PAI-2/SerpinB2 inhibits proteolytic activity in a P. gingivalis-dominated multispecies bacterial consortium.

OBJECTIVE: The aim of this study was to investigate the ability of the serine protease inhibitor plasminogen activator inhibitor type 2 (PAI-2/Serpin B2) to inhibit proteases produced by a multispecies bacterial consortium in vitro. BACKGROUND: Gingival and periodontal inflammation is associated with an increased flow of protein-rich gingival fluid. This nutritional change in the microenvironment favors bacteria with a proteolytic phenotype, triggering inflammation and associated tissue breakdown. PAI-2 is produced by macrophages and keratinocytes and is present in very high concentrations in gingival crevicular fluid; the highest level in the body. DESIGN: A multispecies bacterial consortium comprising nine bacterial strains, resembling the conditions in a periodontal pocket, was grown planktonically and as a biofilm. After seven days PAI-2 was added to the consortium and the proteolytic activity was assayed with fluorogenic protease substrates; FITC-labeled casein to detect global protease activity, fluorescent H-Gly-Pro-AMC for serine protease activity and fluorescent BIKKAM-10 for Porphyromonas gingivalis-associated protease activity. Protease activity associated with biofilm cells was examined by confocal scanning laser microscopy. RESULTS: PAI-2 inhibited proteolytic activity of the bacterial consortium, as seen by decreased fluorescence of all substrates. PAI-2 specifically inhibited P. gingivalis proteolytic activity. CONCLUSION: To our knowledge, this is the first time that PAI-2 has been shown to inhibit bacterial proteases. Given the high concentration of PAI-2 in the gingival region, our results indicate that PAI-2 might play a role for the integrity of the epithelial barrier.

The utility of gingival crevicular fluid matrix metalloproteinase-8 response patterns in prediction of site-level clinical treatment outcome.

BACKGROUND: Different gingival crevicular fluid (GCF) matrix metalloproteinase (MMP)-8 response patterns were studied among non-smoking and smoking patients with chronic periodontitis (CP) and generalized aggressive periodontitis (GAgP) to test the utility of GCF MMP-8 levels predicting the site-level treatment outcome. METHODS: Data from four independent longitudinal studies were combined. Altogether, the studies included 158 periodontal sites from 67 patients with CP and 32 patients with GAgP, and GCF samples were collected at baseline, after the treatment, and during the 6-month maintenance period. All GCF samples were analyzed by immunofluorometric assay for MMP-8. Different site-level MMP-8 response patterns were explored by the cluster analysis. Most optimal MMP-8 cutoff levels were searched with receiver operating characteristic analyses, and the predictive utility of defined levels was tested. RESULTS: Distinct types of MMP-8 response patterns were found in both smokers and non-smokers. MMP-8 levels exceeding the optimal cutoff levels separately defined for smokers and non-smokers indicated increased risk for compromised treatment outcome at baseline and during the maintenance period. Seventy-one percent of non-smokers (positive likelihood ratio of 4.22) and 88% of smokers (positive likelihood ratio of 5.00) with positive test results at both baseline and the maintenance period had compromised treatment outcome. The double-positive result indicated 46% and 39% point risk increase for the compromised outcome, respectively. CONCLUSION: GCF MMP-8 analysis with defined cutoff levels could be used to predict the site-level treatment outcome and for longitudinal monitoring of the disease status during the maintenance period.

Telomerase expression in individuals with chronic and aggressive periodontitis.

BACKGROUND: The aim of this study is to evaluate the expression of human telomerase reverse transcription (hTERT) enzyme in chronic periodontitis (CP) and aggressive periodontitis (AgP) compared with healthy individuals. METHODS: A total of 79 individuals consented to participate in the study. The study sample comprised healthy individuals (n = 30), patients with CP (n = 30), and patients with AgP (n = 19). Gingival tissue was collected and evaluated for hTERT expression by Western blot and immunohistochemical methods. Reverse transcription polymerase chain reaction was performed using the gingival crevicular fluid (GCF) samples. RESULTS: The hTERT messenger RNA (mRNA) and protein expression was significantly higher in AgP compared with CP (P <0.001). In GCF, 53.33% of patients with CP and 68.42% of patients with AgP were showing hTERT mRNA expression, but it was not detected in the control group. The AgP tissue showed higher hTERT expression compared with CP (P <0.001). The hTERT mRNA expression did not show a correlation with gingival index (GI), plaque index (PI), probing depth (PD), and clinical attachment loss (AL) in patients with AgP, whereas hTERT protein expression was strongly correlated with GI, PI, PD, and AL in patients with AgP. The protein expression of hTERT shows significant but moderate correlation with GI and AL in patients with CP. CONCLUSION: High expression of hTERT might be associated with periodontal disease progression, suggesting that hTERT could be a potential prognostic marker.

