Calprotectin In Gingival Crevicular Fluid Research Articles

Calprotectin levels in gingival crevicular fluid and serum of patients with chronic periodontitis and type 2 diabetes mellitus before and after initial periodontal therapy

This study is aimed to compare the total amount of calprotectin in gingival crevicular fluid (GCF) and the concentration of calprotectin in serum among the patients with type 2 diabetes mellitus and chronic periodontitis (DM-P), the patients with chronic periodontitis (CP) and the healthy controls, as well as the variation of these indicators before and three months after the initial periodontal therapy for the DM-P patients. 35 patients with DM-P patients, 32 patients with CP patients, and 43 healthy controls were recruited. Calprotectin levels in serum and GCF, periodontal parameters, fasting blood glucose (FBG), and HbA1c were measured at baseline for all the groups and three months after the initial periodontal therapy for the DM-P patients. At baseline, the calprotectin levels in GCF and serum were the highest in DM-P, followed by CP, and the lowest in healthy controls. GCF calprotectin was significantly and positively correlated with serum calprotectin and probing depth (PD), while serum calprotectin had a significant positive correlation with GCF calprotectin and HbA1c. Periodontal parameters, HbA1c, and serum and GCF calprotectin became significantly reduced after the initial periodontal treatment. The reduction of serum calprotectin was consistent with that of HbA1c, while the decrease of GCF calprotectin was in agreement with that of PD, attachment loss (AL), and bleeding on probing (BOP). The levels of calprotectin in serum and GCF in the DM-P patients are significantly higher than those in CP patients and healthy controls, which significantly reduced 3months after the initial periodontal therapy. Furthermore, it suggests diabetic patients might exhibit more pronounced inflammation periodontally and systemically.

Full-mouth disinfection effects on gingival fluid calprotectin, osteocalcin, and N-telopeptide of Type I collagen in severe periodontitis.

To compare the effects of full-mouth disinfection (FMD) and full-mouth ultrasonic debridement (FMUD) on clinical, microbiological and biochemical parameters with conventional quadrant-wise scaling and root planning (Q-SRP) in severe chronic periodontitis. In the present prospective randomized controlled clinical trial with three parallel arms (#NCT04038801), 60 chronic periodontitis patients were randomly assigned to three study groups by a consecutive number in ascending order: FMD (n=20), FMUD (n=20), and Q-SRP (n=20). All measurements and treatments were performed by the same investigator. At baseline, gingival crevicular fluid (GCF) and subgingival plaque were collected and clinical periodontal parameters were recorded. Ultrasonic debridement was completed within 24hours in FMD and FMUD groups. Chlorhexidine gluconate was used for FMD. Q-SRP was performed by hand instruments per quadrant at 1-week-intervals. Clinical measurements and sampling were repeated at 1, 3, and 6 months after treatment. Real-time PCR was used for quantitative analysis of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum, and total bacteria count. GCF Calprotectin, osteocalcin, and N-telopeptide of type I collagen (NTx) levels were analyzed by ELISA. The changes of GCF biomarker levels after treatment between groups were the primary outcomes. No harm was observed. All treatment strategies resulted in significant improvements in all clinical parameters (P<0.05), with no significant differences between study groups at all time-points (P ˃ 0.05). Aggregatibacter actinomycetemcomitans was significantly decreased in FMD compared to FMUD and Q-SRP at 6 months (P<0.05). Although GCF NTx total amounts increased in all groups during the study period, this increase was less prominent in full-mouth groups at three time points after treatment (P<0.05). Present results represent the short-term effects. Full-mouth treatment approaches offered limited beneficial effects on microbiological and biochemical parameters over quadrant-wise approach. All three treatment strategies can be recommended in the management of severe chronic periodontitis.

Useful immunochromatographic assay of calprotectin in gingival crevicular fluid for diagnosis of diseased sites in patients with periodontal diseases.

