Role of salivary matrix metalloproteinase-8 (MMP-8) in chronic periodontitis diagnosis

Abstract

Periodontitis is an inflammatory disease of the periodontium. Any imbalance between the matrix metalloproteinases (MMPs) secreted by neutrophils and tissue inhibitors initiates the destruction of collagen in gum tissue, leading to chronic periodontitis. This study aimed to correlate salivary levels of MMP-8 and periodontal parameters of chronic periodontitis to establish MMP-8 as a noninvasive marker for the early diagnosis of chronic periodontitis. The study involved 40 subjects visiting the periodontic OPD of Dr. Ziauddin Ahmad Dental College and Hospital, located in Aligarh, U.P., India, from 2011 to 2012. The subjects were divided into two groups: group I consisted of 20 periodontally healthy subjects (controls) while group II consisted of 20 patients with chronic periodontitis. Chronic periodontitis was assessed on the basis of several periodontal parameters, including pocket probing depth (PPD), clinical attachment level (CAL), gingival index (GI), and plaque index (PI). Around 3 ml of unstimulated and whole expectorated saliva was collected for MMP-8 estimation by ELISA using Quantikine human total MMP-8 immunoassay kits. Data were analyzed using STATISTICA (Windows version 6) software. Salivary MMP-8 levels of groups I and II were 190.91±143.89 ng/ml and 348.26±202.1 ng/ml, respectively. The MMP-8 levels and periodontal status (PPD, CAL, GI, and PI) of groups I and II showed positive and significant correlations (for PPD, r = 0.63, P<0.001; for CAL, r = 0.54, P<0.001; for GI, r = 0.49, P<0.001; and for PI, r = 0.63, P<0.001). The results of this study demonstrate elevated concentrations of MMP-8 in individuals with chronic periodontitis.

Keywords

matrix metalloproteinase-8 / chronic periodontitis / pocket probing depth / clinical attachment level / gingival index / plaque index

Introduction

Periodontitis is an inflammatory disease of the periodontium characterized by progressive destruction of the tissue supporting the tooth because of the presence of over 300 species of recognized bacteria in the oral cavity [ 1, 2]. Conventional clinical diagnostic criteria, such as probing depth, attachment level, bleeding on probing, plaque index, and radiographic assessment of the alveolar bone loss, is often insufficient to determine the sites of the active disease, monitor responses to therapy, or measure the degree of susceptibility to future disease progression.

Saliva as a mirror of oral and systemic health is a valuable source of clinically relevant information because it contains biomarkers that are specific to the unique physiological aspects of periodontal diseases. The use of saliva for translational and clinical application has recently been proposed. Salivary proteins and RNAs can be used to detect oral cancer [ 3, 4] and Sjogren’s syndrome [ 5]. Saliva contains locally and systemically derived markers of periodontitis; it offers the basis for a patient-specific diagnostic test for periodontitis. Correlations between salivary biomarkers and the clinical features of periodontal disease have been evaluated in terms of three aspects of periodontitis—inflammation, collagen degradation, and bone turnover. Higher enzyme activities were observed in chronic periodontal patients than in healthy controls [ 6]. Enzymes in the saliva can originate from cells in the salivary glands, microorganisms, epithelial cells, neutrophils, and gingival crevicular fluid and perform important functions in the destruction of the periodontium.

Neutrophils are the most important cells in the host and act as a defense against periodonto-pathogenic bacteria. Their granules contain hydrolytic neutral enzymes (e.g., elastase, cathepsin G, myeloperoxidase, and lysozyme), hydrolases [e.g., cathepsin B, cathepsin D, β-glucuronidase, lactoferrin, neutrophil collagenase (i.e., matrix metalloproteinases-8(MMP-8))], and MMP-9.

Research shows that MMP-8 acts a as key marker in chronic periodontitis; MMP-8 has been highly correlated with bleeding on probing, attachment loss, and probing pocket depth [ 7, 8]. This substance exhibits a unique ability to decompose types I and III collagen [ 9, 10]. MMPs are proteolytic enzymes belonging to the zinc protease super family involved in the physiological degradation of extracellular matrix proteins and basement membranes. MMPs may be categorized into several groups, including collagenase (MMPs-1, -8, and -13), gelatinase (MMPs-2 and -9), stromelysin (MMPs-3, -10, and -11), matrilysins (MMP-7), and other membrane-associated MMPs.

During inflammation, bacterial products, cytokines, and growth factors act on periodontal and inflammatory cells, which then produce and release increasing amounts of MMPs in the cells of the periodontium and decreasing amounts of tissue inhibitors [ 11, 12]. Any imbalance between the MMPs and tissue inhibitors initiates the destruction of collagens present in the gums, leading to chronic periodontitis [ 13, 14]. A simple and noninvasive diagnostic procedure for the detection of chronic periodontitis that allows rapid screening and provides accurate predicted information will thus be of great value to both patients and dentists.

