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Background and Objective Bleeding on probing (BOP) is a widely accepted measure used in periodontal diagnostics. Previous studies suggest that several factors can affect BOP propensity. The aim of this study was to investigate the relative impact of different local and modifying factors on BOP levels. Materials and Methods The oral health of five hundred and forty-four adolescents (two birth cohorts) aged 15-17 years living in Kotka, Finland, was examined including periodontal probing depth, visible plaque index, root calculus, and BOP. Whole saliva samples were collected and measured for active matrix metalloproteinase-8 (aMMP-8) by time-resolved immunofluorometric assay (IFMA). Results Bacterial plaque/calculus accumulation (oral hygiene) had a major influence on BOP levels. The relative impact was several times greater compared with the extent of periodontal pocketing, aMMP-8 levels, smoking, toothbrushing, or gender. Furthermore, BOP levels were significantly elevated among adolescents with poor oral hygiene than good oral hygiene even if adjusted for the extent of periodontal pocketing (P = 4 mm deep periodontal pockets existed. The difference in the extent of periodontal pocketing was not significant between the two birth cohorts of adolescents (P = .731). Conclusions BOP levels can be regarded as an important indicator of the extent of bacterial challenge and its adverse effects on the gingival inflammation. However, the level of oral hygiene may mask the association between the extent of gingival bleeding and the severity of the periodontal inflammatory condition. Thus, relying on BOP levels (below 10% or 20%) may provide insufficient information about the periodontal treatment need of an adolescent depending on his/her level of oral hygiene. Yet, more research is needed to confirm the results, also in adult populations.

Publisher Copyright: © 2022 The Authors. Journal of Periodontal Research published by John Wiley & Sons Ltd. Objective: The aim of this study was to determine the diagnostic utility of an MMP-8 biosensor assay in differentiating periodontal health from gingivitis and periodontitis and compare it with an established time-resolved immunofluorescence assay (IFMA) and enzyme-linked immunosorbent assay (ELISA). Background: Currently available antibody-based assays display a wide variability in their ability to accurately measure matrix metalloproteinase-8 (MMP-8) levels in saliva. Methods: Salivary MMP-8 levels were analyzed in 189 systemically healthy participants using an antibody-based biosensor prototype that operates using a surface acoustic wave technology and compared with IFMA and ELISA antibody assays. Participants were categorized into 3 groups: periodontal health (59), gingivitis (63), and periodontitis (67). A sub-population of participants (n = 20) with periodontitis received periodontal treatment and were monitored for 6 months. Results: All the assays demonstrated significantly higher salivary MMP-8 concentrations in participants with periodontitis versus gingivitis, periodontitis versus health, and gingivitis versus health (all p

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.

The objective of this study was to evaluate the levels of MMP-8, MMP-9, TIMP-1 and interleukin-1beta (IL-1β) in gingival crevicular fluid during the early and late stages of healing in gingival recession sites treated with coronally advanced flap plus platelet-rich fibrin (CAF+PRF) compared with CAF plus connective tissue graft (CAF+CTG).Twenty-four nonsmoking patients with Miller Class I or Class II localized gingival recession defects in bilateral sites received treatment with either CAF+PRF (PRF group) or CAF+CTG (CTG group). Gingival crevicular fluid samples were collected at baseline and at 10 d and 1 mo, 3 mo and 6 mo after surgery. The levels of MMP-8, MMP-9, TIMP-1 and IL-1β in gingival crevicular fluid were measured using a time-resolved immunofluorometric assay and ELISAs.Gingival crevicular fluid levels of IL-1β were significantly elevated in the CTG group at 10 d compared with baseline (p 0.05). At 10 d after surgery, the levels of TIMP-1 in gingival crevicular fluid showed a significant decrease in the CTG group compared with the PRF group (p 0.05). The levels of IL-1β and MMP-8 in gingival crevicular fluid were significantly lower in the PRF group than in the CTG group at 10 d (p 0.05). No significant differences were found in all clinical and biochemical parameters at 1, 3, and 6 mo between study groups (p 0.05).Root coverage with CAF+PRF has a significant effect on increasing gingival crevicular fluid TIMP-1 levels and suppressing gingival crevicular fluid MMP-8 and IL-β levels at 10 d. Gingival crevicular fluid levels of MMP-8, MMP-9, TIMP-1 and IL-1β did not seem to be affected by the technique at later phases of wound healing.