Effect of phase 1 periodontal therapy on gingival crevicular fluid levels of matrix metalloproteinases-3 and -13 in chronic periodontitis patients.

AIM: The aim of the present study was to estimate the gingival crevicular fluid (GCF) levels of matrix metalloproteinases (MMP)-3 and -13 in periodontally-healthy controls and chronic periodontitis (CP) patients, and also to investigate the effect of phase 1 periodontal therapy on MMP-3 and -13 levels in CP patients. METHODS: Fifty-five systemically-healthy patients were divided into two groups: group 1 (healthy) and group 2 (CP). The recording of clinical parameters and GCF sampling was done at baseline for both groups and again at 6 weeks post-therapy for group 2. The MMP level was determined by ELISA. RESULTS: A significant increase in the mean MMP-3 and -13 was found between healthy and CP patients. There was a statistically-significant reduction of GCF MMP-3 and -13 concentration after periodontal therapy in the CP group. A positive correlation was found between clinical parameters and GCF MMP-3 and -13 levels. CONCLUSIONS: A lower concentration of GCF MMP-3 and -13 was found in healthy patients, and a higher concentration was noted for CP patients, which was reduced after periodontal therapy. This indicates the important role played by these MMP in periodontal destruction. Thus, MMP-3 and -13 could be used as inflammatory biomarkers in diagnosing periodontal disease severity.

Immunolocalization of heme oxygenase-1 in periodontal diseases.

CONTEXT: Chronic periodontitis is an inflammatory condition of supporting tissues initiated by organisms in dental plaque. The reactive oxygen species and free radicals mediate connective tissue destruction in periodontitis. In order to counteract the free radical mediated tissue damage, numerous antioxidant mechanisms exist within the host. One such system is heme oxygenase enzymes. Heme oxygenase is the key enzyme involved in catabolism of heme. It cleaves the heme molecule to yield equimolar amounts of biliverdin, carbon monoxide, and iron. These end products act as important scavengers of reactive oxygen metabolites. Increased heme oxygenase expression has been identified in inflammatory condition, such as pancreatitis, diabetes, nephritis, and atherosclerosis. Since chronic periodontitis is one such inflammatory condition, we assessed the expression of heme oxygenase-1, in smokers and periodontitis group using immunohistochemistry technique. AIMS: The aim of this study is to compare the expression of heme oxygenase-1 in patients with healthy periodontium, periodontitis and smokers. MATERIALS AND METHODS: Gingival tissue samples were taken from 30 patients, who were divided into three groups healthy controls (n = 10), chronic periodontitis (n = 10), and smokers with chronic periodontitis (n = 10). All the samples were subjected to immunohistochemical staining using the antiheme oxygenase-1 antibody and were tested for efficiency by staining a positive control (prostate cancer tissue sections) and a negative control. The results were tabulated and analyzed. RESULTS: Our results showed increased expression of heme oxygenase-1 in the gingival tissue samples taken from smokers compared with periodontitis and healthy tissue. CONCLUSION: The results of our study is an increasing evidence of involvement of antioxidant enzymes like heme oxygenase-1 in periodontal inflammation and their implication for treatment of chronic periodontitis.

Gingival crevicular fluid matrix metalloproteinase-8 levels predict treatment outcome among smokers with chronic periodontitis.