Calprotectin, an inflammation-related protein, is present in gingival crevicular fluid (GCF), and the determination of calprotectin is useful for diagnosing periodontal diseases. The authors have recently developed a novel immunochromatographic (IC) chip system to determine calprotectin levels in GCF. In the present study, the usefulness of this diagnostic system is investigated in patients with periodontal diseases. Thirty-six patients with periodontal diseases participated in this clinical test at multiple centers. Periodontitis sites (n=118) and non-periodontitis (healthy) sites (n=120) were selected after periodontal examination. GCF collection and periodontal examination were performed at baseline, after supragingival and subgingival scaling and root planing. Calprotectin levels in GCF were determined using a novel IC chip system and evaluated as a visual score and an IC reader value. Correlations between GCF calprotectin levels, clinical indicators, and changes in calprotectin levels by periodontal treatments were investigated. Receiver operating characteristic (ROC) analysis of IC reader value for GCF calprotectin was performed to predict periodontal diseases. The visual score of GCF calprotectin was highly correlated with the IC reader value. IC reader values of GCF calprotectin in the periodontitis group were higher than those of the healthy group at three dental examination stages, and they significantly decreased with periodontal treatments. Visual scores and IC reader values of GCF calprotectin were correlated to levels of clinical indicators. ROC analysis for GCF calprotectin showed an optimal cutoff value to predict periodontal diseases. Determination of GCF calprotectin using a novel IC chip system is useful for diagnosis of periodontal diseases.

Platelet activation and platelet–leukocyte interaction in generalized aggressive periodontitis

Generalized aggressive periodontitis (GAgP) is an inflammatory disease of host response to bacterial challenge. To explore the role of platelets in host-microbial interactions in patients with periodontitis, 124 patients with GAgP and 57 healthy subjects were enrolled. Reliable indicators of subclinical platelet functional status, platelet count (PLT), platelet large cell ratio (PLCR), and mean platelet volume (MPV), were significantly lower in the GAgP group than in the control group and were negatively correlated with clinical periodontal parameters. The levels of important cytosolic protein in neutrophils, calprotectin (S100A8/A9) in plasma, and gingival crevicular fluid (GCF) were significantly higher in patients with GAgP compared with healthy subjects. Moreover, the GCF calprotectin level was negatively correlated with PLCR and MPV values. To explore the possible mechanisms of changes in platelet indices in periodontitis, flow cytometry analysis was performed, and patients with GAgP were found to have a higher status of platelet activation compared with healthy controls. Porphyromonas gingivalis (P. gingivalis) and recombinant human S100A8/A9 (rhS100A8/A9) induced platelet activation and facilitated platelet-leukocyte aggregate formation in whole blood of healthy subjects. In response to P. gingivalis and rhS100A8/A9, platelets from patients with GAgP increased activation and increased formation of platelet-leukocyte aggregates compared with those from healthy subjects. Platelet aggregates and platelets attached to leukocytes were found on gingival tissues from patients with GAgP, suggesting that decreased platelet size and count in the circulation might be related to consumption of large, activated platelets at inflamed gingiva. Platelets may have a previously unrecognized role in host response to periodontal infection.

The pro-apoptotic and pro-inflammatory effects of calprotectin on human periodontal ligament cells.

Calprotectin, a heterodimer of S100A8 and S100A9 subunits, is associated with inflammatory disorders such as rheumatoid arthritis and cystic fibrosis. Although calprotectin levels are increased significantly in the gingival crevicular fluid (GCF) of periodontitis patients, its effects on periodontal ligament cells (PDLCs) remain largely unknown. The aim of this study was to evaluate calprotectin levels in the GCF of generalized aggressive periodontitis (AgP) patients and to investigate the effects of recombinant human calprotectin (rhS100A8/A9) and its subunits (rhS100A8 and rhS100A9) in PDLCs. Both the concentration and amount of crevicular calprotectin were significantly higher in the AgP group compared with healthy controls. In addition, the GCF calprotectin levels were correlated positively with clinical periodontal parameters including bleeding index, probing depth, and clinical attachment loss. rhS100A8/A9 promoted cell apoptosis, whereas rhS100A8 and rhS100A9 individually exerted little effect on apoptosis in PDLCs. rhS100A9 and rhS100A8/A9 increased the activation of nuclear factor-κB (NF-κB) by promoting the nuclear translocation of p65 in PDLCs, subsequently inducing expression of the pro-inflammatory cytokines IL-6, IL-8, TNFα, and COX2. Treatment with an NF-κB inhibitor partially reversed the rhS100A9- and rhS100A8/A9-induced upregulation of the pro-inflammatory cytokines. rhS100A9, and not rhS100A8, was mainly responsible for the pro-inflammatory role of calprotectin. Collectively, our results suggest that calprotectin promotes apoptosis and the inflammatory response in PDLCs via rhS100A9. These findings might help identify novel treatments for periodontitis.