Emingil G, Han B, Ozdemir G, Tervahartiala T, Vural C, Atilla G, Baylas H, Sorsa T. The effect of azithromycin, as an adjunct to nonsurgical periodontal treatment, on microbiological parameters and gingival crevicular fluid biomarkers in generalized aggressive periodontitis. J Periodont Res 2012; 47: 729–739. © 2012 John Wiley & Sons A/S Background and Objective: To study the effectiveness of azithromycin in combination with nonsurgical periodontal therapy on clinical and microbiological parameters, and on the MMP-8 and TIMP-1 levels in gingival crevicular fluid, over a 6-mo time-period in patients with generalized aggressive periodontitis. Material and Methods: Thirty-two patients with generalized aggressive periodontitis were included in this randomized, double-blind, placebo-controlled, parallel-arm study. They were randomly assigned to azithromycin or placebo groups (500 mg once daily for 3 d). Probing depth, clinical attachment levels, presence of bleeding on probing and plaque were recorded. Gingival crevicular fluid samples were obtained from one single-rooted tooth, while microbiological samples were obtained from two single-rooted teeth, all with a probing depth of ≥ 6 mm. Microbiological parameters were analyzed by quantitative real-time PCR for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Prevotella intermedia and total bacteria. Gingival crevicular fluid biomarkers were determined by immunofluorometric assay and ELISA. Results: All clinical parameters improved, and microbiological parameters and gingival crevicular fluid MMP-8 levels significantly decreased, over the 6-mo period (p

Mantyla P, Buduneli E, Emingil G, Tervahartiala T, Pussinen PJ, Baris N, Akilli A, Atilla G, Sorsa T. Acute myocardial infarction elevates serine protease activity in saliva of patients with periodontitis. J Periodont Res 2012; 47: 345–353. © 2011 John Wiley & Sons A/S Background and Objective: There are indications that acute myocardial infarction (AMI) may have an effect on the oral environment, which is reflected in the expression of salivary and gingival proteinases. According to our knowledge, no studies have been carried out to investigate the effect of AMI on the activities of two major tissue-destructive serine protease and microbial effectors, elastase and cathepsin G, produced by oral fluid polymorphonuclear granulocytes (PMN). Therefore, we compared the activities of elastase and cathepsin G in saliva from patients with AMI and from systemically healthy subjects (non-AMI) with similar periodontal conditions. Material and Methods: A total of 92 patients (47 AMI and 28 non-AMI patients with gingivitis or periodontitis, and 17 systemically and periodontally healthy subjects as a control group) were recruited. Clinical periodontal measurements were recorded, and stimulated whole-saliva samples were collected. The patients with AMI were clinically examined within 3–4 d after admission to the coronary care unit. The activities of saliva neutrophil elastase and cathepsin G were measured after collection, at specific time-points during incubation (from baseline to 23 h) by specific synthetic peptide substrate assays. Results: The saliva of patients with AMI and periodontitis had a significant trend for the highest elastase activities among the study groups. Elastase and cathepsin G activities correlated significantly with each other in the AMI periodontitis group (r = 0.8, p

This work was supported by grants from the Academy of Finland, the Research Foundation of the Helsinki University Central Hospital, and the Scientific and Technologic Investigation Resource FONDECYT 1090461, Santiago, Chile. The authors are thankful to Professor Carlos Lo´ pez- Oti´n for providing MMP-8 KO mice

Background and Objective: Tissue destruction caused by periodontitis may increase the number of cytokines implicated in the pathogenesis of cardiovascular diseases. We measured the concentration of the leukocyte-derived proteolytic enzyme, salivary neutrophil collagenase-2 [matrix metalloproteinase-8 (MMP-8)], as a marker of periodontal disease and assessed its relationship to coronary heart disease (CHD). Our aim was to study whether salivary MMP-8 levels were different among patients with and without CHD. The hypothesis was that patients with heart disease might present higher salivary MMP-8 levels than cardiologically healthy controls. Material and Methods: Saliva samples were taken from 256 patients with CHD and from 250 matched controls with known oral and general health status. The MMP-8 levels in saliva were analyzed by immunofluorometric assay, salivary albumin was assessed by enzyme-linked immunosorbent assay (ELISA) and total protein was determined using the colorimetric method. We further investigated the molecular forms and isoform distribution of salivary MMP-8 by western immunoblotting. The MMP-8 results were adjusted for the number of teeth and salivary protein concentrations. Results: The adjusted logarithmic MMP-8 values were 0.145 ± 0.245 µg/l in patients with CHD and 0.088 ± 0.115 µg/l in controls (p