BACKGROUND: Molecular biomarkers are needed for diagnostic use in periodontal diseases. The aim of this study is to explore different gingival crevicular fluid (GCF) matrix metalloproteinase-8 (MMP-8) patterns in smokers and non-smokers with chronic periodontitis (CP) and test the utility of baseline GCF MMP-8 levels in predicting categorically assessed treatment outcomes. METHODS: The study population comprised 15 patients with CP (five non-smokers and 10 smokers). GCF sampling of five to seven periodontal sites per patient was done at baseline, post-treatment, and bimonthly during the maintenance period from 8 to 12 months. GCF MMP-8 levels were measured with an immunofluorometric assay. MMP-8 response patterns were explored by cluster analysis. The ability of baseline MMP-8 levels to predict categorical treatment outcomes was analyzed with receiver operating characteristic curves. RESULTS: GCF MMP-8 response patterns could be clustered into two different site profiles among both smokers and non-smokers. Smoker site profiles 1 and 2 had significantly different clinical attachment level and gingival recession changes by the end of the maintenance period. In smoker sites, baseline MMP-8 levels significantly predicted the categorical treatment outcome. CONCLUSIONS: Baseline GCF MMP-8 levels strongly predict how MMP-8 levels behave during the maintenance period. In smoker sites, high baseline MMP-8 levels indicate weak treatment response.

Evaluation of systemic levels of neutrophilic enzymes in patients with hypertension and chronic periodontitis.

BACKGROUND: Inflammation stimulates neutrophils to release their enzymes into the extracellular matrix. The aim of the present study is to investigate the serum levels of matrix metalloproteinase (MMP)-8, MMP-9, tissue inhibitor of MMP (TIMP)-1, myeloperoxidase (MPO), and neutrophil elastase (NE) in patients with hypertension and chronic periodontitis (CP). METHODS: A total of 95 patients were included in the study. Patients were categorized into three groups: healthy control (n = 29), hypertensive control (n = 32), and hypertensive CP (n = 34). Periodontal parameters were recorded, and serum samples were collected from each participant. Serum MMP-8, MMP-9, TIMP-1, MPO, and NE levels in circulation were assessed by enzyme-linked immunosorbent assay. RESULTS: The hypertensive CP group had significantly higher serum MMP-8, MMP-9, and NE levels than the healthy control group (P <0.05). All study groups had similar serum TIMP-1 levels (P >0.05). Significantly higher serum MPO levels were detected in patients with hypertension and CP than healthy controls and hypertensive controls (P <0.05); however, the difference in serum MPO levels was not significant between the healthy controls and hypertensive controls (P >0.05). There was no significant difference in MMP-8/TIMP-1 ratio among the study groups (P >0.05). MMP-9/TIMP-1 ratio was significantly higher in patients with hypertension and CP than healthy controls (P <0.05). CONCLUSIONS: The presence of hypertension along with CP has a considerable effect on serum neutrophilic enzyme levels, except TIMP-1. However, the levels of these enzymes do not seem to be affected by the presence of hypertension only. Further studies including patients who have only CP might help illuminate the effect of CP on these enzymes in patients with hypertension.

Evaluation of antioxidant enzymes activity and malondialdehyde levels in patients with chronic periodontitis and diabetes mellitus.

BACKGROUND: The aim of this study is to investigate the impact of diabetes, a known risk factor for periodontitis, on activities of antioxidant enzymes superoxide dismutase (SOD), glutathione reductase (GR), and catalase (CAT) as well as levels of free radical damage marker malondialdehyde (MDA) in blood and saliva of individuals with chronic periodontitis (CP). METHODS: Sixty patients with CP (30 patients with type 2 diabetes mellitus [DMCP] and 30 systemically healthy patients [CP]) and 60 periodontally healthy individuals (30 patients with type 2 diabetes mellitus and 30 systemically healthy patients [PH]) were included in this study. After clinical measurements, blood and saliva samples were collected. SOD, GR, and CAT activities in red blood cell lysate and saliva and MDA levels in plasma and saliva samples were spectrophotometrically assayed. An analysis of variance test followed by a post hoc test was used to compare the intragroup and intergroup variances among the study groups. RESULTS: MDA levels in both the periodontitis groups were higher than in the periodontally healthy groups, but the difference between the CP and DMCP groups did not reach statistical significance (P >0.05). There was a highly significant difference between the CP and PH groups for all the enzymes studied except for SOD in blood. Only salivary SOD and GR activities were significantly different in the CP and DMCP groups. CONCLUSIONS: This study favors the role of oxidative stress in both diabetes and periodontitis. It shows that the compensatory mechanism of the body is partially collapsed because of excessive production of free radicals during periodontitis and is not able to cope with increased free radical generation attributable to diabetes, thereby worsening the situation.

IPLA2 mRNA expression by human neutrophils in type 2 diabetes and chronic periodontitis.

