Päivi Mäntylä

Matrix metalloproteinases: Contribution to pathogenesis, diagnosis and treatment of periodontal inflammation

Matrix metalloproteinases (MMPs) form a family of enzymes that mediate multiple functions both in the tissue destruction and immune responses related to periodontal inflammation. The expression and activity of MMPs in non‐inflamed periodontium is low but is drastically enhanced to pathologically elevated levels due to the dental plaque and infection‐induced periodontal inflammation. Soft and hard tissue destruction during periodontitis and peri‐implantitis are thought to reflect a cascade of events involving bacterial virulence factors/enzymes, pro‐inflammatory cytokines, reactive oxygen species and MMPs. However, recent studies suggest that MMPs can also exert anti‐inflammatory effects in defence of the host by processing anti‐inflammatory cytokines and chemokines, as well as by regulating apoptotic and immune responses. MMP‐inhibitor (MMPI)‐drugs, such as doxycycline, can be used as adjunctive medication to augment both the scaling and root planing‐treatment of periodontitis locally and to reduce inflammation systematically. Furthermore, MMPs present in oral fluids (gingival crevicular fluid (GCF), peri‐implant sulcular fluid (PISF), mouth‐rinses and saliva) can be utilized to develop new non‐invasive, chair/bed‐side, point‐of‐care diagnostics for periodontitis and dental peri‐implantitis.

Scientific Basis of a Matrix Metalloproteinase‐8 Specific Chair‐side Test for Monitoring Periodontal and Peri‐implant Health and Disease

ABSTRACT: Matrix metalloproteinases (MMPs), especially collagenase‐2 (MMP‐8), are key mediators of irreversible tissue destruction associated with periodontitis and peri‐implantitis. MMP‐8 is known to exist in elevated amounts and in active form in the gingival crevicular fluid (GCF) and peri‐im‐plant sulcular fluid (PISF) from progressing periodontitis and peri‐implantitis lesions and sites, respectively. (Sorsa et al. Ann. N.Y. Acad. Sci. 737: 112‐131 [1994]; Teronen et al. J. Dent. Res. 76: 1529‐1537 [1997]). We have developed monoclonal antibodies to MMP‐8 (Hanemaaijer et al. J. Biol. Chem. 272: 31504‐31509 [1997]) that can be used in a chair‐side dipstick test to monitor the course and treatment of periodontitis and peri‐implantitis. Monoclonal and polyclonal antibody tests for MMP‐8 coincided with the classical functional collagenase activity test from GCF and PISF (Sorsa et al. J. Periodont.Res. 22: 386‐393 [1988]) in periodontal and peri‐implant health and disease. In future a chair‐side functional and/or immunological MMP‐test can be useful to diagnose and monitor periodontal and peri‐implant disease and health.

Detection of gingival crevicular fluid MMP‐8 levels with different laboratory and chair‐side methods

OBJECTIVE The aim of the study was to compare four methods for gingival crevicular fluid (GCF) matrix metalloproteinase (MMP)-8 detection. METHODS Matrix metalloproteinase-8 levels from 20 GCF samples from two periodontally healthy subjects, 18 samples from two patients with gingivitis and 45 samples from six patients with moderate to severe periodontitis, altogether 83 samples, were analysed using (1) a time-resolved immunofluorometric assay (IFMA), (2) an MMP-8 specific chair-side dip-stick test, (3) a dentoAnalyzer device and (4) the Amersham ELISA kit. Western immunoblot using same monoclonal anti-MMP-8 as in IFMA and dentoAnalyzer was used to identify molecular forms of MMP-8 in GCFs. RESULTS Correlation between IFMA and dentoAnalyzer results calculated with Spearmans correlation coefficient was 0.95 (P = 0.01). The chair-side dip-stick test results were well in line with these assays. Periodontitis sites with unstable characteristics were differentiated with these methods. The Amersham ELISA results were not in line with the findings by other methods. CONCLUSIONS Immunofluorometric assay and dentoAnalyzer can detect MMP-8 from GCF samples and these methods are comparable. Using Western immunoblot, it was confirmed that IFMA and dentoAnalyzer can detect activated 55 kDa MMP-8 species especially in periodontitis-affected GCF. dentoAnalyzer is among the first quantitative MMP-8 chair-side testing devices in periodontal and peri-implant diagnostics and research.