Determination of calprotectin in gingival crevicular fluid by immunoassay on a microchip

ObjectivesGingival crevicular fluid (GCF) contains calprotectin, which appears to be a useful biomarker for periodontal diseases because of its high level in GCF from periodontally diseased pockets. To determine calprotectin in GCF that has a very small volume, sandwich enzyme-linked immunosorbent assay (ELISA) on a microchip was performed and its utility was estimated. Design and methodsAnti-calprotectin primary antibody was discharged on a microchip using a piezoelectric inkjet printing system. Calprotectin standard and calprotectin in GCF samples from eleven subjects were determined by the ELISA method with the prepared microchip and their values were compared with those obtained by conventional ELISA. ResultsUsing the ELISA on a microchip, a reasonable standard curve of calprotectin protein (1.56–100ng/mL) was obtained. Calprotectin in GCF samples was quantified and showed reasonable values in accordance with the condition of periodontal diseases. The values determined by the microchip method and conventional ELISA showed a significant linear relationship (R2=0.981). ConclusionsCalprotectin in GCF was determined using the ELISA on a microchip with high efficiency and this ELISA method for calprotectin determination may become a useful method for diagnosing periodontal diseases.

Microbiological profile and calprotectin expression in naturally occurring and experimentally induced gingivitis

This study was performed to evaluate the microbiological profile and the calprotectin expression in gingival crevicular fluid (GCF) in spontaneous and experimentally induced gingival inflammation. Thirty-seven periodontally healthy subjects were evaluated in real life conditions (N-O gingivitis) as well as after 21days of experimental gingivitis trial (E-I gingivitis). During the experimental gingivitis trial, in one maxillary quadrant (test quadrant), gingival inflammation was induced by oral hygiene abstention, while in the contralateral (control) quadrant, oral hygiene was routinely continued. The results of the study showed that (1) the microbiological profile of quadrants where gingival inflammation was experimentally induced (i.e., E-I test quadrants) differed significantly from that of either quadrants where gingival inflammation was controlled by proper plaque control (i.e., E-I control quadrants) or quadrants with N-O gingivitis, and (2) GCF calprotectin was significantly higher at E-I test quadrants compared to either E-I control quadrants or quadrants with N-O gingivitis. A positive intrasubject correlation was found between GCF concentration of calprotectin at sites presenting N-O and E-I gingivitis. N-O and E-I gingivitis showed a different microbiological profile of the subgingival environment. GCF calprotectin is a reliable marker of gingival inflammation, and its concentration in N-O gingivitis is correlated with its expression in E-I gingivitis. The modality of plaque accumulation seems to affect the subgingival microbiological profile associated with a gingivitis condition. Calprotectin levels in GCF may be regarded as a promising marker of the individual susceptibility to develop gingival inflammation in response to experimentally induced plaque accumulation.

Gingival Crevicular Fluid Osteocalcin, N‐Terminal Telopeptides, and Calprotectin Levels in Cyclosporin A–Induced Gingival Overgrowth

The aim of this cross-sectional study is to investigate gingival crevicular fluid (GCF) osteocalcin, cross-linked N-terminal telopeptide (NTx), and calprotectin levels in cyclosporin A (CsA)-induced gingival overgrowth (GO). Forty medicated patients with CsA including 20 with GO (CsA GO+), 10 without GO (CsA GO-), 10 with GO and chronic periodontitis (CsA CP) and 60 patients with CP alone, 20 patients with gingivitis, and 20 healthy patients were enrolled. Probing depth, clinical attachment level, plaque index, and papillary bleeding index were recorded. GCF calprotectin, osteocalcin, and NTx levels were analyzed by enzyme-linked immunosorbent assay. Parametric tests were used for statistical analysis. The CsA GO+ and CP groups had significantly lower GCF osteocalcin levels and osteocalcin/NTx ratio than the healthy group, whereas GCF osteocalcin levels and osteocalcin/NTx ratio in the gingivitis group were higher than the CsA GO+, CsA GO-, CsA CP, and CP groups (P <0.05). The CP group had elevated GCF calprotectin levels compared to the other study groups (P <0.05). The CsA GO+ and CsA GO- groups also had higher GCF calprotectin levels compared to the CsA CP, gingivitis, and healthy groups (P <0.05). Increased GCF calprotectin and decreased GCF osteocalcin levels in the CsA GO+ and CsA GO- groups might suggest that CsA plays a role on the levels of these markers. The similarity of GCF osteocalcin, NTx, and calprotectin levels in the CsA GO+ and CsA GO- groups might suggest that these molecules are not involved in the pathogenesis of GO.

