat periodontitis in animal models, studies ...globally analyzing inflamed tissue proteins to glean insight into possible mechanisms of action are missing. Using quantitative shotgun proteomics, we aimed to investigate differences in composition of inflammatory gingival tissue exudate (“gingival crevicular fluid”; GCF), before and after local administration of an inhibitor of the central complement component, C3, in nonhuman primates. The C3 inhibitor, Cp40 (also known as AMY-101) was administered locally in the maxillary gingival tissue of cynomolgus monkeys with established periodontitis, either once a week (1×-treatment; n = 5 animals) or three times per week (3×-treatment; n = 10 animals), for 6 weeks followed by another 6 weeks of observation in the absence of treatment. 45 GCF samples were processed for FASP digestion and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. Data were processed using the ProgenesisQI software. The statistical significance of differences between the groups was determined by RM-ANOVA, and a protein expression change was considered as a true regulation at >2-fold and p < 0.05. The human orthologues were subjected to Gene Ontology analyses using PANTHER. Data are available via ProteomeXchange with identifier PXD009502. 573 proteins with >2 peptides were longitudinally quantified. Both 3× and 1× administration of Cp40 resulted in significant down-regulation of dozens of proteins during the 6-week course of treatment as compared to baseline. Following drug withdrawal at 6 weeks, more than 50% of the down-regulated proteins showed increased levels at week 12. The top scored pathway was “complement activation, alternative pathway”, and several proteins involved in this pathway were down-regulated at 6 weeks. We mapped the proteomic fingerprint changes in local tissue exudate of cynomolgus monkey periodontitis in response to C3 inhibition and identified the alternative pathway of complement activation and leukocyte degranulation as main targets, which are thus likely to play significant roles in periodontal disease pathogenesis. Label-free quantitative proteomics strategies utilizing GCF are powerful tools for the identification of treatment targets and providing insights into disease mechanisms.
Matrix metalloproteinase-8 is a promising candidate biomarker for oral fluid (gingival crevicular fluid, peri-implant sulcular fluid and saliva) and mouthrinse chair-side/point-of-care diagnostics to ...predict, diagnose and determine the progressive phases of episodic periodontitis and peri-implantitis, as well as to monitor the treatments and medications. Matrix metalloproteinase-8 can be used alone or together with interleukin-1beta and Porphyromonas gingivalis to calculate cumulative risk score at the subject level as a successful diagnostic tool, especially in large-scale public health surveys, in which a thorough periodontal examination is not feasible.
Objective. To investigate the effect of a chewing gum containing probiotic bacteria on gingival inflammation and the levels of selected inflammatory mediators in gingival crevicular fluid (GCF). ...Material and Methods. Forty-two healthy adults with moderate levels of gingival inflammation entered a double-blind placebo-controlled study design. The subjects were randomly assigned to one of three parallel arms: Group A/P was given one active and one placebo gum daily, Group A/A received two active chewing gums, and Group P/P two placebo gums. The chewing gums contained two strains of Lactobacillus reuteri: ATCC 55730 and ATCC PTA 5289 (1×108 CFU/gum, respectively). The subjects were instructed to chew the gums for 10 min over the course of 2 weeks. Bleeding on probing (BOP) and GCF sampling were conducted at baseline and after 1, 2 and 4 weeks. The levels of IL-1 , TNF- , IL-6, IL-8 and IL-10 were determined using luminex technology and multiplex immunoassay kits. Results. BOP improved and GCF volume decreased in all groups during the chewing period, but the results were statistically significant (p<0.05) only in Groups A/P and A/A. The levels of TNF- and IL-8 decreased significantly (p<0.05) in Group A/A compared with baseline after 1 and 2 weeks, respectively. A non-significant decreasing tendency was also observed concerning IL-1 during the chewing period. The levels of IL-6 and IL-10 were unaffected in all groups after 1 and 2 weeks. Conclusions. The reduction of pro-inflammatory cytokines in GCF may be proof of principle for the probiotic approach combating inflammation in the oral cavity.
The methodologies applicable for the evaluation of periodontal associated diseases are constantly evolving to provide quick, realistic, and scientifically proven results. Trends in the past followed ...a clinical evaluation of periodontal tissues and radiographic-based reports that formed the foundation for detection of diseases involving the structures supporting the teeth. As the confines and limitations of conventional strategies became obvious over the passage of time, hand in hand variety of techniques have evolved and experimentally justified. These improvisations are based on an improved understanding of the periodontal-pathogenic cascade. Periodontal pathogenesis and a paradigm shift from disease understanding to disease prevention and treatment entail few prerequisites that demand the objectivity of diagnostics procedure that includes sensitivity and specificity along with an explanation of the intensity of the disease, Gingival crevicular fluid an oral bio-fluid resides in the close proximity with gingival tissues have been widely used to understand and differentiate the periodontal health and diseased status. The biomarkers present in the GCF can be a reliable tool to detect the minute changes seen in the disease processes. The GCF consists of various host and bacterial-derived products as well as biomarkers which in turn can be evaluated for the diagnosis, prognosis as well as management of the periodontal disease. Thus, the review aims at describing GCF as a potential oral biofluid helpful in differentiating periodontal health and disease status.
