Bacterial profiles and proteolytic activity in peri-implantitis versus healthy sites.

Peri-implantitis is a biofilm-induced destructive inflammatory process that, over time, results in loss of supporting bone around an osseointegrated dental implant. Biofilms at peri-implantitis sites have been reported to be dominated by Gram-negative anaerobic rods with a proteolytic metabolism such as, Fusobacterium, Porphyromonas, Prevotella and Tannerella, as well as anaerobic Gram-positive cocci. In this study, we hypothesized that protease activity is instrumental in driving bone destruction and we therefore compared the microbial composition and level of protease activity in samples of peri-implant biofluid (PIBF) from 25 healthy subjects (H group) and 25 subjects with peri-implantitis (PI group). Microbial composition was investigated using culture techniques and protease activity was determined using a FITC-labelled casein substrate. The microbial composition was highly variable in subjects both in the H and PI groups but one prominent difference was the prevalence of Porphyromonas/Prevotella and anaerobic Gram positive cocci which was significantly higher in the PI than in the H group. A subgroup of subjects with peri-implantitis displayed a high level of protease activity in the PIBF compared to healthy subjects. However, this activity could not be related to the presence of specific bacterial species. We propose that a high level of protease activity may be a predictive factor for disease progression in peri-implantitis. Further longitudinal studies are however required to determine whether assessment of protease activity could serve as a useful method to identify patients at risk for progressive tissue destruction.

Gingival status, crevicular fluid tissue-type plasminogen activator, plasminogen activator inhibitor-2 levels in pregnancy versus post-partum.

BACKGROUND: This study was conducted to evaluate a possible link between periodontal status of pregnant women and the plasminogen activator system in gingival crevicular fluid (GCF). METHODS: GCF samples were obtained from four interproximal sites of anterior teeth in 43 women during the second trimester and also after delivery. Full mouth dental plaque, bleeding on probing (BOP) and probing depth (PD) values were recorded at six sites/tooth in each subject. GCF levels of tissue type plasminogen activator (t-PA) and its inhibitor, plasminogen activator-inhibitor-2 (PAI-2) were determined by ELISA. Data comparisons between pregnancy and post-partum were made by Wilcoxon signed rank test. RESULTS: The number of pockets with a PD>4 mm and total volume of GCF sampled were reduced significantly after delivery (p=0.000 and p=0.013, respectively). No significant differences were detected in GCF concentrations of t-PA or PAI-2 between pregnancy and post-partum. CONCLUSIONS: Our results suggest that GCF t-PA and PAI-2 concentrations are not affected by pregnancy. Reductions in PD values and GCF volume following delivery indicate a resolution of oedema in gingival tissues, possibly related to hormonal changes due to the ending of pregnancy.

Mucins MUC5B and MUC7 in minor salivary gland secretion of children and adults.

OBJECTIVE: The study was designed to investigate the relative amount of MUC5B and MUC7 in minor salivary glands in children and adults, in order to test the hypothesis that secretion of salivary mucins changes between childhood and adulthood. METHODS: Ninety individuals in the age-groups 3-year-olds, 14-year-olds, and young adults 20-25 year-olds were recruited. Sialopapers were applied on the labial and the buccal mucosa and then placed in the Periotron 8,000 (Proflow ) for calculation of the amount of saliva. The assessment of MUC5B and MUC7 was carried out in an ELISA using the LUM5B-2 and the LUM7-1 antiserum, respectively. RESULTS: MUC5B and MUC7 were detected in the labial minor gland saliva in all age groups. In buccal gland saliva, only a few individuals in each age group showed detectable amounts of the mucins. In the labial area, a significantly lower level of MUC7 was noted in 3-year-olds compared with adults. CONCLUSION: The results indicate a site-dependent difference in minor gland mucin secretion and an age-related difference in the labial gland secretion of MUC7.

Relationship between fibrinolytic activity and gingival inflammatory reaction in young individuals.