Background and objectives: Application of capsaicin on alveolar mucosa provokes pain and neurogenic vasodilatation in the adjacent gingiva. Pain-associated inflammatory reactions may initiate expression of several pro-inflammatory mediators. Collagenase-2 (matrix metalloproteinase-8: MMP-8) is the major destructive protease, especially in the periodontitis-affected gingival crevice fluid (GCF). With this background, we wished to study whether capsaicin stimulation of alveolar mucosa can induce changes in the GCF MMP-8 levels. Material and methods: For 10 generally and periodontally healthy human volunteers, capsaicin (3%)-moistened filter paper was applied unilaterally to the buccal alveolar mucosa on the anterior maxilla. GCF samples were collected from the tooth at the stimulation site and from several other incisors in the upper jaw. MMP-8 levels and molecular forms in GCF samples were determined by immunofluorometric assay (IFMA) and western immunoblotting, respectively. Results: Capsaicin stimulation of the alveolar mucosa induced significant local elevations in levels and activation of MMP-8 in GCF of the adjacent teeth. Western immunoblot revealed that both neutrophil- and mesenchymal-type MMP-8 isoforms were elevated and activated, together with 110 kDa high-molecular size MMP-8 species. This capsaicin-evoked MMP-8 elevation lasted several minutes after stimulation. During the experiments, no marked changes occurred in MMP-8 levels in the GCF of distantly located teeth. Conclusions: These results suggest that capsaicin-evoked neurogenic gingival inflammation can trigger the expression and activation of MMP-8 in GCF of the adjacent teeth.

OBJECTIVES Matrix metalloproteinases (MMPs) play crucial role in various tissue destructive inflammatory processes by degrading almost all peri-cellular and basement membrane components. MMP-8 (collagenase-2) is the major MMP in periodontitis. MMP-7 (matrilysin-1), in addition to its ability to degrade matrix and basement membrane components, activates other latent pro-MMPs and defensins, host cell-derived antimicrobial cryptidins. The aim of the present study was to characterize the relationship, levels and molecular forms of MMP-8 and MMP-7 in diseased peri-implant sulcular fluid (PISF). MATERIALS AND METHODS Seventy-two human dental implant fluid samples were collected with filter paper strips from peri-implant sulci from healthy and untreated diseased implant sites. Gingival index (GI) and/or bone resorption (BR) were also recorded. Western immunoblot method with polyclonal anti-human-MMP-8 and monoclonal anti-human-MMP-7 antibodies was used, and immunoreactivities were quantified with computer scanning program. The effects of MMP inhibitors (doxycycline, chemically modified tetracycline-3, clodronate, CTT-peptide and marimastat) were studied on the activity of recombinant human matrilysin-1 (MMP-7) using beta-casein degradation assay. RESULTS The levels of active forms of MMP-8 and MMP-7 were significantly elevated in diseased PISF in relation to healthy PISF. Furthermore, MMP-8 and MMP-7 levels correlated significantly to each other and GI. MMP-8 was present not only as bands corresponding to 75-kDa polymorphonuclear leukocyte (PMN) -type pro- and 65-kDa active forms, but also as 55-kDa non-PMN-type pro- and 45-kDa active forms. Immunoreactivities > 80 kDa most likely represented dimeric and/or inhibitor-bound MMP-8 complexes and the low molecular weight (< 30 kDa) species were apparently degraded fragments. In diseased PISF, 19-21-kDa active MMP-7 and 28-30-kDa pro-MMP-7 species were detected, and the active 19-21-kDa forms of MMP-7 predominated in diseased PISF. Doxycycline (50 micro m and 250 micro m), chemically modified non-antimicrobial tetracycline (CMT-3) (50 micro m and 100 micro m), clodronate (a bisphosphonate, 20 micro m and 500 micro m) and the cyclic CTT (CTTHWGFTLC)-peptide (125 micro m and 250 micro m), all known broad-spectrum or selective MMP-inhibitors, did not inhibit the activity of human recombinant MMP-7; only marimastat (1 micro m and 5 micro m) inhibited MMP-7. DISCUSSION Increased immunoreactivities of the active MMP-8 and MMP-7 species in PISF from diseased peri-implantitis lesions eventually reflect the stage and course of peri-implantitis; MMP-7 may potentially act as MMP-8 and defensin activator in diseased PISF. CONCLUSION The elevated levels of MMP-8 and matrilysin-1/MMP-7 were identified in active forms in diseased PISF, but MMP-7 was less prominent. MMP inhibitors, potential future tissue protective drugs, seemingly do not interfere with the defensive antibacterial action of MMP-7.

