Methodological evaluation of gingival crevicular fluid volume and neutrophil elastase levels: sequential sampling, length of sampling time and two different sampling methods.

Objectives: The mechanisms underlying the formation and composition of gingival crevicular fluid (GCF) and its flow into and from periodontal pockets are not understood very well. The aim of this study was to evaluate the length of sampling time and sequential sampling of GCF neutrophil elastase (NE) enzyme levels by using intracrevicular and orifice methods.Material and methods: Twenty adults (mean age of 41.8 years, ranged 31-60 years, 18 males and 2 females) with chronic periodontitis were enrolled and all completed the 3-d study. GCF was collected by both intracrevicular and intrasulcular methods, 720 samples of GCF were collected. In first, second and third day, the length of sampling time in seconds (s) and order were '5- 10-30-s'; '10- 30- 5-s' and '30- 5- 10-s,' respectively. GCF elastase levels were determined by hydrolysis of neutrophil specific substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide.Results: NE activity (µU) and NE activity/volume (µU/µl) were significantly different for order of sampling (p < .05), but not for the length of sampling time (p>.05).Conclusions: Within the limits of this study, the choice of sampling technique in GCF-profile studies seems to be a critical decision as it has the potential to affect the GCF volume and NE activity.

The level of salivary lactate dehydrogenase as an indicator of the association between gingivitis and related factors in Japanese university students.

The aim of this study was to investigate the association between the presence of gingivitis estimated using the salivary level of lactate dehydrogenase (LD) and related factors in young Japanese adults. Data from 1,915 participants (21.4 ± 2.5 years) were analyzed. Unstimulated saliva was collected from each participant and the salivary LD level was evaluated using a commercially available test kit with an integer scale ranging from 1 to 10. Gingivitis was defined as the LD level of ≥8. The number of permanent teeth, the simplified oral hygiene index (OHI-S), the presence of partially erupted molars and body mass index were recorded. Additionally, participants answered a questionnaire. The percentage of male participants, the number of permanent teeth, the OHI-S and the presence of partially erupted molars were higher, whereas the proportion receiving dental check-ups was lower in the gingivitis group (n = 88, 4.6%) than in the healthy group. Logistic regression analysis showed that gingivitis was significantly associated with OHI-S (OR: 2.68, 95% CI: 1.94-3.69) and receiving dental checkups (OR: 0.31, 95% CI: 0.10-0.99). The present findings indicated that the OHI-S and receiving dental checkups were significantly associated with gingivitis, as assessed by the salivary LD level, in this cohort.

Gingival Inflammation and Salivary or Serum Granulocyte-Secreted Enzymes in Patients With Polycystic Ovary Syndrome.

BACKGROUND: The objective of this cross-sectional study is to investigate levels of salivary and serum matrix metalloproteinase (MMP)-9, myeloperoxidase (MPO), neutrophil elastase (NE), and MMP-9/tissue inhibitor of MMP-1 (TIMP)-1 ratio in patients with polycystic ovary syndrome (PCOS) and systemically healthy controls in the presence or absence of gingivitis. METHODS: Serum and salivary levels of these biomarkers were evaluated in the following: 1) periodontally healthy women with PCOS (n = 45); 2) women with PCOS and gingivitis (n = 35); 3) systemically and periodontally healthy women (n = 25); and 4) systemically healthy women with gingivitis (n = 20). Enzyme-linked immunosorbent assay was used to determine levels of these biomarkers. A full-mouth clinical periodontal evaluation was performed for each patient. RESULTS: Salivary MMP-9 and NE levels, as well as MMP-9/TIMP-1 ratios, were higher in the systemically healthy women with gingivitis compared with periodontally healthy women with PCOS (P <0.001; P <0.01; and P <0.0001, respectively). Serum MMP-9 and MPO levels were higher in women with PCOS and gingivitis compared with periodontally healthy women with PCOS (P <0.05). Serum MMP-9 levels were lower in healthy women with gingivitis than systemically and periodontally healthy women or women with PCOS and gingivitis (P <0.05). PCOS groups exhibited a positive correlation among clinical periodontal parameters and serum MMP-9 levels or salivary MPO, NE levels, and MMP-9/MMP-1 ratio. Correlation was negative among clinical periodontal parameters and serum MMP-9 levels and MMP-9/TIMP-1 ratio in systemically healthy patients (P <0.05). CONCLUSIONS: The present findings emphasize that PCOS and gingival inflammation are associated with each other, as evidenced by salivary and serum levels of neutrophilic enzymes. This interaction may contribute to the perturbation of ovarian remodeling in PCOS.

