Proteolytic roles of matrix metalloproteinase (MMP)‐13 during progression of chronic periodontitis: initial evidence for MMP‐13/MMP‐9 activation cascade

Aim: Matrix metalloproteinases (MMP)‐13 can initiate bone resorption and activate proMMP‐9 in vitro, and both these MMPs have been widely implicated in tissue destruction associated with chronic periodontitis. We studied whether MMP‐13 activity and TIMP‐1 levels in gingival crevicular fluid (GCF) associated with progression of chronic periodontitis assessed clinically and by measuring carboxy‐terminal telopeptide of collagen I (ICTP) levels. We additionally addressed whether MMP‐13 could potentiate gelatinase activation in diseased gingival tissue. Materials and Methods: In this prospective study, GCF samples from subjects undergoing clinical progression of chronic periodontitis and healthy controls were screened for ICTP levels, MMP‐13 activity and TIMP‐1. Diseased gingival explants were cultured, treated or not with MMP‐13 with or without adding CL‐82198, a synthetic MMP‐13 selective inhibitor, and assayed by gelatin zymography and densitometric analysis. Results: Active sites demonstrated increased ICTP levels and MMP‐13 activity (p<0.05) in progression subjects. The MMP‐9 activation rate was elevated in MMP‐13‐treated explants (p<0.05) and MMP‐13 inhibitor prevented MMP‐9 activation. Conclusions: MMP‐13 could be implicated in the degradation of soft and hard supporting tissues and proMMP‐9 activation during progression of chronic periodontitis. MMP‐13 and ‐9 can potentially form an activation cascade overcoming the protective TIMP‐1 shield, which may become useful for diagnostic aims and a target for drug development.     

Enamel matrix derivative induces the expression of tissue inhibitor of matrix metalloproteinase‐3 in human gingival fibroblasts via extracellular signal‐regulated kinase

Zeldich E, Koren R, Dard M, Weinberg E, Weinreb M, Nemcovsky CE. Enamel matrix derivative induces the expression of tissue inhibitor of matrix metalloproteinase‐3 in human gingival fibroblasts via extracellular signal‐regulated kinase. J Periodont Res 2010; doi: 10.1111/j.1600‐0765.2009.01218.x © 2009 John Wiley & Sons A/S Background and Objective: Periodontal disease is characterized by increased expression and activity of matrix metalloproteinases (MMPs) and insufficient expression/activity of their inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). This altered MMP–TIMP balance results in progressive destruction of gingival and periodontal extracellular matrix. Enamel matrix derivative (EMD), clinically used for periodontal regeneration in a device called Emdogain^®, has been suggested to enhance gingival healing following periodontal procedures in humans. We previously showed that EMD increases the proliferation of human and rat gingival fibroblasts and protects them from tumor necrosis factor‐induced apoptosis. In the present study, the modulation of MMP and TIMP expression by EMD was investigated. Material and Methods: Primary human gingival fibroblasts were treated in vitro with tumor necrosis factor, EMD or both in serum‐free conditions, and RNA was analyzed with an extracellular matrix‐focused microarray and quantitative real‐time polymerase chain reaction. Results: Microarray analysis showed detectable expression of MMP‐1, MMP‐2, MMP‐3, MMP‐7 and MMP‐13, as well as TIMP‐1 and TIMP‐3 in untreated cells. There was no apparent regulation of the expression of MMP‐2, MMP‐7, MMP‐13 and TIMP‐1 by either tumor necrosis factor or EMD. In contrast, tumor necrosis factor significantly increased MMP‐1 expression, and EMD reduced it when both agents were present. Also, EMD significantly induced TIMP‐3 expression, an effect which was dependent on activation of extracellular signal‐regulated kinase 1/2, since it was totally abolished by a selective extracellular signal‐regulated kinase pathway inhibitor. Conclusion: These data suggest that EMD may affect gingival health by ways other than cell proliferation/survival, i.e. by stimulation of TIMP‐3 production, which could improve the MMP–TIMP balance in gingival tissue and curb extracellular matrix destruction.     