Gursoy M, Kononen E, Tervahartiala T, Gursoy UK, Pajukanta R, Sorsa T. Longitudinal study of salivary proteinases during pregnancy and postpartum. J Periodont Res 2010; 45: 496–503. © 2010 John Wiley & Sons A/S Background and Objective: Matrix metalloproteinases (MMPs) and their regulators are connected to periodontal inflammation and destruction. However, the presence and role of the salivary MMPs in pregnancy-related gingivitis are not well known. Our longitudinal study aimed to monitor salivary proteinase levels and possible changes, and relate them to periodontal status during pregnancy and postpartum. Material and Methods: Salivary samples were collected from 30 periodontally healthy pregnant women five times (once during each trimester, 4–6 wk after delivery and after lactation) and, as their controls, from 24 non-pregnant women three times (during successive months). Periodontal examination included visible plaque index, bleeding on probing, probing pocket depth and clinical attachment level measurements. Matrix metalloproteinase-8 levels were measured by immunofluorometric assay, and MMP-2 and MMP-9 levels and molecular forms by gelatin zymography. Salivary elastase, myeloperoxidase and tissue inhibitor of matrix metalloproteinase-1 levels were measured by ELISA. Results: Elastase concentrations maintained stable during the follow-up, while myeloperoxidase concentrations increased significantly after delivery. During pregnancy, MMP-8 concentrations were significantly lower than postpartum concentrations, being lowest during the second trimester and highest after delivery, and varying inversely to pregnancy gingivitis, observed as elevated percentages of bleeding on probing and probing pocket depth during the second and third trimester. In pregnant women, the highest MMP-2 and MMP-9 levels were found in saliva after lactation. In the control group, both clinical and enzymological findings remained stable during the follow-up period. Conclusion: Our results suggest that hormonal changes during pregnancy induce or enhance susceptibility to gingivitis, while salivary proteinase and myeloperoxidase levels are reduced.

Matrix metalloproteinases (MMPs) play crucial role in various tissue destructive inflammatory processes by degrading almost all peri-cellular and basement membrane components. MMP-8 (collagenase-2) is the major MMP in periodontitis. MMP-7 (matrilysin-1), in addition to its ability to degrade matrix and basement membrane components, activates other latent pro-MMPs and defensins, host cell-derived antimicrobial cryptidins. The aim of the present study was to characterize the relationship, levels and molecular forms of MMP-8 and MMP-7 in diseased peri-implant sulcular fluid (PISF).Seventy-two human dental implant fluid samples were collected with filter paper strips from peri-implant sulci from healthy and untreated diseased implant sites. Gingival index (GI) and/or bone resorption (BR) were also recorded. Western immunoblot method with polyclonal anti-human-MMP-8 and monoclonal anti-human-MMP-7 antibodies was used, and immunoreactivities were quantified with computer scanning program. The effects of MMP inhibitors (doxycycline, chemically modified tetracycline-3, clodronate, CTT-peptide and marimastat) were studied on the activity of recombinant human matrilysin-1 (MMP-7) using beta-casein degradation assay.The levels of active forms of MMP-8 and MMP-7 were significantly elevated in diseased PISF in relation to healthy PISF. Furthermore, MMP-8 and MMP-7 levels correlated significantly to each other and GI. MMP-8 was present not only as bands corresponding to 75-kDa polymorphonuclear leukocyte (PMN) -type pro- and 65-kDa active forms, but also as 55-kDa non-PMN-type pro- and 45-kDa active forms. Immunoreactivities80 kDa most likely represented dimeric and/or inhibitor-bound MMP-8 complexes and the low molecular weight (30 kDa) species were apparently degraded fragments. In diseased PISF, 19-21-kDa active MMP-7 and 28-30-kDa pro-MMP-7 species were detected, and the active 19-21-kDa forms of MMP-7 predominated in diseased PISF. Doxycycline (50 micro m and 250 micro m), chemically modified non-antimicrobial tetracycline (CMT-3) (50 micro m and 100 micro m), clodronate (a bisphosphonate, 20 micro m and 500 micro m) and the cyclic CTT (CTTHWGFTLC)-peptide (125 micro m and 250 micro m), all known broad-spectrum or selective MMP-inhibitors, did not inhibit the activity of human recombinant MMP-7; only marimastat (1 micro m and 5 micro m) inhibited MMP-7.Increased immunoreactivities of the active MMP-8 and MMP-7 species in PISF from diseased peri-implantitis lesions eventually reflect the stage and course of peri-implantitis; MMP-7 may potentially act as MMP-8 and defensin activator in diseased PISF.The elevated levels of MMP-8 and matrilysin-1/MMP-7 were identified in active forms in diseased PISF, but MMP-7 was less prominent. MMP inhibitors, potential future tissue protective drugs, seemingly do not interfere with the defensive antibacterial action of MMP-7.