The fibrinolytic system (the plasminogen activating system) is involved in several physiological and pathological processes. Through the transformation of plasminogen to the aggressive broad spectrum protease plasmin, potent enzymatic activity is released. Plasmin acts directly on connective tissue components, and indirectly by activating proforms of the metalloproteinases. The destructive potential of the fibrinolytic system may thus be of importance for the initiation and progression of periodontal diseases. Earlier studies have shown high concentrations of the plasminogen activator t-PA and its inhibitor PAI-2 in gingival crevicular fluid (GCF) as well as enhanced concentrations in areas of gingival inflammation. The aim of this study was to investigate a possible relationship between the gingival inflammatory reactivity and the fibrinolytic activity in gingival crevicular fluid. Thirty-one young individuals took part in the study. Gingival Index scores and Plaque Index scores were assessed and used to formulate a score expressing an individuals' inflammatory response to microbial plaque levels (Relative G/P score). The fibrinolytic activity of GCF was assessed with a fibrin gel lysis assay, and the levels of t-PA and PAI-2 were assayed with ELISAs. All samples showed fibrinolytic activity. A positive correlation between the fibrinolytic activity and Relative G/P score was found. Thus, in individuals with an enhanced reactivity to dental plaque, a higher plasminogen activating activity in GCF was seen. This indicates a higher potential for tissue proteolysis in these individuals, possibly facilitating spread and deeper involvement of the lesions.

The localization of the relaxed form of plasminogen activator inhibitor type 2 in human gingival tissues.

The plasminogen activating system is important in extracellular proteolysis. Plasmin degrades tissues and activates proteases. Plasminogen activators (tissue type; t-PA and urokinase type; u-PA) and plasminogen activator inhibitors (PAI-1, PAI-2) are found in high concentrations in gingival crevicular fluid (GCF). Previous findings indicate the significance of PAI-2 in gingival inflammation. When PAI-2 inhibits a plasminogen activator its conformation relaxes and neoepitopes can be detected with a monoclonal antibody (#2H5). Our aim was to study if and where in the gingival region PAI-2 has acted as an inhibitor. Methodological studies were performed on GCF with western blotting. Frozen sections of human gingiva were studied immunohistochemically. The methodological studies showed that our antibody #2H5 selectively detects relaxed low molecular weight non-glycosylated PAI-2. Total PAI-2 and relaxed PAI-2 were found in all gingival epithelia with a honeycomb-like staining. Relaxed PAI-2 showed the most pronounced staining in the cell layers near the surface of the epithelium and no staining in the suprabasal layers, while total PAI-2 was found throughout the epithelium, often more pronounced suprabasally. The results showed that PAI-2 indeed has acted as an inhibitor of a protease in gingival tissues, primarily in the epithelia. The results also suggest primarily an intracellular localization and thus the interaction of PAI-2 with a protein other than t-PA.

Increasing expression of tissue plasminogen activator and plasminogen activator inhibitor type 2 in dog gingival tissues with progressive inflammation.

Urokinase and tissue-type plasminogen activators (u--PA and t--PA) are serine proteases that convert plasminogen into plasmin, which degrades matrix proteins and activates metalloproteinases. The PAs are balanced by specific inhibitors (PAI--1 and PAI--2). Local production of t--PA and PAI--2 was recently demonstrated in human gingival tissues. The aim now was to investigate the production and localization of t--PA and PAI--2 in gingival tissues from dogs in three well-defined periodontal conditions; clinically healthy gingiva, chronic gingivitis and an initial stage of ligature-induced loss of attachment. At the start of the experiment the gingiva showed clear signs of inflammation. Clinically healthy gingiva were obtained after 21 days period of intense oral hygiene. Attachment loss was induced by placing rubber ligatures around the neck of some teeth. Biopsies were taken from areas representing the different conditions and prepared for in situ hybridization and immunohistochemistry. In clinically healthy gingiva both t--PA mRNA and antigen were expressed in a thin outer layer of the sulcular and junctional epithelia. No t--PA signals or staining were seen in connective tissue. Both mRNA signaling and immunostaining for t--PA were stronger in chronic gingivitis. In areas with loss of attachment, t--PA mRNA as well as antigen were found in the sulcular and junctional epithelia to a similar degree as in gingivitis. Occasionally the connective tissue was involved, especially in connection with vessels. PAI--2 mRNA was seen in a thin outer layer of the sulcular and junctional epithelia in clinically healthy gingiva, but no signals were seen in connective tissue. PAI--2 antigen was found primarily in the outer layer of the sulcular and junctional epithelia. Some cells in the connective tissue were stained. In gingivitis, PAI--2 signals were mainly found in the same locations, but more intense and extending towards the connective tissue. Immunostaining was seen in the outer half of the sulcular and junctional epithelia as well as in the upper part of the connective tissue, close to the sulcular epithelium. In sites with loss of attachment, PAI--2 mRNA was found throughout the sulcular and junctional epithelia, as was the antigen, which stained intensely. No PAI--2 mRNA was seen in connective tissue; the antigen was found scattered, especially near vessels. This study shows that the expression of both t--PA and PAI--2 increases with experimental gingival inflammation in the dog, and furthermore, the two techniques demonstrate a strong correlation between the topographical distribution of the site of protein synthesis and the tissue location of the antigens for both t--PA and PAI--2. The distribution correlates well with previous findings in humans.