This study was undertaken to determine the role of MMP-8 as a noninvasive marker for chronic periodontitis.

Materials and methods

The research protocol for the present study was approved by the ethical committee of our institution, and informed consent was obtained from each subject prior to involvement in the study. This study was conducted from 2011 to 2012. A total of 40 subjects, aged between 30 and 55 years, who had visited the OPD of the periodontics department of Dr. Ziauddin Ahmad Dental College and Hospital, Aligarh, U.P., India, were selected for the study. The patients were divided into two groups. Group I consisted of 20 subjects who were systematically and periodontally healthy, above 30 years of age, and had at least 20 teeth. Group II consisted of 20 subjects with generalized chronic periodontitis. These patients were also above 30 years of age and had at least 20 teeth. The probing pocket depth of these patients was≥5 mm, and their clinical attachment level was≥2mm. Smokers, pregnant and lactating women, and individuals with either acute or chronic medical illness or on oral medication for the last three months were not included in the study.

Periodontal disease status was evaluated at four sites of each tooth (i.e., mesiobuccal, buccal, distobuccal, lingual/palatal) using the same periodontal probe (University of North Carolina-15 probe, Hu-Freidy’s, USA) and conducted by the same examiner to avoid bias. The parameters tested included probing pocket depth (PPD), clinical attachment level (CAL), gingival index (GI), and plaque index (PI). PPD represents the distance between the base of the pocket and the gingival margin. The distance was calculated with a UNC-15 probe held parallel to the vertical axis of the tooth. The pocket depth was measured at four sites of each tooth. CAL represents the distance between the bases of the pocket to the cement-enamel junction. GI [ 15] was calculated using the formula: gingival index score= total score/number of surface examined, and the different scores used were: score 0 — normal gingiva, score 1 — mild inflammation, score 2 — moderate inflammation, and score 3 — severe inflammation.

PI [ 16] was determined using the formula: mean plaque index score= total score / number of surface examined, and the different scores used were: score 0 — no plaque on the tooth surface, score 1 — no plaque observed by the naked eye, score 2 — thin plaque visible to the naked eye, and score 3 — abundance of soft matter in the gingival pocket. The mean gingival and plaque index scores of each tooth were obtained by dividing the total score by the number of teeth examined.

The clinical parameters described above were recorded and saliva samples necessary for MMP-8 estimation were taken from both groups. Subjects were required to fast for 2 h prior to examination. About 3 ml of unstimulated and whole expectorated saliva was collected from each subject and placed in sterile 5 ml saliva-collecting tubes according to the method described by Navazesh [ 17]. Samples were stored at –80 °C until use. Salivary MMP-8 levels of each subject were estimated in duplicate using Quantikine human total MMP-8 immunoassay kits (R and D Systems, Inc., USA) by ELISA [ 18]. Absorbance was read at 450 nm using a correction wavelength that was set at 540 nm or 570 nm. Data were analyzed using STATISTICA (Windows version 6) software, and correlations between MMP-8 levels and periodontal status were assessed by Pearson correlation analysis.

Results

The present study evaluated correlations between periodontal status and salivary MMP-8 levels in chronic periodontitis patients. Changes in periodontal parameters, namely, PPD, CAL, GI, and PI, as a function of salivary MMP-8 levels were assessed for both group I (controls) and group II (chronic periodontitis). The mean±SD values of periodontal parameters are shown in Table 1.

ANOVA revealed significant differences (P<0.001) in the periodontal parameters obtained between groups I and II. Salivary MMP-8 levels in both groups I and II are shown in Table 2. Significant differences (P<0.001) were observed when the mean (±SD) values of salivary MMP-8 levels obtained from the two groups were compared. Salivary MMP-8 levels and all periodontal parameters (PPD, CAL, GI, and PI) of groups I and II showed positive and significant correlations (Table 3).

Discussion

The saliva reflects the general health of an individual and helps in monitoring the onset of a specific disease. Our study was conducted using unstimulated saliva. The majority of previous investigations involved gingival crevicular fluid (GCF) and used represented pooled concentrations from all sites of the mouth to provide an overall assessment of the disease status and severity. In these studies, GCF sample collection involved sampling of a minute amount of fluid on filter paper strips, which requires a longer sampling time. In addition, contamination of blood and plaque products was often encountered [ 19]. These problems are nonexistent in a method that involves easy, noninvasive, and rapid saliva collection. Its measurement requires no special equipment and expertise. Our study was conducted using unstimulated saliva; stimulated whole saliva is less suitable for diagnostic applications than unstimulated saliva because foreign substances used to stimulate saliva tend to modulate the fluid pH and induce the water phase of saliva secretion, thereby diluting the concentrations of proteins of interest [ 20].