Type 2 diabetes mellitus (T2D) is becoming increasingly prevalent worldwide and complications of T2D cause significant systemic and dental morbidity in the susceptible individual. Although T2D has been linked as a significant risk factor for chronic periodontitis (CP), molecular mechanisms explaining the pathogenesis and inflammatory impact of CP in T2D are lacking. iPLA2 is the calcium-independent form of phospholipase A2. In previous studies, we demonstrated that iPLA2 enzyme activity is altered in T2D. The purpose of this study was to elucidate the level of the iPLA2 abnormality in T2D by measuring messenger RNA levels in T2D-associated CP. A total of 53 healthy and T2D subjects with CP were recruited for this study. The clinical periodontal exam included probing pocket depth, clinical attachment levels and bleeding on probing. Peripheral venous blood was collected and neutrophils were isolated. Real time polymerase chain reaction was used to quantify iPLA2 mRNA in neutrophils from healthy controls and people with diabetes. Results revealed that the prevalence of moderate to severe CP was increased in people with T2D. The iPLA, mRNA levels in diabetics with different severity of CP were not significantly different compared to healthy controls; 1.07 vs 0.97 (mild CP), 1.07 vs 0.85 (moderate CP) and 1.07 vs 1.05 (severe CP). Collectively, the data suggest that levels of iPLA2 mRNA in T2D are not different than in health and are not directly influenced by periodontal disease status. The impact of inflammation on iPLA2 regulation is at the level of activation of the enzyme rather than expression at the mRNA level.

Differential expression of mitogen activating protein kinases in periodontitis.

AIM: Following toll-like receptor (TLR) engagement, lipopolysaccharide (LPS) can stimulate the expression of pro-inflammatory cytokines thus activating the innate immune response. The production of inflammatory cytokines results, in part, from the activation of kinase-induced signalling cascades and transcriptional factors. Of the four distinct classes of mitogen-activated protein kinases (MAPK) described in mammals, p38, c-Jun N-terminal activated kinases (JNK1-3) and extracellular activated kinases (ERK1,2) are the best studied. Previous data have established that p38 MAPK signalling is required for inflammation and bone loss in periodontal disease pre-clinical animal models. MATERIALS & METHODS: In this study, we obtained healthy and diseased periodontal tissues along with clinical parameters and microbiological parameters. Excised fixed tissues were immunostained with total and phospho-specific antibodies against p38, JNK and ERK kinases. RESULTS: Intensity scoring from immunostained tissues was correlated with clinical periodontal parameters. Rank correlations with clinical indices were statistically significantly positive (p-value < 0.05) for total p38 (correlations ranging 0.49-0.68), phospho-p38 (range 0.44-0.56), and total ERK (range 0.52-0.59) levels, and correlations with JNK levels also supported association (range 0.42-0.59). Phospho-JNK and phospho-ERK showed no significant positive correlation with clinical parameters of disease. CONCLUSION: These data strongly implicate p38 MAPK as a major MAPK involved in human periodontal inflammation and severity.

Periodontal treatment reduces matrix metalloproteinase levels in localized aggressive periodontitis.

BACKGROUND: Matrix metalloproteinases (MMPs) are a family of host-derived proteinases reported to mediate multiple functions associated with periodontal breakdown and inflammation. High MMP levels in African-American children with localized aggressive periodontitis (LAgP) have been reported previously by the present authors. However, little is known about MMP reductions in gingival crevicular fluid (GCF) after therapy. This study aims to evaluate MMP levels in the GCF after treatment of LAgP and to correlate these levels with clinical response. METHODS: GCF samples were collected from 29 African-American individuals diagnosed with LAgP. GCF was collected from one diseased site (probing depth [PD] >4 mm, bleeding on probing [BOP], and clinical attachment level ≥ 2 mm) and one healthy site (PD ≤ 3 mm, no BOP) from each individual at baseline and 3 and 6 months after periodontal treatment, which consisted of full-mouth scaling and root planing (SRP) and systemic antibiotics. The volume of GCF was controlled using a calibrated gingival fluid meter, and levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-12, and MMP-13 were assessed using fluorometric kits. RESULTS: MMP-1, MMP-8, MMP-9, MMP-12, and MMP-13 levels were reduced significantly up to 6 months, comparable to healthy sites at the same point. Significant correlations were noted between MMP-2, MMP-3, MMP-8, MMP-9, MMP-12, and MMP-13 levels and percentage of sites with PD >4 mm. MMP-3, MMP-12, and MMP-13 levels also correlated with mean PD of affected sites. CONCLUSION: Treatment of LAgP with SRP and systemic antibiotics was effective in reducing local levels of specific MMPs in African-American individuals, which correlated positively with some clinical parameters.