Association between serum and oral matrix metalloproteinase-8 levels and periodontal health status.

BACKGROUND AND OBJECTIVE: Matrix metalloproteinase-8 (MMP-8) is involved in a wide range of pathologies including periodontitis and cardiovascular diseases (CVD). The association between periodontitis and CVD has been repeatedly recognized. The aim of the study was to analyze to what extent circulating active MMP-8 (aMMP-8) is associated with periodontal disease status and oral fluid aMMP-8 levels in otherwise healthy subjects. MATERIAL AND METHODS: In a cross-sectional study, aMMP-8 was measured in serum of 59 volunteers, comprising 19 periodontally healthy subjects, 20 patients with gingivitis as well as 20 with periodontitis. All study subjects were characterized regarding aMMP-8 concentrations in different oral fluids as well as clinically and microbiologically with respect to periodontal disease. aMMP-8 levels in gingival crevicular fluid were measured using the enzyme-linked immunosorbent assay. Saliva enzyme levels as well as circulating aMMP-8 were determined by a time-resolved immunofluorometric assay. Both methods utilized the same monoclonal antibodies. Correlation and regression analyses were performed to study the potential association between serum aMMP-8 and oral parameters. RESULTS: Oral aMMP-8 levels were significantly higher in patients with periodontitis compared to periodontally healthy or gingivitis subjects. Highest serum aMMP-8 concentration was also found in the periodontitis group. The serum levels correlated significantly with oral aMMP-8 as well as with clinical parameters in a dose-dependent manner. These results were confirmed in a multivariate regression analysis. After adjusting for potential confounders, saliva aMMP-8 concentrations as well as periodontitis severity were significant predictors of serum aMMP-8. CONCLUSION: The associations between circulating aMMP-8 and oral aMMP-8 as well as periodontal findings in a dose-dependent manner may contribute to linking periodontal disease with increased CVDsusceptibility.

Structural insights unravel the zymogenic mechanism of the virulence factor gingipain K from Porphyromonas gingivalis, a causative agent of gum disease from the human oral microbiome.

Skewing of the human oral microbiome causes dysbiosis and preponderance of bacteria such as Porphyromonas gingivalis, the main etiological agent of periodontitis. P. gingivalis secretes proteolytic gingipains (Kgp and RgpA/B) as zymogens inhibited by a pro-domain that is removed during extracellular activation. Unraveling the molecular mechanism of Kgp zymogenicity is essential to design inhibitors blocking its activity. Here, we found that the isolated 209-residue Kgp pro-domain is a boomerang-shaped all-ß protein similar to the RgpB pro-domain. Using composite structural information of Kgp and RgpB, we derived a plausible homology model and mechanism of Kgp-regulating zymogenicity. Accordingly, the pro-domain would laterally attach to the catalytic moiety in Kgp and block the active site through an exposed inhibitory loop. This loop features a lysine (Lys129) likely occupying the S1 specificity pocket and exerting latency. Lys129 mutation to glutamate or arginine led to misfolded protein that was degraded in vivo Mutation to alanine gave milder effects but still strongly diminished proteolytic activity, without affecting the subcellular location of the enzyme. Accordingly, the interactions of Lys129 within the S1 pocket are also essential for correct folding. Uniquely for gingipains, the isolated Kgp pro-domain dimerized through an interface, which partially overlapped with that between the catalytic moiety and the pro-domain within the zymogen, i.e. both complexes are mutually exclusive. Thus, pro-domain dimerization, together with partial rearrangement of the active site upon activation, explains the lack of inhibition of the pro-domain in trans. Our results reveal that the specific latency mechanism of Kgp differs from those of Rgps.

Role of indoleamine 2,3-dioxygenase in an inflammatory model of murine gingiva.