Crevicular fluid matrix metalloproteinase‐8, ‐13, and TIMP‐1 levels in type 2 diabetics

Oral Diseases (2010) 16 , 476–481 Objectives: To evaluate whether type 2 diabetes mellitus (DM) enlarged and if so the quantum of such increase in the gingival crevicular fluid (GCF) levels of matrix metalloproteinase‐8 (MMP‐8), MMP‐13 and tissue inhibitor of metalloproteinases‐1 (TIMP‐1). Methods: Subjects (n = 73) were divided into five groups as follows: 12 DM patients with gingivitis (DM‐G), 12 DM patients with periodontitis (DM‐P), 12 systemically healthy patients with gingivitis (H‐G), 13 systemically healthy patients with periodontitis (H‐P) and 24 periodontally, systemically healthy volunteer subjects (H‐C). Full‐mouth clinical periodontal measurements were performed at six sites per tooth. Gingival crevicular fluid samples were obtained from two sites representing the clinical periodontal diagnosis in single‐rooted teeth. Gingival crevicular fluid levels of MMP‐8, MMP‐13 and TIMP‐1 were analysed by immunofluorometric MMP assay (IFMA), enzyme‐linked immunosorbent assay (ELISA). Data were tested statistically by parametric tests. Results: All clinical periodontal measurements were similar in both diabetic and systemically healthy patients with periodontal disease (all P > 0.05). Total amounts of MMP‐8 in GCF samples were significantly lower in H‐C group than DM‐G, DM‐P, H‐P groups (all P < 0.05). Matrix metalloproteinase‐13, TIMP‐1 total amounts were similar in study groups (P > 0.05). Diabetes mellitus patients exhibited similar levels of MMP‐8, MMP‐13, TIMP‐1 with systemically healthy gingivitis/periodontitis patients (P > 0.05). Conclusions: Within the limits of this study, DM does not seem to significantly affect GCF levels of MMP‐8, MMP‐13, TIMP‐1 or clinical periodontal status.     

Induction of MMP‐2 at the interface between epithelial cells and fibroblasts from human periodontal ligament

Shimonishi M, Takahashi I, Terao F, Komatsu M, Kikuchi M. Induction of MMP‐2 at the interface between epithelial cells and fibroblasts from human periodontal ligament. J Periodont Res 2010; 45: 309–316. © 2009 John Wiley & Sons A/S Background and Objective: MMP‐2 can degrade type IV collagen and MMP‐14 can activate pro MMP‐2. The present study was undertaken to examine the expression of MMP‐2 and MMP‐14 with respect to interaction between the cells of the epithelial rests of Malassez and fibroblasts from human periodontal ligament. Material and Methods: Explants of human periodontal ligament tissues produced outgrowths containing both putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts after incubation in a modified serum‐free medium. The distribution and expression of MMP‐2 and MMP‐14 were analysed using immunohistochemistry, in situ hybridization and RT‐PCR analysis. The conditioned media and cell extracts were collected for western blot analysis for MMP‐2. Results: Putative epithelial rests of Malassez cells at the interface between the cells of the epithelial rests of Malassez and fibroblasts expressed MMP‐2 and MMP‐14 strongly. However, in situ hybridization analysis revealed that human periodontal ligament fibroblasts expressed MMP‐2 mRNA while putative epithelial rests of Malassez cells expressed MMP‐14 mRNA at the interface. The RT‐PCR analysis showed that the expression of MMP‐2 mRNA was significantly higher when putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts were cultured together than when cultured alone. Western blot analysis showed that the active form of MMP‐2 was detected at higher levels in the conditioned medium of the co‐cultured cells. Conclusion: These findings indicate that putative epithelial rests of Malassez cells stimulate the production of MMP‐2 in human periodontal ligament fibroblasts. Up‐regulated proMMP‐2 bound by MMP‐14 expressed in epithelial rests of Malassez cells can degrade matrix molecules, such as type IV collagen, in the basal membrane between putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts.     

Investigation of matrix metalloproteinase‐1 −1607 1G/2G polymorphism in a Turkish population with periodontitis

Aim: Matrix metalloproteinase‐1 (MMP‐1) is a proteolytic enzyme that degrades extracellular matrix and plays a fundamental role during destruction of periodontal tissues. The aim of this study was to examine the association between MMP‐1 ?1607 1G/2G polymorphism and chronic periodontitis susceptibility in a Turkish population. Material and Methods: A total of 180 subjects were enrolled in this study. All the subjects received a periodontal examination including full‐mouth clinical attachment loss measurements, probing depths, plaque index scores, gingival index scores and radiographic bone loss ratios. Three groups formed according to periodontal conditions were healthy, moderate periodontitis and severe periodontitis groups. MMP‐1 ?1607 1G/2G gene promoter polymorphism was genotyped using a polymerase chain reaction‐restriction fragment length polymorphism method. Results: Analysis of the polymorphism showed no differences in distribution of the MMP‐1 ?1607 1G/2G polymorphism among healthy, moderate periodontitis and severe periodontitis groups (p>0.05). When the groups were further stratified by smoking status, we found no significant differences in genotype distributions, allele frequencies and carriage rates among any groups either (p>0.05). Conclusions: On the basis of the results, no significant association is found for the MMP‐1 ?1607 1G/2G polymorphism with susceptibility to periodontitis. Moreover, smoking status did not seem to affect this result.     