Calprotectin levels in gingival crevicular fluid predict disease activity in patients treated for generalized aggressive periodontitis

Clinical parameters such as probing depth and bleeding on probing are commonly used for monitoring after periodontal treatment. However, these parameters have poor prognostic utility. The biomarker calprotectin is used to monitor conditions such as inflammatory bowel disease because of its ability to predict disease activity. Levels of calprotectin in gingival crevicular fluid correlate with periodontal disease severity and treatment outcome. The validity of calprotectin as predictor for future periodontal disease activity has not yet been investigated. Thirty-six subjects with generalized aggressive periodontitis were treated with scaling and root planing (SRP), and with adjunctive antimicrobial medications. Probing depth, clinical attachment level and bleeding on probing were assessed at baseline, and 3 and 6mo after SRP. A gingival crevicular fluid sample was collected from the initially deepest site in each patient 3mo after SRP and analysed for calprotectin levels. Activity was defined as a probing depth increase of >0.5mm between 3 and 6mo at the sample site. The ability of individual parameters to predict activity was analysed by construction of receiver operating characteristic curves. Nine active sites were identified. Clinical attachment level, probing depth, bleeding on probing and gingival crevicular fluid volume showed no predictive utility [area under the curve (AUC) <0.6, p>0.05]. However, calprotectin concentration (AUC=0.793, p=0.01) and the total amount/sample of calprotectin (AUC=0.776, p=0.02) significantly predicted activity. Patients with calprotectin levels above calculated cut-off values had significantly more active sites than patients with negative results. Calprotectin levels were predictors of disease activity at both site and subject levels. The calculated cut-off values provide a dichotomous basis for prospective evaluation of calprotectin as a diagnostic marker for monitoring periodontal treatment.

Gingival Crevicular Fluid Calprotectin, Osteocalcin and Cross-Linked N-Terminal Telopeptid Levels in Health and Different Periodontal Diseases

Aim:The aim of the present study was to investigate gingival crevicular fluid (GCF) calprotectin, osteocalcin and cross-linked N-terminal telopeptide (NTx) levels in health along with different periodontal diseases.Material and methods:Twenty chronic periodontitis (CP), 20 generalized aggressive periodontitis (G-AgP), 20 gingivitis and 20 healthy subjects were included. Probing depth, clinical attachment level, plaque index and papillary bleeding index was recorded. GCF calprotectin, osteocalcin and NTx levels were analyzed by enzyme-linked immunosorbent assay (ELISA).Results:CP, G-AgP and gingivitis groups had higher GCF calprotectin total amount compared to healthy subjects (p&lt; 0.008). CP and G-AgP groups had similar, but higher levels compared to gingivitis groups (p&lt; 0.008). CP and G-AgP groups had lower GCF osteocalcin total amount compared to gingivitis and healthy groups (p&lt; 0.008). CP group had higher GCF NTx but lower osteocalcin total amount and osteocalcin/NTx ratio than the G-AgP group (p&lt; 0.008)Conclusions:Our results suggest that elevated GCF calprotectin levels play a role as a reliable inflammatory marker in the pathogenesis of periodontal disease. Fluctuating GCF levels of osteocalcin and NTx might point out to the abnormal bone turnover in periodontitis. Our data document for the first time the role of NTx in the pathogenesis of different periodontal diseases.

Gingival crevicular fluid calprotectin, osteocalcin and cross-linked N-terminal telopeptid levels in health and different periodontal diseases.