BACKGROUND AND OBJECTIVE: Indoleamine 2,3-dioxygenase (IDO) is one of the major pathways for metabolism of tryptophan in a variety of cells, including immune cells. Increasing evidence indicates that IDO is a critical player in establishing the balance between immunity and tolerance and ultimately in the maintenance of homeostasis. By inducing inflammation in gingival tissue, we tested the hypothesis that IDO is a pivotal player in regulating the immune and inflammatory responses of gingiva. MATERIAL AND METHODS: We utilized the IDO knockout mouse model in conjunction with lipopolysaccharide (LPS)-induced inflammation. Accordingly, wild-type and IDO knockout mice were injected with LPS or vehicle in the anterior mandibular gingiva, twice over a 2-wk period, which was followed by procurement of gingival tissue for histopathology and preparation of tissue for flow cytometry-based studies. RESULTS: Clinical and histological examinations revealed a marked adverse impact of IDO deficiency on gingival inflammation. These observations were consistent with a more marked increase in the number of cells positive for the proinflammatory cytokine interleukin (IL)-17, but no significant change in the number of cells positive for the anti-inflammatory cytokine IL-10, in LPS-treated IDO knockout mice. Consistent with the more marked proinflammatory impact of IDO deficiency, the percentage of regulatory T cells was much reduced in gingival tissue of LPS-treated IDO knockout mice than in gingival tissue of wild-type mice. These proinflammatory changes were accompanied with a prominent increase in apoptotic and necrotic cell death in gingival tissue of IDO knockout mice compared with wild-type mice. CONCLUSION: Collectively, our findings support a major role for IDO in the development of gingival inflammation, as an example of an inflammatory condition, and lay the foundation for subsequent studies to explore it as a novel immunotherapy target.

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.

Proteolytic Mediators in Gestational Diabetes Mellitus and Gingivitis.

BACKGROUND: This study evaluates levels of matrix metalloproteinase (MMP)-8, MMP-9, and tissue inhibitor of MP-1 (TIMP-1) in biofluids of women with gestational diabetes mellitus (GDM) and systemically healthy counterparts with different statuses of periodontal health. METHODS: Seventy-one women with GDM and gingivitis (Gg), 30 women with GDM and healthy periodontium (Gh), 28 systemically and periodontally healthy women (Hh), and 37 systemically healthy women with gingivitis (Hg) were evaluated. MMP-8, MMP-9, and TIMP-1 levels were determined in gingival crevicular fluid (GCF), saliva, and serum by immunofluorometric and enzyme-linked immunosorbent assays. Full-mouth clinical periodontal parameters were recorded. RESULTS: GCF and serum MMP-8 concentrations, serum MMP-9 concentrations, and serum MMP-8/MMP-1 and MMP-9/MMP-1 molar ratios were significantly higher in Gg compared with Hg group (P <0.05). Serum MMP-8 levels and salivary TIMP-1 levels were higher in Gh compared with Hg group (P <0.05) whereas salivary MMP-8/TIMP-1 molar ratio was lower in Gh compared with Hg group (P <0.05). Elevated concentrations of GCF MMP-8 and MMP-9 were found in Gg compared with Gh group (P <0.05). Significant correlations were found between local levels of biomarkers and clinical periodontal parameters in only GDM group. CONCLUSION: GDM may modulate both local and circulating levels of MMP-8 especially when associated with gingivitis.

Production of endogenous hydrogen sulfide in human gingival tissue.

OBJECTIVE: Endogenous hydrogen sulfide (H2S) has recently been shown to play an important role in inflammation, but the role of endogenous H2S in the human gingival tissue is unknown. The aim of this study was to investigate whether gingiva had enzymes for H2S synthesis, and whether the effect of these enzymes for H2S production changed with periodontal inflammation. DESIGN: Gingival tissues were collected from patients undergoing periodontal operation including gingivitis, moderate chronic periodontitis, severe chronic periodontitis and normal controls. RT-PCR and western blotting were performed to measure mRNA and protein levels of cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) for H2S production. Immunohistochemistry was carried out to detect the location of the enzymes. H2S levels and synthesis in gingival tissue were evaluated with modified methylene blue method. RESULTS: The mRNA and protein of CBS and CSE were both expressed in human gingiva and raised significantly in moderate and severe periodontitis compared of that in healthy control. CBS, but not CSE, increased in gingivitis (p<0.05). However, there was no significant difference of H2S level and synthesis among these groups (p>0.05). CONCLUSIONS: Both CBS and CSE were expressed in human gingival tissue. The mRNA and protein levels of CBS and CSE were up-regulated in periodontitis.