The effect of smoking on the mRNA expression of MMPs and TIMP-1 in untreated chronic periodontitis patients: a cross-sectional study

Mouzakiti E, Pepelassi E, Fanourakis G, Markopoulou C, Tseleni‐Balafouta S, Vrotsos I. The effect of smoking on the mRNA expression of MMPs and TIMP‐1 in untreated chronic periodontitis patients: a cross‐sectional study. J Periodont Res 2011; 46: 576–583.©2011 John Wiley & Sons A/S Background and Objective: Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important for extracellular matrix. Expression of MMPs has been evaluated in gingiva without studying smoking. The aim of this study was to explore the effect of smoking on mRNA expression of MMP‐1, ‐3, ‐8, ‐9 and ‐13 and TIMP‐1 in untreated chronic periodontitis and in periodontal health. Material and Methods: Gingival samples were harvested from 30 subjects with untreated chronic periodontitis (15 nonsmokers and 15 smokers) and 30 periodontally healthy subjects (15 nonsmokers and 15 smokers). Full‐mouth plaque score, gingival index, bleeding on probing, probing depth and clinical attachment level were recorded. Total RNA was isolated, and the mRNA expression of MMPs and TIMP‐1 was assessed by RT‐PCR. Results: Periodontitis groups were comparable in clinical measurements. Nonsmoker subjects with periodontitis had statistically significantly higher MMP‐1, lower MMP‐9 and TIMP‐1 expression and higher MMP‐1/TIMP‐1 ratio than smokers; and higher MMP‐8 expression and MMP‐8/TIMP‐1 and MMP‐1/TIMP‐1 ratios than healthy nonsmokers. Healthy nonsmokers had statistically significantly higher MMP‐13 expression than healthy smokers. Smoker periodontitis and healthy subjects had similar expression levels of MMPs and TIMP‐1 and MMPs/TIMP‐1 ratios. There was correlation among the MMPs only for smoker periodontitis subjects. Expression of MMP‐13 was correlated with mean clinical attachment level. Conclusion: Within its limits, this study demonstrated that smoking affected mRNA expression of MMPs and TIMP‐1, MMPs/TIMP‐1 ratios and relationships among MMPs in untreated chronic periodontitis and expression of MMPs in health. In the absence of smoking, chronic periodontitis affected expression of MMPs and MMPs/TIMP‐1 ratios.     

Cigarette smoke condensate affects the collagen-degrading ability of human gingival fibroblasts

Background and Objective: Cigarette smoke condensate, the particulate matter of cigarette smoke, is composed of thousands of chemicals, including nicotine. Cigarette smoking is a risk factor for periodontal disease. This study investigated the influence of cigarette smoke condensate on the collagen‐degrading ability of human gingival fibroblasts and its mechanism. Material and Methods: Human gingival fibroblasts were exposed for 72 h to various concentrations of total particulate matter cigarette smoke condensate. Cell proliferation and cytotoxicity were evaluated using water‐soluble tetrazolium‐1 and lactate dehydrogenase, respectively. The collagen‐degrading ability of human gingival fibroblasts was evaluated in collagen‐coated six‐well plates. Conditioned media and membrane extracts were collected for zymography and western blot analyses of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Results: Cell proliferation decreased and cytotoxicity increased in human gingival fibroblasts with increasing concentrations of cigarette smoke condensate. Cell proliferation decreased by more than 50% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 200 μg/mL, and cytotoxicity increased to more than 30% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 400 μg/mL. Cigarette smoke condensate increased the collagen‐degrading ability of human gingival fibroblasts, especially at a concentration of 100 μg/mL (1.5‐fold increase, p < 0.05) compared with the control. Cigarette smoke condensate increased the production of proMMP‐1, proMMP‐2, MMP‐14 and TIMP‐1, and decreased the production of TIMP‐2, in conditioned media. Furthermore, compared with the control group, cigarette smoke condensate increased the production of MMP‐2, MMP‐14 and TIMP‐2 in membrane extracts, especially at concentrations of 50–100 μg/mL. Conclusion: Cigarette smoke condensate affects human gingival fibroblast proliferation and is toxic at total particulate matter cigarette smoke condensate concentrations of ≥ 400 μg/mL. Cigarette smoke condensate can increase the collagen‐degrading ability of human gingival fibroblasts by altering the production and localization of MMPs and TIMPs.     