Collagenase-2 (MMP-8) as a point-of-care biomarker in periodontitis and cardiovascular diseases. Therapeutic response to non-antimicrobial properties of tetracyclines

Neutrophil collagenase or collagenase-2 (matrix metalloproteinase [MMP]-8) belongs to the collagenase subgroup of the MMP superfamily of calcium- and zinc-dependent neutral proteinases. MMP-8 is catalytically the most competent proteinase to initiate type I collagen and extracellular matrix degradation associated with periodontal and peri-implant tissue destruction leading to tooth and dental implant loss. Regarding cardiovascular diseases, pathologically excessive MMP-8 has been implicated in atherosclerotic plaque destabilization and rupture probably through its proteolytic ability to thin the protecting collagenous fibrous cap lining coronary and other arteries. During the initiation and course of inflammatory responses in periodontitis, peri-implantitis and cardiovascular diseases, proinflammatory mediators including especially MMP-8 are up-regulated not only in affected tissues but also in the secreted, disease-affected, oral fluids (gingival crevicular fluid [GCF], peri-implant sulcular fluid [PISF], mouthrinse and saliva) as well as in serum and plasma. Regarding periodontitis, peri-implantitis and cardiovascular diseases, the oral fluid and serum MMP-8 analysis has proven to be a sensitive and an objective biomarker as an indicator of health, pathologic processes and pharmacologic response to therapeutic intervention including doxycycline medication as an MMP inhibitor. Oral fluids, i.e., GCF, PISF, mouthrinse and saliva are easily and non-invasively collected for the site- and patient-specific diagnostic analysis in periodontitis and peri-implantitis, whereas serum and/or plasma sample collection is required for diagnosis and monitoring of cardiovascular diseases. Research in periodontology and cardiology has identified a need for the development of innovative point-of-care diagnostic tests for MMP-8. We summarize and review the recent studies on these topics.

Serum microbial- and host-derived markers of periodontal diseases: a review.

Periodontitis is bacterial infection of tooth-supporting tissues leading to inflammation and, subsequently, to loss of teeth. It is one of the most common infections worldwide. Recent studies have shown that periodontal infection may pose a threat to general health by increasing the risk of cardiovascular and lung diseases, and preterm labour. Thus, useful markers of systemic exposure to periodontitis are needed. Markers of periodontitis in serum include those derived directly from periodontopathic pathogens and those originating from the host defence and immune mechanisms. Periodontitis is associated with endotoxemia, which can be directly measured as elevated concentrations of lipopolysaccharide (LPS) in periodontitis patients compared with healthy subjects. Also indirect methods determining endotoxemia, such as elevated concentrations of serum LPS binding protein, soluble CD14, and antibodies to LPS of periodontal pathogens have been reported. Surrogate measures of the host response against periodontal infection, such as matrix metalloproteinases, cytokines, chemokines, inflammation markers, antiphospholipid antibodies, and antibodies to periodontal pathogens, have been used. Of these, however, only antibodies to periodontal pathogens may be seen as specific markers of systemic exposure to periodontopathic pathogens. In this paper we describe and discuss serum markers of periodontitis that have been used for research purposes and/or to support diagnostics. Based on literature review, we encourage research and development of serum screening methods for periodontitis that could be used by general physicians.

Associations Between Matrix Metalloproteinase-8 and -14 and Myeloperoxidase in Gingival Crevicular Fluid From Subjects With Progressive Chronic Periodontitis: A Longitudinal Study

BACKGROUND Matrix metalloproteinase (MMP)-8 is a central mediator in chronic periodontitis. MMP-8 can be activated by the cooperative action of other MMPs such as MMP-14, reactive oxygen species, and microbial proteases. The aim of this study is to associate the levels, molecular forms, isoenzyme distribution, and degree of activation of MMP-8 and -14, myeloperoxidase (MPO), and tissue inhibitor of MMP (TIMP)-1 in gingival crevicular fluid (GCF) from patients with progressive periodontitis at baseline and after periodontal therapy. METHODS In this longitudinal study, GCF samples from active (n = 25) and inactive (n = 25) sites of subjects with periodontitis were screened at baseline for GCF levels of MMP-8 by immunofluorometric assay, of MMP-14 by specific activity assay, and of MPO and TIMP-1 by enzyme-linked immunosorbent assay. MMP-8 and MPO were also measured after periodontal treatment. Molecular forms were determined by immuno-Western blot analyses and subjected to densitometric scanning and statistical analyses. RESULTS High MMP-8 and MPO levels and a strong MPO/MMP-8 positive correlation were found in active and inactive sites at baseline. After treatment, decreases in MPO and MMP-8 were seen, except for active sites in which MMP-8 differences were not significant (P <0.05). CONCLUSIONS We present initial data that show that GCF levels and associations between MPO and MMP-8 are related to progression episodes and treatment responses in patients with chronic periodontitis. Our results suggest an interaction between the MPO oxidative pathway and MMP-8 activation, and this cascade might be useful as a potential biomarker for treatment outcomes.