Immunohistochemical analysis of the gingiva with periodontitis of type I plasminogen deficiency compared to gingiva with gingivitis and periodontitis and healthy gingiva.

OBJECTIVE: Type I plasminogen deficiency (Plgdef) is an uncommon chronic inflammation of mucous membranes. Gingival enlargements usually proceed with progressive periodontal destruction and tooth-loss. Plasmin(ogen)-independent enzymatic mechanisms for fibrin clearance have already been discussed in the literature. Our primary objective was to verify, immunohistochemically, the occurrence of different enzymatic factors involved in tissue breakdown of inflamed compared to healthy gingiva. Secondly, we tried to find out, if these patients have a similar microbiological profile to the patients with known gingivitis and periodontitis. MATERIALS AND METHODS: Immunohistochemical analysis of enzymes elastase, plasminogen (plg), cathepsin G, matrix-metalloproteinase (MMP)-3 and MMP-7 and of glycoprotein fibrinogen were performed with gingival tissues from 3 healthy controls, 8 patients with Plgdef and 3 patients with gingivitis and periodontitis. Furthermore, plaque from 5 patients with plasminogen deficiency were also obtained to determine the microbiological profile. RESULTS: Significantly high numbers of elastase positive leukocytes were detected in all samples. Staining for MMP-3 and MMP-7 was seen in samples with gingivitis and periodontitis with a stronger staining in samples with periodontitis by Plgdef. Fibrinogen was detectable in all samples. Staining for plg was stronger in samples with periodontitis than in other samples. Staining for cathepsin G was weak in gingivitis and periodontitis. Subgingival microbial flora showed elevated colony forming units of Prevotella intermedia/nigrescens, Fusobacterium spp., Eikenella corrodens, Porphyromonas gingivalis and viridans streptococci. CONCLUSION: Strong staining of elastase, MMP-3 and MMP-7 and weak staining of plg in Plgdef samples supports the plasmin(ogen) - independent fibrin clearance. Similar subgingival microbiological flora was observed in periodontitis with Plgdef as in other periodontal diseases. Further investigations should determine the exact pathomechanism and focus on effective treatment methods of this entity.

Differential Matrix Metalloprotease (MMP) Expression Profiles Found in Aged Gingiva.

The periodontium undergoes age-related cellular and clinical changes, but the involved genes are not yet known. Here, we investigated age-related genetic changes in gingiva at the transcriptomic level. Genes that were differentially expressed between young and old human gingiva were identified by RNA sequencing and verified by real-time PCR. A total of 1939 mRNA transcripts showed significantly differential expression between young and old gingival tissues. Matrix metalloprotease (MMP) regulation was the top pathway involved in gingival aging. MMP3, MMP9, MMP12, and MMP13 were upregulated in old gingival tissues, concomitantly with interleukin-1 beta (IL1B) expression. In vitro experiments using human gingival fibroblasts (hGFs) showed that MMP12 was upregulated in old hGFs compared to young hGFs. Moreover, the MMP3, MMP9 and IL1B levels were more highly stimulated by infection with the oral bacterium, Fusobacterium nucleatum, in old hGFs compared to young hGFs. Collectively, these findings suggest that, in gingiva, the upregulation of MMP12 may be a molecular hallmark of natural aging, while the upregulations of MMP3, MMM9, and IL1B may indicate externally (e.g., infection)-induced aging. These findings contribute to our understanding of the molecular targets involved in gingival aging.

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.

Decreased salivary matrix metalloproteinase-8 reflecting a defensive potential in juvenile parotitis.