Expression of MMPs and TIMP-1 in smoker and nonsmoker chronic periodontitis patients before and after periodontal treatment

Mouzakiti E, Pepelassi E, Fanourakis G, Markopoulou C, Tseleni‐Balafouta S, Vrotsos I. Expression of MMPs and TIMP‐1 in smoker and nonsmoker chronic periodontitis patients before and after periodontal treatment. J Periodont Res 2012; 47: 532–542. © 2012 John Wiley & Sons A/S Background and Objective: Nonsurgical periodontal treatment controls periodontal inflammation. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are implicated both in the destruction and in the healing of periodontal tissues. The aim of the present study was to compare the mRNA expression of MMP‐1, ‐3, ‐8, ‐9 and ‐13 and TIMP‐1 in chronic periodontitis before and after initial periodontal treatment. Material and Methods: Ninety gingival samples were harvested from 30 patients with chronic periodontitis (15 nonsmokers and 15 smokers) before and after nonsurgical treatment and from 30 periodontally healthy control subjects (15 nonsmokers and 15 smokers). Clinical parameters were assessed before and after treatment. Total RNA was isolated, and mRNA expression of MMPs and TIMP‐1 was assessed by RT‐PCR. Results: Periodontal treatment significantly increased TIMP‐1 expression and decreased the ratios of MMPs/TIMP‐1. Post‐treatment, nonsmokers with periodontitis had significantly higher MMP‐8 and TIMP‐1 expression than healthy nonsmokers, and smokers with periodontitis had significantly higher MMP‐13 and TIMP‐1 expressions than healthy smokers. Post‐treatment, smokers had significantly higher TIMP‐1 expression and lower MMP‐8/TIMP‐1 ratio than nonsmokers. Post‐treatment, there was no correlation among MMPs, and the expression of MMPs and TIMP‐1 was not correlated with clinical measurements. Conclusion: Periodontal treatment increased TIMP‐1 expression and decreased the ratios of MMPs/TIMP‐1 in chronic periodontitis. The post‐treatment increase in TIMP‐1 expression was higher for smokers. The TIMP‐1 expression was higher post‐treatment than in health. Post‐treatment, MMP‐8 expression was higher in nonsmokers with periodontitis than in healthy nonsmokers, whereas MMP‐13 expression was higher in smokers with periodontitis than in healthy smokers.     

Smoking and matrix metalloproteinases,neutrophil elastase and myeloperoxidase in chronic periodontitis

Oral Diseases (2010) 17 , 68–76 Objectives: To investigate possible relationship between smoking and serum concentrations of matrix metalloproteinase‐8,‐9 (MMP‐8, MMP‐9), tissue inhibitor of matrix metalloproteinases‐1 (TIMP‐1), neutrophil elastase (NE), myeloperoxidase (MPO) in chronic periodontitis (CP) patients relative to periodontally healthy subjects. Methods: Serum samples were obtained from 111 subjects before initiation of any periodontal intervention. Fifty‐five CP patients (39 non‐smokers, 16 smokers) and 56 periodontally healthy subjects (39 non‐smokers, 17 smokers) were recruited. Serum concentrations of MMP‐8 were determined by IFMA and MPO, MMP‐9, TIMP‐1, NE concentrations by ELISA. ANCOVA and Pearson correlation analysis was utilized for statistical analysis. Results: Serum MPO, NE concentrations were higher in smoker CP than non‐smoker CP patients (P = 0.002 and P < 0.001, respectively), whereas these were similar in smoker, non‐smoker periodontally healthy groups (P > 0.05). TIMP‐1 concentration was higher in non‐smoker CP than smoker CP group (P < 0.05). MMP‐9/TIMP‐1 ratios were higher in smoker CP than non‐smoker CP group (P = 0.01). MMP‐8 concentrations, MMP‐8/TIMP‐1 and MMP‐9/TIMP‐1 ratios in CP group were not significantly different from those in periodontally healthy group (P > 0.05). Conclusions: Our findings of significantly elevated serum MMP‐9, MPO, NE together with decreased TIMP‐1 in smoker CP patients than non‐smokers support that smoking together with periodontal destruction may expose/predispose to cardiovascular diseases.     