Aim: The aim of the present study was to investigate gingival crevicular fluid (GCF) calprotectin, osteocalcin and cross-linked N-terminal telopeptide (NTx) levels in health along with different periodontal diseases. Material and methods: Twenty chronic periodontitis (CP), 20 generalized aggressive periodontitis (G-AgP), 20 gingivitis and 20 healthy subjects were included. Probing depth, clinical attachment level, plaque index and papillary bleeding index was recorded. GCF calprotectin, osteocalcin and NTx levels were analyzed by enzyme-linked immunosorbent assay (ELISA). Results: CP, G-AgP and gingivitis groups had higher GCF calprotectin total amount compared to healthy subjects (p < 0.008). CP and G-AgP groups had similar, but higher levels compared to gingivitis groups (p < 0.008). CP and G-AgP groups had lower GCF osteocalcin total amount compared to gingivitis and healthy groups (p < 0.008). CP group had higher GCF NTx but lower osteocalcin total amount and osteocalcin/NTx ratio than the G-AgP group (p < 0.008) Conclusions: Our results suggest that elevated GCF calprotectin levels play a role as a reliable inflammatory marker in the pathogenesis of periodontal disease. Fluctuating GCF levels of osteocalcin and NTx might point out to the abnormal bone turnover in periodontitis. Our data document for the first time the role of NTx in the pathogenesis of different periodontal diseases.

Gingival crevicular fluid levels of calprotectin and myeloperoxidase during therapy for generalized aggressive periodontitis

Levels of the inflammation marker calprotectin in gingival crevicular fluid correspond to clinical and biochemical parameters of periodontal inflammation. Neutrophil granulocytes (polymorphonuclear neutrophils: PMNs) are supposed to be the main source of calprotectin in gingival crevicular fluid, but evidence is still lacking. The influence of periodontal therapy on gingival crevicular fluid levels of calprotectin has not yet been determined. Gingival crevicular fluid levels of calprotectin were monitored during therapy for generalized aggressive periodontitis. Interrelations between calprotectin and the PMN marker myeloperoxidase (MPO) were evaluated. Gingival crevicular fluid samples were collected from 23 patients with generalized aggressive periodontitis before and 3 months after non-surgical therapy with an adjunctive antimicrobial medication. Clinical parameters were recorded with a pressure-calibrated electronic probe. Levels of calprotectin and MPO in gingival crevicular fluid were analysed by enzyme-linked immunosorbent assay (ELISA) procedures. At baseline, levels of calprotectin and MPO were highly correlated. Bleeding and suppurating sites showed significantly higher levels of calprotectin and MPO than non-bleeding, non-suppurating sites. Therapy significantly decreased levels of both biomarkers. These changes of calprotectin and MPO were highly correlated and also related to probing-depth reduction. Three months after therapy, the levels of both markers still showed significant correlations in initially deep sites, whereas in initially shallow sites no significant correlation was found. After therapy, levels of markers in bleeding and non-bleeding sites were comparable. The correlations between calprotectin and MPO indicate that PMNs are a major contributor to the calprotectin content in gingival crevicular fluid of severely affected sites. Calprotectin levels in gingival crevicular fluid and their changes reflect periodontal inflammation as well as the clinical treatment outcome. A prognostic potential of this marker substance remains to be determined.

Effect of Porphyromonas gingivalis Lipopolysaccharide, Tumor Necrosis Factor-α, and Interleukin-1β on calprotectin release in human monocytes.