Localization of plasminogen activators and plasminogen-activator inhibitors in human gingival tissues demonstrated by immunohistochemistry and in situ hybridization.

The plasminogen-activating system plays an important part in tissue proteolysis in physiological as well as pathological processes. Plasminogen activators u-PA (urokinase) and t-PA (tissue) as well as the inhibitors PAI-1 and PAI-2 are present in gingival crevicular fluid in concentrations significantly greater than in plasma. This fact, and the finding that the concentrations of t-PA and PAI-2 are higher in areas with gingival inflammation, indicate local production of these components. The present study describes, by means of in situ hybridization and immunohistochemistry, the localization of the plasminogen activators and their inhibitors in gingival tissues from patients undergoing periodontal surgery. t-PA mRNA and t-PA antigen were primarily found in the epithelial tissues, predominantly in the sulcular and junctional regions, although occasionally in the oral epithelium and in blood vessels of the connective tissue. u-PA and u-PA-receptor signals were seen in single cells within the junctional and sulcular epithelia and adjacent to blood vessels close to the junctional epithelium, but rarely in the oral epithelium. Similar to t-PA, the predominant location of PAI-2 mRNA was the gingival epithelia. In the junctional and sulcular epithelia, PAI-2 mRNA was seen throughout the thickness, while in the oral epithelium the strongest signals were seen in stratum granulosum and stratum spinosum. PAI-1 mRNA was invariably found in the connective tissue associated with blood vessels. The present study confirms earlier indications of local production of plasminogen activators and their inhibitors in gingival tissues. In addition, the results demonstrate that t-PA and PAI-2 in these patients are produced predominantly in the epithelial tissues. Furthermore, the presence of t-PA and PAI-2 seems to be most pronounced in the areas likely to be subjected to bacterial assault.

Aggravation of gingival inflammatory symptoms during pregnancy associated with the concentration of plasminogen activator inhibitor type 2 (PAI-2) in gingival fluid.

Gingival inflammatory symptoms are aggravated during pregnancy. In vitro studies suggest a hormonal influence on the plasminogen activator inhibitor type 2 (PAI-2), and a disturbed balance of the fibrinolytic system could help to explain pregnancy gingivitis. Gingival crevicular fluid (GCF) was sampled in 14 women in pregnant and post-pregnant states. The gingival condition was assessed by the gingival index of Løe & Silness (GI) and the amount of bacterial plaque by the plaque index of Silness & Løe (PI). The ratio of sites with gingivitis to sites with bacterial plaque was calculated (G/P-ratio). Antigen levels of tissue plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitors type 1 (PAI-1) and PAI-2 in GCF were determined with ELISAs and 17 beta-oestradiol and progesterone in serum with radioimmunoassays. For each individual the differences (delta) in hormone levels and PAs and PAIs between pregnancy and post-pregnancy were calculated. Based on differences in G/P-ratio between pregnancy and post-pregnancy, subgrouping was done into a high-reacting and a low-reacting group. For the total group, the mean G/P-ratio was 2.0 during and 1.2 after pregnancy (p = 0.064). A statistically significant correlation between delta progesterone and delta PAI-2 was noted: the higher delta progesterone, the lower delta PAI-2. No other significant correlations between hormone levels and components of the fibrinolytic system were found. For the total group of women, the concentrations of PAI-2, PAI-1 and t-PA were significantly higher during than after pregnancy. The individuals in the high-reacting group, however, showed a lower or unchanged production of PAI-2 during pregnancy, while those in the low-reacting group showed a greatly increased production. The lower inhibitory capacity in terms of a low production of PAI-2 during pregnancy in women with a higher inflammatory reaction indicates that the components of the fibrinolytic system may be involved in the development of pregnancy gingivitis and implies that PAI-2 serves as an inhibitor of importance for tissue proteolysis. The present finding contributes to the explanation of pregnancy gingivitis.