Identification of Quercitrin as a Potential Therapeutic Agent for Periodontal Applications

Background: Flavonoids are natural phenolic compounds with antioxidant, anti‐inflammatory, and antimicrobial capacity. This study aims to investigate the effects of different flavonoids for potential use in periodontal applications. Methods: Cultures of Staphylococcus epidermidis or primary human gingival fibroblasts (HGFs) were treated with different doses of chrysin, diosmetin, galangin, quercitrin, and taxifolin. The effect of these molecules was evaluated on S. epidermidis growth rate and HGF viability, gene expression, collagen production, reactive oxygen species (ROS) levels, wound healing, and production of matrix metalloproteinase (MMP)‐1 and tissue inhibitor of MMP‐1 (TIMP1). Results: Among all the screened flavonoids, quercitrin showed the most promising biologic effects, in both HGFs and S. epidermidis. Thus, quercitrin was not toxic for HGFs; increased collagen IIIα1 and decorin levels; downregulated interleukin‐6 messenger RNA levels; decreased the expression of profibrotic markers during wound healing; decreased ROS levels in basal and stimulated conditions; and decreased the MMP1/TIMP1 ratio. Quercitrin also decreased the bacterial growth rate. Conclusions: Results suggest that quercitrin could contribute to protect and recover the integrity of gingival tissues, thus displaying a potential use for periodontal disease treatment or to functionalize dental implant abutments to improve soft tissue integration. Further studies are required to confirm the role of quercitrin in gingival tissues.     

Assessment of the involvement of the macrophage migration inhibitory factor–glucocorticoid regulatory dyad in the expression of matrix metalloproteinase‐2 during periodontitis

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine and counter‐regulator of endogenous glucocorticoids (GCs). It is implicated in acute and chronic inflammatory diseases. This study investigated the role of the MIF–GC regulatory dyad in the expression and release of matrix metalloproteinase‐2 (MMP‐2) during periodontitis, in vivo and in vitro. In a Mif‐knockout (KO) mouse model of ligature‐induced periodontitis, gingival tissues and blood were collected and analysed for levels of interleukin‐6 (IL‐6), MIF, MMP‐2, and corticosterone. In addition, human gingival fibroblasts (HGFs) were tested for production of IL‐6 and MMP‐2 after stimulation with hydrocortisone (HC), MIF, tumour necrosis factor‐alpha (TNF‐α), or Fusobacterium nucleatum, a pathogen known to elicit immune responses during periodontitis. Wild‐type (WT) mice showed a local and systemic increase of MIF levels during inflammation, which was confirmed by increased local IL‐6 concentrations. Systemic GC levels were reduced in WT and Mif‐KO mice during inflammation, with overall lower concentrations in Mif‐KO mice. In vivo and in vitro, MMP‐2 production was not dependent on MIF or inflammatory stimuli, but was inhibited by HC. Therefore, MIF does not appear to stimulate expression of MMP‐2 in the gingival tissues, whereas GC upregulates MIF and downregulates MMP‐2. Our findings further suggest that MIF may regulate systemic GC levels.     

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

Oral Diseases (2010) 17 , 115–122 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 (4–5 mm) and deep (≥6 mm) 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.     

Effects of a vegetable extract from Lupinus albus (LU105) on the production of matrix metalloproteinases (MMP1, MMP2, MMP9) and tissue inhibitor of metalloproteinases (TIMP1, TIMP2) by human gingival fibroblasts in culture

This study examined the effects of a vegetable extract from Lupinus albus (LU105) on MMPs and TIMPs secreted by human gingival fibroblasts in culture. LU105 was extracted from seeds of L. albus and is freely soluble in water. Gelatin zymography showed that control human gingival fibroblasts maintained in culture for 48 h express pro-MMP2 (progelatinase A) in the culture medium while the active form of MMP2 (gelatinase A), the active form of MMP9 (gelatinase B), and pro-MMP9 (progelatinase B) are not detected. Fibroblasts derived from inflamed gingiva expressed in the culture medium increased amounts of pro-MMP2 (progelatinase A) compared with controls and significant amounts of pro-MMP9 (progelatinase B). LU105 diminished the expression by gingival fibroblasts derived from inflamed tissue of both pro-MMP2 and pro-MMP9. Furthermore LU105 did not modify the amount of TIMP2 expressed in culture by controls or by gingival fibroblasts derived from inflamed tissue. TIMP1 and MMP1 significantly decreased when LU105 was added in the culture media of gingival fibroblasts derived from inflamed tissue compared with control fibroblasts. Thus LU105 seems to offer an opportunity to restore a correct balance between MMP2, MMP9, MMP1, and their natural inhibitors, i.e., TIMP1 and TIMP2 in human inflamed gingiva.