OBJECTIVE: Matrix metalloproteinases MMP-2 and MMP-9 have been associated with juvenile parotitis. However, the role of MMP-8 has not been addressed previously. This work focuses on salivary MMP-8 and -9 levels in juvenile parotitis. METHODS: During a five-year period at Helsinki University Hospital, a tertiary care hospital, 41 patients aged 17 or under, were identified as having parotitis; from 36 of these patients, saliva samples were collected for MMP-8 IFMA (time-resolved immunofluorometric assay) analyses. Control saliva samples were collected from 34 age- and gender-matched children admitted for an elective surgery who had no history of parotitis. For comparison, salivary levels of MMP-9, tissue inhibitor of matrix metalloproteinase (TIMP-1), MMP-8/TIMP-1 ratio, human neutrophil elastase (HNE), and myeloperoxidase (MPO) were analyzed by ELISA. Additionally, salivary MMP-8 levels were compared to historical saliva samples from 18 adult gingivitis patients as well as to 10 healthy adult controls. RESULTS: The median (25%, 75% percentile) MMP-8 concentration in saliva of parotitis patients was significantly lower than MMP-8 concentration in saliva of their controls [50.4ng/ml (37.5, 72.9) vs. 148.5ng/ml (101.2, 178.5) p<0.0001] and lower than in patients with gingivitis [347.9ng/ml (242.6, 383.2) p<0.0001] or healthy adult controls [257.2ng/ml (164.9, 320.7) p<0.0001]. The MMP-8/TIMP-1 ratio was lower than in controls [0.13 (0.05-0.02) vs. 0.3 (0.17-0.46) p<0.0001]. The median MMP-9 concentration in saliva of parotitis patients was significantly higher than in controls [143.9ng/m (68.8-189.0) vs. 34.9ng/ml (16.3-87.6) p<0.0001]. Neither HNE, MPO, nor TIMP-1 alone separated the patients from the control groups. CONCLUSIONS: MMP-9 was up-regulated in juvenile parotitis saliva, suggesting that MMP-9 may play a destructive role in juvenile parotitis, as others have suggested. The present novel findings reveal a decreased salivary MMP-8 concentration, suggesting that MMP-8 may reflect in juvenile parotitis down-regulated or anti-inflammatory immune characteristics.

Association of thalassemia major and gingival inflammation: A pilot study.

OBJECTIVES: This cross-sectional study aimed to investigate the relationship between thalassemia major (TM) and gingival inflammation through the salivary, serum, and gingival crevicular fluid (GCF) levels of matrix metalloproteinase (MMP)-8, MMP-9 and tissue inhibitor of MMP (TIMP)-1. METHODS: Biofluid samples and full-mouth clinical periodontal recordings were obtained from 29 otherwise healthy patients with TM and 25 systemically healthy (SH) individuals. Biofluid samples were evaluated by immunofluorometric assay (IFMA) and enzyme-linked immunoassays (ELISAs). Data were tested statistically by Kolmogorov Simirnov, Mann-Whitney U tests, Spearman correlation analysis. RESULTS: Age, smoking status, bleeding on probing, plaque index were similar in the study groups, but probing depth, gender data exhibited significant differences (p=0.037 for both). Salivary MMP-8, MMP-9, TIMP-1 concentrations were significantly higher in the TM than SH group (p=0.014; p<0.001; p=0.042, respectively). Serum TIMP-1 concentrations were significantly higher; MMP-8/TIMP-1, MMP-9/TIMP-1 molar ratios were significantly lower in the TM than SH group (p<0.001; p=0.005; p=0.022, respectively). Very few GCF samples revealed biochemical data above the detection limits. Numerous correlations were found between clinical periodontal parameters and biochemical data. CONCLUSIONS: It may be suggested that TM may exacerbate the local inflammatory response as manifested in salivary MMP-8, MMP-9, TIMP-1 levels.

Expression of key executioner of apoptosis caspase-3 in periodontal health and disease.

AIM: A highly-regulated form of programmed cell death is apoptosis, and its perturbation has been associated with periodontal disease. Caspase-3 is one of the key executioners of apoptosis. The present study was designed to evaluate and correlate the levels of caspase-3 in gingival crevicular fluid (GCF) and serum in participants with clinically-healthy periodontium, gingivitis, and chronic periodontitis (CP). METHODS: Forty-four sex- and age-matched participants were enrolled into three groups based on clinical parameters. Group 1 participants had clinically-healthy periodontium, group 2 participants had gingivitis, and group 3 participants had CP. GCF and serum samples were collected to evaluate the levels of caspase-3. RESULTS: The mean caspase-3 concentration in GCF and serum was highest in group 3, followed by group 2, and was significantly correlated with gingival index, probing depth (PD), and clinical attachment level (CAL). CONCLUSION: GCF and the serum concentration of caspase-3 proportionally increases with the progression of periodontal disease, that is, gingival inflammation, PD, and CAL.