Periodontal disease and gene-expression levels of metalloendopeptidases in human buccal mucosal epithelium.

BACKGROUND AND OBJECTIVE: Endopeptidases, such as neutral endopeptidase (NEP), endothelin-converting enzyme-1 (ECE-1) and a disintegrin and metalloprotease 17 (ADAM17), are believed to have various important roles in oral mucosal and epidermal tissue for the regulation of defensive biological responses in the oral cavity, and their expression and activity are influenced by various factors, including oral diseases. However, knowledge concerning these endopeptidases in the oral cavity has been minimal until now. This study focused on three metalloendopeptidases - NEP, ECE-1 and ADAM17 - in the oral buccal mucosal epithelium of patients with periodontal diseases and investigated the relationship between their gene-expression levels and periodontal disease. MATERIAL AND METHODS: The levels of expression of NEP, ECE-1 and ADAM17 mRNAs in tissue samples collected from the oral buccal mucosal epithelium of 61 patients were investigated by relative quantification using real-time RT-PCR analysis. information on oral and systemic health was obtained from the clinical record of each patient. RESULTS: Among the three groups, classified based on the diagnosis of periodontal diseases (healthy/gingivitis, early periodontitis and moderate/advanced periodontitis), the relative expression level of NEP mRNA was significantly increased in the early periodontitis group and in the moderate/advanced periodontitis group compared with that in the healthy/gingivitis group. Moreover, the relative expression levels of ECE1 and ADAM17 mRNAs were significantly increased in the moderate/advanced periodontitis group compared with those in the healthy/gingivitis group. The correlation coefficients between the mean relative expression levels of NEP and ECE1 mRNAs, NEP and ADAM17 mRNAs, and ECE1 and ADAM17 mRNAs were r = 0.758, r = 0.707 and r = 0.934, respectively (p < 0.001). Furthermore, among the oral-related factors, there was a significant correlation between the number of sites with probing pocket depths of more than 4 mm and of more than 6 mm and the relative expression levels of NEP, ECE1 and ADAM17 mRNAs. In stepwise logistic regression models, high relative expression levels of ECE1 and ADAM17 mRNAs were significantly associated with moderate/advanced periodontitis. CONCLUSION: The present study suggests that the severity of periodontal disease may be associated with the expression of metalloendopeptidase genes, including NEP, ECE1 and ADAM17, in the buccal mucosal epithelium.

Altered relationship between MMP-8 and TIMP-2 in gingival crevicular fluid in adolescents with Down's syndrome.

BACKGROUND AND OBJECTIVE: Periodontitis is more frequently found in subjects with Down's syndrome. The aim was to investigate whether the relationship between MMPs and TIMPs) in the gingival crevicular fluid of subjects with Down's syndrome is altered compared with controls. MATERIAL AND METHODS: Twenty-one adolescents with Down's syndrome and gingivitis (DS-G), 12 subjects with Down's syndrome and periodontitis (DS-P), 26 controls with gingivitis (HC-G) and eight controls with periodontitis (HC-P) were clinically examined. All patients were between 11 and 20 years of age. Gingival crevicular fluid was collected from each subject and the concentrations of MMPs (2, 3, 8, 9 and 13) and TIMPs (1, 2 and 3) (expressed as pg/µL adjusted for volume of gingival crevicular fluid) were determined using multianalyte kits from R&D Systems. RESULTS: The concentrations of MMP-2, MMP-3, MMP-8, MMP-9 and TIMP-2 in gingival crevicular fluid were significantly higher (p < 0.005) in the DS-G group compared with the HC-G group. The correlation coefficient between MMP-8 and TIMP-2 differed significantly (p = 0.006) between the DS-G group and the HC-G group. On the contrary, the correlation coefficients between MMPs and TIMPs did not differ significantly between the DS-P group and the HC-P group. However, the DS-P group exhibited a significantly lower concentration of TIMP-2 in the gingival crevicular fluid compared with the HC-P group. CONCLUSION: Down's syndrome subjects with gingivitis exhibit higher concentrations of MMPs in gingival crevicular fluid with an altered relationship between MMP-8 and TIMP-2, which might impair the periodontal tissue turnover.