MMP-8 is released from neutrophils and considered a superior marker of inflammation present in oral fluid. This substance can decompose types I and III collagen, which is an important function during periodontal destruction in periodontitis. This results in increased levels of MMP-8 during periodontitis. Various studies have demonstrated increased MMP-8 levels related to periodontitis severity and course [ 21– 23].

The results of the present study are in agreement with findings in other studies. Kraft et al. [ 8] showed a statistically significant correlation between increasing mean pocket depth and MMP-8 concentration. Another study by Sorsa et al. [ 9] showed that MMPs (especially MMPs-8, -9, and -13) are involved in destructive periodontal disease and that MMP-8 or neutrophil collagenase is the major interstitial collagenase present in periodontitis affecting gingival tissue, GCF, saliva, and mouth rinse samples. Herr et al. [ 10] confirmed that, during progressive periodontal breakdown, gingival and periodontal ligament collagens are cleaved by the host’s cell-derived interstitial collagenases. MMP-8 levels have also been demonstrated to be highly elevated in saliva taken from patients with periodontal disease.

The results of a study conducted by Tonetti et al. [ 24] showed that MMP-3 and MMP-8 transcripts are not detectable in healthy sites but may be specifically detected in periodontitis sites. Miller et al. [ 19] stated that MMP-8, a key enzyme in extracellular collagen matrix degradation, is derived predominantly from PMNs during the acute stages of periodontal disease and correlates well with periodontal parameters. Moreover, the presence of the enzyme significantly increases the risk of periodontal disease. Kuula et al. [ 25] observed that MMP-8 is the major collagenolytic MMP in gingival tissue and oral fluids, and elevated levels of this enzyme have been associated with the severity of periodontal inflammation and disease; basal physiological levels may be associated with tissue homeostasis and even act as protection against diseases. A recent study by Rathnayake et al. [ 26] concluded that patients with severe periodontitis presented elevated salivary concentrations of IL-1 and MMP-8, and IL-1 and MMP-8 can be used as markers of periodontal disease in a larger patient population. Another study conducted by Gursoy et al. [ 27] proved that active MMP-8 is a strong biomarker candidate for detecting alveolar bone destruction in saliva. The concentrations of MMP-8, MMP-9, and MMP-13 in saliva are higher in subjects with generalized periodontitis than in the corresponding controls. Recently, Javedet al. [ 28] showed that salivary IL-6 and MMP-8 levels are elevated in patients with chronic periodontitis with and without prediabetes.

Conclusions

The results of this study demonstrate elevated concentrations of MMP-8 in individuals with chronic periodontitis. While many questions remain unanswered, the potential advantages of salivary analysis for the diagnosis of periodontal diseases suggest that further studies are necessary. Integrating new salivary diagnostic methods into clinical practice is important in helping dental professionals conduct essential health-related decisions for patients. More studies must be performed to determine the longitudinal relationship of salivary MMP-8 and disease progression.

References

[1] Nunn ME. Understanding the etiology of periodontitis: an overview of periodontal risk factors. Periodontol 2000 2003; 32(1): 11–23

[2] Offenbacher S. Periodontal diseases: pathogenesis. Ann Periodontol 1996; 1(1): 821–878

[3] Hu S, Arellano M, Boontheung P, Wang J, Zhou H, Jiang , Elashoff D, Wei R, Loo JA, Wong DT. Salivary proteomics for oral cancer biomarker discovery. Clin Cancer Res 2008; 14(19): 6246–6252

[4] Li Y, St John MA, Zhou X, Kim Y, Sinha U, Jordan RC, Eisele D, Abemayor E, Elashoff D, Park NH, Wong DT. Salivary transcriptome diagnostics for oral cancer detection. Clin Cancer Res 2004; 10(24): 8442–8450

[5] Hu S, Wang J, Meijer J, Ieong S, Xie Y, Yu T, Zhou H, Henry S, Vissink A, Pijpe J, Kallenberg C, Elashoff D, Loo JA, Wong DT. Salivary proteomic and genomic biomarkers for primary Sjögren’s syndrome. Arthritis Rheum 2007; 56(11): 3588–3600

[6] Armitage GC. Periodontal diagnoses and classification of periodontal diseases. Periodontol 2000 2004; 34(1): 9–21

[7] Giannobile WV, Beikler T, Kinney JS, Ramseier CA, Morelli T, Wong DT. Saliva as a diagnostic tool for periodontal disease: current state and future directions. Periodontol 2000 2009; 50(1): 52–64

[8] Kraft-Neumärker M, Lorenz K, Koch R, Hoffmann T, Mäntylä P, Sorsa T, Netuschil L. Full-mouth profile of active MMP-8 in periodontitis patients. J Periodontal Res 2012; 47(1): 121–128