Matrix metalloproteinases and myeloperoxidase in gingival crevicular fluid provide site‐specific diagnostic value for chronic periodontitis

AIM To identify the diagnostic accuracy of gingival crevicular fluid (GCF) candidate biomarkers to discriminate periodontitis from the inflamed and healthy sites, and to compare the performance of two independent matrix metalloproteinase (MMP)-8 immunoassays. MATERIALS AND METHODS Cross sectional study. GCF (N = 58 sites) was collected from healthy, gingivitis and chronic periodontitis volunteers and analysed for levels of azurocidin, chemokine ligand 5, MPO, TIMP-1 MMP-13 and MMP-14 by ELISA or activity assays. MMP-8 was assayed by immunofluorometric assay (IFMA) and ELISA. Statistical analysis was performed using linear mixed-effects models and Bayesian statistics in R and Stata V11. RESULTS MMP-8, MPO, azurocidin and total MMP-13 and MMP-14 were higher in periodontitis compared to gingivitis and healthy sites (p < 0.05). A very high correlation between MPO and MMP-8 was evident in the periodontitis group (r = 0.95, p < 0.0001). MPO, azurocidin and total levels of MMP-8, MMP-13 and MMP-14 showed high diagnostic accuracy (≥0.90), but only MMP-8 and MPO were significantly higher in the periodontitis versus gingivitis sites. MMP-8 determined by IFMA correlated more strongly with periodontal status and showed higher diagnostic accuracy than ELISA. CONCLUSIONS MPO and collagenolytic MMPs are highly discriminatory biomarkers for site-specific diagnosis of periodontitis. The comparison of two quantitative MMP-8 methods demonstrated IFMA to be more accurate than ELISA.

Analysis of matrix metalloproteinases, especially MMP-8, in gingival creviclular fluid, mouthrinse and saliva for monitoring periodontal diseases.

Matrix metalloproteinase-8 is a promising candidate biomarker for oral fluid (gingival crevicular fluid, peri-implant sulcular fluid and saliva) and mouthrinse chair-side/point-of-care diagnostics to predict, diagnose and determine the progressive phases of episodic periodontitis and peri-implantitis, as well as to monitor the treatments and medications. Matrix metalloproteinase-8 can be used alone or together with interleukin-1beta and Porphyromonas gingivalis to calculate cumulative risk score at the subject level as a successful diagnostic tool, especially in large-scale public health surveys, in which a thorough periodontal examination is not feasible.

Salivary biomarkers of bacterial burden, inflammatory response, and tissue destruction in periodontitis

AIM Chronic periodontitis has an episodic and multifactorial character, with fluctuations in bacterial burden, inflammatory response, and tissue destruction. We investigated the association of selected salivary biomarkers with periodontal parameters and validated the use of a novel salivary diagnostic approach, the cumulative risk score (CRS), in detection of periodontitis in subjects with angiographically verified coronary artery disease diagnosis. MATERIALS AND METHODS The concentrations of matrix metalloproteinase (MMP)-8, interleukin (IL)-1β, and Porphyromonas gingivalis were analysed from saliva of 493 subjects. The subjects participated in a detailed clinical and radiographic oral examination. The CRS index, combining the three salivary biomarkers, was calculated for each subject. RESULTS High salivary concentrations of MMP-8, IL-1β, and P. gingivalis were associated with deepened periodontal pockets and alveolar bone loss, and MMP-8 and IL-1β with bleeding on probing. The CRS index had a stronger association with moderate to severe periodontitis (OR 6.13; 95% CI 3.11-12.09) than any of the markers alone. CONCLUSIONS Salivary concentrations of MMP-8, IL-1β, and P. gingivalis are associated with various clinical and radiographic measures of periodontitis. The CRS index, combining the three salivary biomarkers, is associated with periodontitis more strongly than any of the markers alone regardless of the coronary artery disease status of the patients.

Oral rinse MMP-8 point-of-care immuno test identifies patients with strong periodontal inflammatory burden

OBJECTIVE To determine whether oral rinse matrix metalloproteinase (MMP)-8 levels, measured by three different methods, tissue inhibitor of matrix metalloprotease-1 (TIMP-1) levels and elastase activity differentiate subjects with different periodontal condition; and second, to find out if MMP-8 levels were comparable among the methods used. METHODS MMP-8 levels were analysed with an immunofluorometric method (IFMA), dentoELISA and commercial ELISA. Also TIMP-1 levels and elastase activity were measured. For statistical analysis 214 study subjects were categorized into four groups, specified by the presence and number of moderate (4-5mm) and deep (≥6mm) periodontal pockets, and bleeding on probing percentage. RESULTS MMP-8 levels especially measured by dentoELISA and adjusted to the number of teeth per subject differentiated the study group with strong periodontal inflammatory burden from groups with lower levels. This was also verified with receiver operating characteristic (ROC) analysis. Elastase activity associated with higher IFMA and dentoELISA MMP-8 levels. IFMA MMP-8/TIMP and dentoELISA MMP-8/TIMP-1 tended to be higher with the increasing level of periodontal inflammatory burden. TIMP-1 levels decreased with increasing age. CONCLUSIONS Oral rinse MMP-8 together with TIMP-1 analysis may have potential in complementary periodontal diagnostics. dentoELISA can be applied in quantitative oral rinse chair side biomarker diagnostics.