Treponema denticola associates with increased levels of MMP-8 and MMP-9 in gingival crevicular fluid.

OBJECTIVES: The aim was to assess the association between the presence of site-specific subgingival micro-organisms and the levels of matrix metalloproteinases-8 and matrix metalloproteinases-9 (MMP-8 and MMP-9) in gingival crevicular fluid (GCF). MATERIALS AND METHODS: The patient group consisted of 56 subjects with periodontitis and the control group of 43 subjects without periodontitis. GCF samples from four test sites for each subject were collected. Polymerase chain reaction was used to detect the presence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, and Treponema denticola. MMP-8 concentrations were analyzed by a time-resolved immunofluorometric assay, and MMP-9 levels were determined by enzyme-linked immunosorbent assay. Student's unpaired t-test, chi-square test, and Fisher's exact P-value were calculated. RESULTS: The presence of T. denticola in the test sites was significantly higher in the patient group than in the control group. The presence of T. forsythia and T. denticola was associated with increased levels of MMP-8 in the test sites. Respectively, site-specific presence of T. denticola was associated with an increase in MMP-9 levels in three of the four test sites. CONCLUSIONS: The presence of subgingival micro-organisms in GCF, particularly T. denticola, appeared to induce a host response with an increased release of MMP-8 and MMP-9 in the test sites.

Evaluation of periimplant crevicular fluid prostaglandin E2 and matrix metalloproteinase-8 levels from health to periimplant disease status: a prospective study.

OBJECTIVE: To compare the levels of prostaglandin E2 (PGE2) and matrix metalloproteinases-8 (MMP-8) in periimplant crevicular fluid (PICF) after osseointegration and loading. MATERIALS AND METHODS: PICF was collected at the 3rd, 6th, 12th, and 18th months after implantation of 72 implants. PGE2 and MMP-8 levels besides clinical parameters were evaluated. RESULTS: Plaque and gingival index at the 6th, 12th, and 18th months presented higher values. Probing depth showed an increase after the 12th month. PGE2 presented a higher value at the 18th month. MMP-8 level demonstrated higher values after the sixth month. PGE2 and MMP-8 demonstrated positive correlations with gingival index and probing depth. CONCLUSION: The detection of PGE2 and MMP-8 in PICF serve to be useful for monitoring the course of periimplant disease. MMP-8 promises to be an early signal of periimplant inflammation.

MMP3 and TIMP1 variants contribute to chronic periodontitis and may be implicated in disease progression.

AIM: Matrix metalloproteinases (MMPs) play a key role in the tissue destruction characteristic of chronic periodontitis. The purpose of this study was to investigate the association of MMP and TIMP polymorphisms with chronic periodontitis in two populations. MATERIAL AND METHODS: A total of 34 polymorphisms spanning 12 MMP and 2 TIMP genes were genotyped in 401 individuals from Brazil (99 cases with chronic periodontitis and 302 controls), and 274 individuals from the US (70 cases and 204 controls). Individuals were considered cases if presenting at least three teeth exhibiting sites of clinical attachment loss ≥ 5 mm in two different quadrants. Controls were characterized by absence of clinical attachment loss and no sites with probing depth >3 mm. MMP3 and TIMP1 mRNA expression was evaluated in healthy and diseased periodontal tissues. RESULTS: TIMP1 showed association with chronic periodontitis in the Brazilian population (for rs5906435, p = 0.0004), whereas MMP3 showed association in the US population (for rs679620, p = 0.0003; and rs650108, p = 0.002) and in the Brazilian population (for rs639752, p = 0.005). MMP3 and TIMP1 mRNA expression was significantly higher in diseased tissues when compared to control tissues. CONCLUSIONS: Our results further support a role for variations in MMP3 in chronic periodontitis and report a novel association with TIMP1. These genes may be considered additional candidate genes for chronic periodontitis.

Analysis of proteins in human gingival crevicular fluid by mass spectrometry.