[9] Sorsa T, Tjäderhane L, Konttinen YT, Lauhio A, Salo T, Lee HM, Golub LM, Brown DL, Mäntylä P. Matrix metalloproteinases: contribution to pathogenesis, diagnosis and treatment of periodontal inflammation. Ann Med 2006; 38(5): 306–321

[10] Herr AE, Hatch AV, Throckmorton DJ, Tran HM, Brennan JS, Giannobile WV, Singh AK. Microfluidic immunoassays as rapid saliva-based clinical diagnostics. Proc Natl Acad Sci USA 2007; 104(13): 5268–5273

[11] Ingman T, Tervahartiala T, Ding Y, Tschesche H, Haerian A, Kinane DF, Konttinen YT, Sorsa T. Matrix metalloproteinases and their inhibitors in gingival crevicular fluid and saliva of periodontitis patients. J Clin Periodontol 1996; 23(12): 1127–1132

[12] Heikkinen AM, Sorsa T, Pitkäniemi J, Tervahartiala T, Kari K, Broms U, Koskenvuo M, Meurman JH. Smoking affects diagnostic salivary periodontal disease biomarker levels in adolescents. J Periodontol 2010; 81(9): 1299–1307

[13] Sorsa T, Tjäderhane L, Salo T. Matrix metalloproteinases (MMPs) in oral diseases. Oral Dis 2004; 10(6): 311–318

[14] Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 2010; 141(1): 52–67

[15] Löe H. The Gingival Index, the Plaque Index and the Retention Index Systems. J Periodontol 1967; 38(6): 610–616

[16] Löe H, Silness J. Periodontal disease in pregnancy. Prevelance and severity. Acta Odontol Scand 1963; 21(6): 533–551

[17] Navazesh M. Methods for collecting saliva. Ann N Y Acad Sci 1993; 694: 72–77

[18] Quantikine Human Total MMP-8 Immunnoassay; Catalog Number DMP800: R&D Systems, Inc. 614 McKinley Place NE, Minneapolis, MN55413, USA

[19] Miller CS, King CP Jr, Langub MC, Kryscio RJ, Thomas MV. Salivary biomarkers of existing periodontal disease: a cross-sectional study. J Am Dent Assoc 2006; 137(3): 322–329

[20] Oberg SG, Izutsu KT, Truelove EL. Human parotid saliva protein composition: dependence on physiological factors. Am J Physiol 1982; 242(3): G231–G236

[21] Sorsa T, Hernández M, Leppilahti J, Munjal S, Netuschil L, Mäntylä P. Detection of gingival crevicular fluid MMP-8 levels with different laboratory and chair-side methods. Oral Dis 2010; 16(1): 39–45

[22] Sorsa T, Tervahartiala T, Leppilahti J, Hernandez M, Gamonal J, Tuomainen AM, Lauhio A, Pussinen PJ, Mäntylä P. Collagenase-2 (MMP-8) as a point-of-care biomarker in periodontitis and cardiovascular diseases. Therapeutic response to non-antimicrobial properties of tetracyclines. Pharmacol Res 2011; 63(2): 108–113

[23] Salminen A, Gursoy UK, Paju S, Hyvärinen K, Mäntylä P, Buhlin K, Könönen E, Nieminen MS, Sorsa T, Sinisalo J, Pussinen PJ. Salivary biomarkers of bacterial burden, inflammatory response, and tissue destruction in periodontitis. J Clin Periodontol 2014; 41(5): 442–450

[24] Tonetti MS, Freiburghaus K, Lang NP, Bickel M. Detection of interleukin-8 and matrix metalloproteinases transcripts in healthy and diseased gingival biopsies by RNA/PCR. J Periodontal Res 1993; 28(6): 511–513

[25] Kuula H, Salo T, Pirilä E, Tuomainen AM, Jauhiainen M, Uitto VJ, Tjäderhane L, Pussinen PJ, Sorsa T. Local and systemic responses in matrix metalloproteinase 8-deficient mice during Porphyromonas gingivalis-induced periodontitis. Infect Immun 2009; 77(2): 850–859

[26] Rathnayake N, Akerman S, Klinge B, Lundegren N, Jansson H, Tryselius Y, Sorsa T, Gustafsson A. Salivary biomarkers of oral health: a cross-sectional study. J Clin Periodontol 2013; 40(2): 140–147

[27] Gursoy UK, Könönen E, Huumonen S, Tervahartiala T, Pussinen PJ, Suominen AL, Sorsa T. Salivary type I collagen degradation end-products and related matrix metalloproteinases in periodontitis. J Clin Periodontol 2013; 40(1): 18–25

[28] Javed F, Ahmed HB, Saeed A, Mehmood A, Bain C. Whole salivary interleukin-6 and matrix metalloproteinase-8 levels in patients with chronic periodontitis with and without prediabetes. J Periodontol 2014; 85(5): e130–e135

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