Site-specific dental plaque pH in 13-year-old Thai schoolchildren.

OBJECTIVE: The aim of this paper was to study pH conditions between dental sites, taking account the presence of caries, calculus, and microbial composition and alkali production. MATERIALS AND METHODS: One hundred 13-year-old Thai schoolchildren were recorded for caries experience (DMFT, DT), calculus, plaque, and gingivitis. Ex vivo urease activity was measured on 11, 26, 31, and 46 (distal aspect) with the rapid urease test and pH at baseline and after rinse with 0.25 % urea solution on mesial site in vivo. Interproximal plaque from contralateral teeth was microbiological analysed with the checkerboard technique. RESULTS: Thirty-four children were caries free. Plaque and calculus were abundant; all children showed a high resting plaque pH and the mandibular incisor showed significantly (p < 0.01) higher pH at baseline, max pH and AOC7.0 after urea challenge, ex vivo urease activity and calculus but lower caries experience than other teeth. A significant inverse correlation (p < 0.02) was found between caries frequency and ex vivo urease activity for tooth 11. Anaerobes predominated over streptococci, but no significant differences between dental sites were found. CONCLUSIONS: The study group had a high baseline plaque pH, in vivo and ex vivo urease activity, and calculus but low caries experience, which was best reflected in the lower incisor region. CLINICAL RELEVANCE: Urease activity and pH on site level may be important determinants for individuals at caries risk.

Gelatinases and extracellular matrix metalloproteinase inducer are associated with cyclosporin-A-induced attenuation of periodontal degradation in rats.

BACKGROUND: The present study aims to examine the inhibitory effect of cyclosporin-A (CsA) on periodontal breakdown and to further explore the correlations of CsA-induced attenuation of periodontal bone loss with the expressions of gelatinases (i.e., matrix metalloproteinase [MMP]-2 and MMP-9) and extracellular matrix metalloproteinase inducer (EMMPRIN). METHODS: Forty Sprague-Dawley rats were randomly divided into four groups: 1) control; 2) CsA; 3) ligature (Lig); and 4) ligature plus CsA (Lig + CsA). The CsA group received 10 mg ⋅ Kg(-1) ⋅ d(-1) CsA for 8 days. The Lig group received silk ligature on selected molars. The Lig + CsA group received silk ligature and CsA treatment. The inhibitory effects of CsA on the ligature-induced periodontal breakdown was examined with microcomputed tomography (micro-CT) and histometric analyses to analyze the amount of attachment loss, crestal bone loss, connective tissue attachment, and the surface area with inflammatory cell infiltration. The effects of CsA on ligature-induced expressions of gelatinases and EMMPRIN in gingival tissues were examined with Western blotting and zymography, respectively. RESULTS: By micro-CT and histology, the Lig + CsA group had significantly more periodontal breakdown than the control and CsA groups but less periodontal breakdown than the Lig group. Consistent results were found for the expressions of gelatinases and EMMPRIN among the groups demonstrating that the Lig + CsA group had significantly less gingival protein expression of gelatinases and EMMPRIN than the Lig group. CONCLUSIONS: CsA inhibited the expressions of gelatinase MMPs and EMMPRIN and partially prevented the periodontal breakdown in ligature-induced experimental periodontitis. The CsA-induced attenuation of periodontal bone loss was strongly correlated positively with the expressions of MMP-2, MMP-9, and EMMPRIN in gingiva.

Expression of cathepsin K in periodontitis and in gingival fibroblasts.

OBJECTIVE: To study non-osteoclastic sources of cathepsin K in periodontitis. MATERIALS AND METHODS: Tissue samples were obtained from 10 otherwise healthy periodontitis pati-ents during routine periodontal flap operations and 10 systemically and periodontally healthy individuals who underwent extraction operations for retained third molars. Methods used were immunohistochemistry, image analysis, immunofluorescence double-staining, gingival fibroblast culture, tumour necrosis factor-α (TNF-α) stimulation and Western blotting. RESULTS: Macrophage-like cells, fibroblast-like cells, vascular endothelial cells and gingival epithelial cells were more intensively stained for cathepsin K and also more frequent in periodontitis than in controls (665 ± 104 vs 258 ± 40 cells mm(-2) , P < 0.01). Some cathepsin K(+) cells in periodontal tissues were CD68(+) , but some were CD68(-) and probably fibroblasts. Indeed, in gingival fibroblast culture, resting fibroblasts released cathepsin K, more 43 kD procathepsin K than 29 kD active cathepsin K. TNF-α increased the release of the activated cathepsin K 4- to 5-fold. CONCLUSIONS: Results suggest that GCF-cathepsin K is not only osteoclast-derived, but in periodontitis, also other cells contribute to it. GCF-cathepsin K, perhaps together with intracellular, lysosomal collagenolytically active cathepsin K in fibroblasts, macrophages and gingival epithelial cells, can contribute to the loss of attachment and destruction of the periodontal ligament.