The plasminogen-activating system in gingival fluid from adults. An intra-individual study before and after treatment of gingivitis.

High concentrations of tissue plasminogen activator (t-PA) and placental type plasminogen activator inhibitor (PAI-2) have previously been found in gingival crevicular fluid (GCF) of adults and children. In the present study, intra-individual comparisons were made of the concentrations of t-PA, urokinase type plasminogen activator (u-PA), PAI-1, and PAI-2 in GCF from the same sites before and after periodontal treatment in eight healthy male volunteers aged 35-46 yr. The gingival state was assessed by exudate measurement, bleeding on standardized probing, and the gingival index of Löe & Silness 3 days before the start of the trial and on the day after completing a 21-day preventive program consisting of instruction and professional cleaning once a week. Eight sites per subject were selected for enzyme analyses, all showing improvement in gingival state during the period. Sampling of GCF at the start and at the end of the trial was done with small disks of Millipore-filter. t-PA and PAI-2 were analyzed with enzyme-linked immunosorbent assays with low method errors. The mean concentrations of t-PA were 0.73 mg/l before treatment and 0.49 mg/l after treatment. The mean concentrations of u-PA were 84.4 micrograms/l before treatment and 101.6 micrograms/l after treatment. PAI-1 was found in three subjects at the detection level. The mean PAI-2 concentrations were 2.19 mg/l before and 1.13 mg/l after treatment. The mean molar ratio PAs/PAI-2 was 0.47 before and 0.48 after treatment. This insignificant change implies a maintained proteolytic balance and indicates that PAI-2 is an important inhibitor of tissue proteolysis.

Tissue plasminogen activator (t-PA) and placental plasminogen activator inhibitor (PAI-2) in gingival fluid from 8-9-year-old children.

High concentrations of tissue plasminogen activator (t-PA) and placental type plasminogen activator inhibitor (PAI-2) have previously been found in gingival crevicular fluid (GCF) of adults. In the present study, the levels were examined in 16 children aged 8-9 yr. Sampling of GCF was performed with small disks of Millipore-filter. t-PA and PAI-2 were analyzed with enzyme-linked immunosorbent assays with low method errors. The mean concentration of t-PA was slightly higher than in adults, while the mean PAI-2-concentration was slightly lower. An intraindividual study comparing healthy and inflamed sites in the children showed slightly higher concentrations in GCF from inflamed sites. No change was observed in the balance between t-PA and PAI-2.

Irritant and sensitizing potential of copper, mercury and tin salts in experimental contact stomatitis of rat oral mucosa.

HgCl2, CuSO4, SnCl2, SnCl4 or sodium lauryl sulphate (SLS) were openly applied to rat oral mucosa for 1 min, followed 6 h later by histologic examination of the tissue response. Granulocytes were the predominant inflammatory cells and no lymphocytic infiltration could be seen with any of the substances tested. Irritant threshold levels were defined histologically for each of the substances. CuSO4 was found to be non-irritant at all concentrations. The addition of non-irritant concentrations of SLS lowered the threshold levels for HgCl2 and SnCl2, but CuSO4/SLS was non-irritant at all concentrations tested. Preapplication to the mucosa of SLS at non-irritant concentrations gave results with HgCl2, SnCl2 and CuSO4 similar to those with SLS added to the metal salt solutions. Lesions of allergic contact type could not be induced in the oral mucosa to any of the metal salt preparations.

Immunogenic potential of some mercury compounds in experimental contact allergy of the rat oral mucosa.

The contact allergenic potential of substances included in a "dental screening test" has been investigated in a previously established animal model for producing contact allergy. We were unable to produce contact hypersensitivity with any of the original formulas. HgCl2 was selected for further studies and, in order to improve its penetration, butanol was used as a solvent. Sensitization was, however, not obtained with any of the HgCl2/butanol formulas. Our results are discussed in relation to the requirements of reaching immunogenic threshold levels of mercury in the tissue.