Background and Objective
Recent emerging evidence has shown that microRNA (miRNAs) is involved in several epigenetic processes linked with periodontitis, increased oxidative stress and cardiovascular ...disease (CVD). The present study aimed to assess the impact of periodontitis on gingival crevicular fluid (GCF) miRNAs expression associated with CVD risk and to evaluate possible confounders that influenced this association.
Materials and Methods
For the present study, healthy controls (n = 28) and subjects with CVD (n = 28), periodontitis (n = 30) and periodontitis + CVD (n = 29) were enrolled. All subjects underwent regular periodontal examinations and blood sampling. In addition, GCF sampling was performed, and miRNAs 7a‐5p, 21‐3p, 21‐5p, 100‐5p, 125‐5p, 200b‐3p, and 200b‐5p expression was analyzed using a real‐time quantitative polymerase chain reaction (RT‐PCR).
Results
The results showed that periodontitis and periodontitis + CVD subjects presented significantly different GCF miRNAs expression compared to healthy controls and CVD subjects. More specifically, compared to healthy controls and CVD, subjects with periodontitis and periodontitis + CVD showed higher GCF miRNA 7a‐5p, miRNA 21‐3p, miRNA 21‐5p, miRNA 200b‐3p, and miRNA 200b‐5p (p < .05) and lower miRNA 100‐5p, miRNA 125‐5p levels (p < .05). Furthermore, the multivariate regression analysis evidenced that periodontitis (miRNA 21‐3p, 100‐5p) and periodontal inflamed surface area (PISA) (miRNA 7a‐5p, 21‐3p, 21‐5p, 100‐5p, 125‐5p, 200b‐3p) were significant predictors of GCF miRNAs concentration (p < .05).
Conclusion
The results of the study highlighted that the periodontitis and periodontitis + CVD group showed higher GCF miRNAs expression than healthy controls and CVD subjects. Furthermore, periodontitis and its extent (PISA) were revealed as significant predictors of GCF miRNAs associated with CVD risk.
Aim
To identify the diagnostic accuracy of gingival crevicular fluid (GCF) candidate biomarkers to discriminate periodontitis from the inflamed and healthy sites, and to compare the performance of ...two independent matrix metalloproteinase (MMP)‐8 immunoassays.
Materials and Methods
Cross sectional study. GCF (N = 58 sites) was collected from healthy, gingivitis and chronic periodontitis volunteers and analysed for levels of azurocidin, chemokine ligand 5, MPO, TIMP‐1 MMP‐13 and MMP‐14 by ELISA or activity assays. MMP‐8 was assayed by immunofluorometric assay (IFMA) and ELISA. Statistical analysis was performed using linear mixed‐effects models and Bayesian statistics in R and Stata V11.
Results
MMP‐8, MPO, azurocidin and total MMP‐13 and MMP‐14 were higher in periodontitis compared to gingivitis and healthy sites (p < 0.05). A very high correlation between MPO and MMP‐8 was evident in the periodontitis group (r = 0.95, p < 0.0001). MPO, azurocidin and total levels of MMP‐8, MMP‐13 and MMP‐14 showed high diagnostic accuracy (≥0.90), but only MMP‐8 and MPO were significantly higher in the periodontitis versus gingivitis sites. MMP‐8 determined by IFMA correlated more strongly with periodontal status and showed higher diagnostic accuracy than ELISA.
Conclusions
MPO and collagenolytic MMPs are highly discriminatory biomarkers for site‐specific diagnosis of periodontitis. The comparison of two quantitative MMP‐8 methods demonstrated IFMA to be more accurate than ELISA.
The effect of non-surgical periodontal treatment on oral and systemic inflammatory mediators in subjects with periodontitis and hyperglycemia remains largely unknown. Therefore, the aim of this ...clinical study was to compare the short-term effect of non-surgical periodontal treatment on serum, saliva and GCF inflammatory markers levels in GP subjects with or without hyperglycemia.
Sixty subjects divided into four groups of equal size were selected to participate: type 2 diabetics with generalized periodontitis (T2DM + GP), pre-diabetics with GP (PD + GP), normoglycemic subjects with GP (NG + GP), and healthy controls. GCF, serum, and saliva samples were obtained at baseline and 30 days after scaling and root planning (SRP) and the levels of interleukin-1β (IL-1 β), IL-8, IL-6, IL-2, IL-5, IL-4, IL-10, Interferon gamma (IFN-γ), Granulocyte macrophage colony-stimulating factor (GM-CSF) and Tumor necrosis factor-alpha (TNF-α) were determined by ultrasensitive multiplex assay. Clinical periodontal measurements were recorded.
SRP yielded significant improvement of all periodontal parameters for all GP groups (p < 0.01). A significant reduction in GCF levels of several cytokines were observed; however, only IL-1B and IFN-γ were consistently reduced post-treatment across all GP groups. Salivary levels of IL-1β were significantly reduced in all GP groups following treatment. No significant differences were observed for serum levels after SRP.
Periodontal treatment reduced local inflammatory markers, specifically IL-1B and IFN-γ, irrespective of the diabetes status. Periodontal treatment had no significant effect on serum levels of the inflammatory markers evaluated in this study.
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