Calprotection is a major cytosolic protein of monocytes, granulocytes, and epithelial cells. It is known that calprotectin is released in inflammatory tissues and detected in gingival crevicular fluid (GCF) of periodontitis patients at high levels. The origin of calprotectin in GCF and its regulation in periodontal disease are unknown. In this study, we investigated the distribution of calprotectin in gingiva tissues with inflammation and the induction of calprotectin release from human monocytes by lipopolysaccharide of Porphyromonas gingivalis (P-LPS), tumor necrosis factor-α (TNF-α), or interleukin -1β(IL-1β) Methods: Gingival tissues were obtained from a healthy donor and a periodontitis patient and calprotectin in gingival tissues was examined by immunohistochemical staining. Monocytes were isolated from the peripheral blood of healthy donors and cultured with P-LPS, TNF-α for 30 minutes to 4 hours. The content of calprotectin in the cell and medium fractions was determined by enzyme-linked immunosorbent assay (ELISA) Results: Calprotectin was markedly detected at the epithelial and adjacent connective tissue with many inflammatory cells in the gingival tissue from the periodontitis patient. P-LPS increased calprotectin release from monocytes to the maximum level after 30 minutes of treatment and its level was elevated to about 2- to 3-fold of the control level in a dose-dependent manner (1 to 1,000 ng/ml). When the effect of TNF-α and IL-Iβ on calprotectin release was investigated, calprotectin release significantly increased to about 2.2- and 1.5-fold that of the control level, respectively. These results demonstrate that calprotectin release from monocytes is induced by P-LPS, TNF-α, and IL-lβ, which in turn, cause and aggravate periodontal disease. J Periodontol 2003;74:1719-1724.

The association of calprotectin level in gingival crevicular fluid with gingival index and the activities of collagenase and aspartate aminotransferase in adult periodontitis patients.

Calprotectin, a major cytosol protein of leukocytes, exists in plasma and other body fluids of healthy human subjects. Since the calprotectin concentration rises markedly in some inflammatory diseases including rheumatoid arthritis, this protein has been thought to be a marker of inflammatory disease. Recently, we identified calprotectin in human dental calculus and gingival crevicular fluid (GCF), and found that the calprotectin concentration in GCF from patients with periodontitis was significantly higher than that in GCF from healthy subjects. In the present study, the association of GCF calprotectin level with GCF volume, gingival index (GI), and levels of biochemical markers including collagenase and aspartate aminotransferase (AST) in GCF was investigated to clarify the relationship between GCF calprotectin level and periodontal inflammation. Ninety GCF samples collected from periodontal pockets with a probing depth of more than 4 mm in 54 patients with adult periodontitis were used for these assays. The GCF volume was measured, and GI in each site was recorded. The calprotectin content in GCF samples was determined by ELISA using a specific antibody. The activity of collagenase or AST was measured by a respective assay kit. The total amount of calprotectin and GCF volume showed a highly significant correlation (r = 0.64, P <0.0001), whereas the calprotectin concentration had no correlation with the GCF volume (r = 0.01, P= 0.924). The mean calprotectin concentration in GCF increased with the degree of GI, and the concentration in individual samples was significantly correlated with the GI score (r = 0.56, P<0.0001). Significant positive correlations were observed in GCF calprotectin versus collagenase (r = 0.57, P <0.0001) and GCF calprotectin versus AST levels (r = 0.40, P <0.005). From the present results and our previous findings, it is shown that the GCF calprotectin level significantly correlates not only with clinical indicators but also with current biochemical marker levels and that calprotectin may be a useful marker for periodontal inflammation.

Calprotectin in gingival crevicular fluid correlates with clinical and biochemical markers of periodontal disease.

Clinical and biochemical markers of periodontal disease have been used for precise objective diagnosis of periodontal inflammation. Interleukin 1beta (IL-1beta) and prostaglandin E2 (PGE2), inflammatory factors, levels in gingival crevicular fluid (GCF) of patients with periodontal disease are elevated and have been studied as biochemical markers. The levels of calprotectin, a leukocyte protein, in body fluids of patients with some inflammatory diseases are raised. Recently, we detected calprotectin in GCF and its concentrations in periodontal pockets were higher than those in healthy gingival crevices. In this study, we investigated the correlations between GCF calprotectin levels and clinical indicators (probing depth and bleeding on probing, BOP), and the IL-1beta or PGE2 levels in GCE Probing depth and BOP at 130 sites of 110 subjects with periodontal or other oral diseases were examined, then GCF samples were collected and their calprotectin, IL-1beta and PGE2 were determined by ELISA. The calprotectin level correlated positively with the probing depth and was significantly higher at BOP-positive than BOP-negative sites. There were significant, positive correlations between the calprotectin and IL-1beta or PGE2 concentrations. These results indicate that the calprotectin level in GCF correlates well with clinical and biochemical markers of periodontal disease and suggest that calprotectin may be useful for evaluating the extent of periodontal inflammation.