BACKGROUND AND OBJECTIVE: Gingival crevicular fluid is a bodily fluid transuded from periodontal tissues into the gingival crevice and periodontal pocket, and contains many species of components. Proteins in gingival crevicular fluid have been studied as markers for periodontal diseases. Mass spectrometric analysis is used for the analyses of proteins, lipids, saccharides and metals, and expected as an approach for disease diagnosis. For better analysis of the protein components in gingival crevicular fluid, we investigated proteins in gingival crevicular fluid samples from the healthy gingival crevice and periodontal pocket using mass spectrometry. MATERIAL AND METHODS: Gingival crevicular fluid samples were collected from subjects who gave their informed consent and were periodontally healthy or had diseased pockets. These samples were electrophoretically separated, and each fraction on the gels was analysed by nano liquid chromatography coupled with tandem mass spectrometry. Antimicrobial peptides detected in gingival crevicular fluid were confirmed by western blotting. RESULTS: One hundred and four proteins were detected in gingival crevicular fluid samples from both healthy sites and sites of periodontitis; 64 proteins were contained only in gingival crevicular fluid from healthy sites and 63 proteins were observed only in gingival crevicular fluid from periodontitis sites. These proteins were blood-, cytoskeleton-, immunity-, inflammation- and lipid-related proteins and enzymes. Some proteins, including ceruloplasmin, glycogen phosphorylase, glutathione S-transferase, phosphoglycerate mutase, psoriasin, S100A11 and resistin, were identified for the first time in gingival crevicular fluid. Antimicrobial peptides, such as lactoferrin, α1-antitrypsin, lipocalin, S100A7, S100A8, S100A9 and cathelicidin, were observed by mass spectrometry and western blotting. CONCLUSION: Multiple protein components in gingival crevicular fluid were analysed at the same time using mass spectrometry, and this approach may be useful for the diagnosis of periodontal 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.

Presence of aspartate aminotransferase in peri-implant crevicular fluid with and without mucositis.

The aim of this study was to assess the presence of aspartate aminotransferase (AST) in peri-implant crevicular fluid, with or without clinical signs of mucositis, to determine its predictive diagnostic value, sensitivity, and specificity. The AST levels were determined (at a threshold of 1200 µIU/mL) for 60 clinically successful implants in 25 patients with or without peri-implant mucositis. Samples were taken prior (AST1) to peri-implant probing with a manual constant-pressure probe (0.2 N) and 15 minutes after probing (AST2). Clinical assessments included radiographic determination of preexisting bone loss, probing, and the evaluation of mucositis, plaque, and bleeding upon probing. Analysis was performed at both the level of the implant and the patient as a unit. We detected a significant difference between AST1 and AST2 at both levels. A significant difference was observed at AST1 between implants that bled upon probing and those that did not. However, when we considered the patient as a unit, there were no significant differences. The plaque index was not significant at either level. AST1 had high specificity and positive predictive diagnostic value (80%) for bleeding upon probing. Probing induces a greater release of AST from inflamed tissues compared with healthy tissues in situ but not at the systemic level. At the implant level, the implant position could be responsible for this difference. Aspartate aminotransferase was a reliable predictor of patients with mucositis.

Analysis of cathepsin-K activity at tooth and dental implant sites and the potential of this enzyme in reflecting alveolar bone loss.

BACKGROUND: Cathepsin-K is an enzyme involved in bone metabolism which may make this feature important for both natural teeth and dental implants. The aims of the present study are to comparatively analyze the gingival crevicular fluid (GCF)/peri-implant sulcus fluid (PISF) cathepsin-K levels of natural teeth and dental implants, and to assess the potential relationship between this biochemical parameter and alveolar bone loss around natural teeth and dental implants. METHODS: Probing depth, bleeding on probing, gingival index, and plaque index clinical parameters were assessed, and GCF/PISF samples were obtained from natural teeth/dental implants presenting with either clinical health, gingivitis/peri-implant mucositis, or chronic periodontitis/peri-implantitis. Cathepsin-K activity levels of 42 GCF samples and 54 PISF samples were determined, and marginal bone loss (MBL) measures were calculated from digitalized standardized intraoral periapical radiographs obtained from natural teeth and dental implants by using cemento-enamel junction and the actual distance between two consecutive threads of the dental implant as reference points for natural teeth and dental implants, respectively. RESULTS: Comparing the natural teeth group with dental implant group with regard to MBL measure, cathepsin-K activity, and GCF/PISF volume revealed no significant differences. In both natural teeth and dental implant groups, despite higher MBL measures, cathepsin-K activity, and GCF/PISF volumes with the presence of inflammation, it was the presence of alveolar bone loss that lead to significantly higher values for these parameters. CONCLUSION: We suggest cathepsin-K as a biochemical parameter for monitoring periodontal/peri-implant alveolar bone loss.