The effect of adjunctive chlorhexidine mouthrinse on GCF MMP-8 and TIMP-1 levels in gingivitis: a randomized placebo-controlled study.

BACKGROUND: The aim of the present study was to evaluate the effect of adjunctive chlorhexidine (CHX) mouthrinse on gingival crevicular fluid (GCF) MMP-8 and TIMP-1 levels in plaque-associated gingivitis. METHODS: A total of 50 gingivitis patients were included in the present study. In addition to daily plaque control, CHX group rinsed with CHX, while placebo group rinsed with placebo mouthrinse for 4 weeks. GCF samples were collected, and clinical parameters including plaque index, papillary bleeding index, calculus index and pocket depth were recorded at baseline and 4 weeks. GCF MMP-8 and TIMP-1 levels were determined by immunofluorometric assay (IFMA) and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: In both groups, GCF MMP-8 levels of anterior and posterior sites at four weeks were not different from baseline (p > 0.05). There were no significant differences in GCF MMP-8 levels between the study groups at four weeks (p > 0.05). GCF TIMP-1 levels of anterior and posterior sites at four weeks were higher compared to baseline in both groups (p < 0.05). There was no significant difference in GCF TIMP level between the study groups at four weeks (p > 0.05). CONCLUSIONS: CHX usage had no significant effects on the GCF MMP-8 and TIMP-1 levels in plaque-associate gingivitis. However, daily plaque control resulted in the increase of GCF TIMP-1 levels regardless of CHX usage.

The relationship between matrix metalloproteinases (MMP-3, -8, -9) in serum and peripheral lymphocytes (CD8+ , CD56+ ) in Down syndrome children with gingivitis.

BACKGROUND AND OBJECTIVE: Altered immune response may be a major contributor to periodontal disease in Down syndrome. This study investigated the relationship between peripheral lymphocytes and matrix metalloproteinases (MMPs) in serum in Down syndrome children with gingivitis. MATERIAL AND METHODS: Children with Down syndrome (n = 10) and healthy controls (n = 10) were clinically and radiographically examined during dental treatment under general anaesthesia. Peripheral blood and gingival crevicular fluid were collected from each subject and concentrations were determined: serum MMP-2, -3, -8 and -9; serum tissue inhibitors of metalloproteinases (TIMP) -1, -2 and -3; and gingival crevicular fluid. Leukocytes were isolated from peripheral blood and the relative amounts (%) of the various cell phenotypes were analysed using flow cytometry. In addition, peripheral blood cells were treated with Porphyromonas gingivalis lipopolysaccharide and levels of MMPs and TIMPs measured. RESULTS: Concentrations of MMP-3, MMP-8 and TIMP-1 in serum were significantly higher (p < 0.05) in the Down syndrome group compared to the controls. When peripheral blood leukocytes were cultured in the presence or absence of P. gingivalis lipopolysaccharide, MMP-8 levels were significantly (p < 0.05) higher in the Down syndrome group compared to controls. Children with Down syndrome exhibited significant positive correlations between CD8(+) T cells and MMP-8 (r = 0.630; p = 0.050), between CD8(+) T cells and MMP-9 (r = 0.648; p = 0.043), and between CD56(+) NK cells and MMP-3 (r = 0.828; p = 0.003) compared to controls. CONCLUSIONS: The positive relationship of serum MMP-3, -8 and -9 with immune cells in children with Down syndrome may facilitate migration of CD8(+) T cells and CD56(+) NK cells into the periodontal tissue, which may contribute to the increased degradation of periodontal tissue in individuals with Down syndrome.