Effects of phase I periodontal treatment on gingival crevicular fluid levels of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1

BACKGROUND: Matrix metalloproteinase-1 (MMP-1) is a member of a family of enzymes which are capable of degrading most extracellular matrix macromolecules. Extracellular control of MMPs is exerted by tissue inhibitors of metalloproteinases (TIMP) and by mechanisms of pro-MMP activation. Levels of MMPs and TIMPs change during healing, inflammation, and normal tissue turnover. The aim of this study was to evaluate the effects of phase I periodontal treatment on gingival crevicular fluid (GCF) levels of MMP-1 and TIMP-1. METHODS: Ten patients with chronic periodontitis who had sites with probing depths > or = 4 mm and 10 periodontally healthy persons (controls) were included in this study. Clinical measurements including plaque (PI) and gingival (GI) indexes, probing depths (PD), and clinical attachment loss (CAL) were recorded both at baseline (before treatment, BT) and after phase I periodontal treatment (AT). Assays for MMP-1 and TIMP-1 were performed by an enzyme-linked immunosorbent assay (ELISA) method. RESULTS: All of the clinical conditions significantly improved and GCF volume decreased AT (P<0.05). Levels of MMP-1 were higher in patients BT than in controls (C) (P<0.05). Levels of MMP-1 were reduced AT compared to BT (P<0.05). In addition, TIMP-1 levels were lower at BT than AT and in C (P<0.05). Statistically significant differences were found between levels of TIMP-1 at BT and AT (P<0.05). The ratio of MMP-1 to TIMP-1 was significantly lower in C than patients at BT; this ratio was also significantly lower at AT than BT (P<0.05). CONCLUSIONS: These results suggest that levels of MMP-1 in GCF decreased and total levels of TIMP-1 in GCF increased after phase I periodontal therapy. The ratio of MMP-1 to TIMP-1 changed after phase I periodontal therapy, becoming close to that of the controls.     

Cytokines, matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 in gingival crevicular fluid from patients with Papillon-Lefèvre syndrome

The aim of the present study was to compare concentrations of cytokines, matrix metalloproteinases (MMPs) and a metalloproteinase inhibitor (TIMP-1) in gingival crevicular fluids (GCF) from sites with gingival inflammation in 28 young patients with Papillon-Lefèvre syndrome (PLS), and in age- and gender-matched controls. Each group consisted of 17 females and 11 males with a mean age of 11.0 years (range 4-22 years). In both groups, anterior upper sites with a clinical diagnosis of gingival inflammation and with pockets < or = 3 mm were selected for sampling of GCF, which was carried out with filter disks inserted into the gingival crevice until saturated. The concentrations of cytokines (IL-1alpha, IL-1beta, TNF-alpha, and IL-8), matrix metalloproteinases (MMP-1, MMP-3, MMP-8, and MMP-9), and their tissue inhibitor (TIMP-1) were analysed using commercial ELISA kits. Significantly higher levels of IL-1beta (P < 0.001) and MMP-8 (P < 0.05) were disclosed among the PLS patients compared with their controls, while the opposite was found for IL-8 (P < 0.05) and MMP-1 (P < 0.001). The individual variations were considerable in both groups. When comparing the expression of cytokines, MMPs, and TIMP-1 in PLS patients with clinically active and non-active periodontitis, the non-active PLS patients showed significantly higher values of IL-1beta than the patients with active periodontal disease (ANOVA, P < 0.01). In conclusion, this study was unable to demonstrate a clear-cut pathognomonic expression of cytokines or MMPs in patients with PLS, but further studies on cytokine and MMP output are warranted.     

Matrix metalloproteinase-7, -8, -9, -25, and -26 and CD43, -45, and -68 cell-markers in HIV-infected patients'' saliva and gingival tissue

BACKGROUND: Matrix metalloproteinases (MMPs) process the extracellular matrix and act in tissue remodelling in many physiological and pathological conditions. Certain MMPs can also exert protective anti-inflammatory properties. The levels and expression of MMPs and tissue inhibitors of MMPs (TIMPs) in saliva and gingival tissues of human immunodeficiency virus-seropositive (HIV+) patients are unclear. METHODS: Enzyme-linked immunosorbent assay methods and Western blots were used to study levels and molecular forms of MMP-7, -8, -9, -25, and -26 and TIMP-1 from salivary samples of HIV+ patients (n = 55) and healthy controls (n = 10). The expression of MMPs was also studied by immunohistochemical means in gingival tissue specimens (n = 11, HIV+ patients; n = 10, healthy controls). RESULTS: The HIV+ patients' MMP-8 levels in saliva were statistically significantly higher only in the acquired immunodeficiency syndrome (AIDS)-phase. MMP-9 levels in ASX- and AIDS-phases showed increased expression. TIMP-1 levels were significantly decreased in lymphadenopathy syndrome (LAS)- and AIDS-related complex (ARC)-phases, while MMP-8/TIMP-1 and MMP-9/TIMP-1 molar ratios were increased in all phases in comparison with controls. The molecular forms of MMP-7, -25, and -26 were different between patients and controls as assessed by Western blot. Immunohistochemical studies showed slightly enhanced MMP-7, -8, -9, -25, and -26 staining in HIV+ gingival tissue samples in comparison with controls. CONCLUSIONS: This study confirmed and further demonstrated differences in salivary amounts and molecular forms of MMPs and TIMP-1 in HIV+ patients. The results may reflect alterations in host defence reactions associated with HIV infection.     

Hydrocortisone affects the expression of matrix metalloproteinases (MMP-1, -2, -3, -7, and -11) and tissue inhibitor of matrix metalloproteinases (TIMP-1) in human gingival fibroblasts

BACKGROUND: There is a positive correlation between the course of periodontal disease and psychosocial stress status. Stress leads to activation of the hypothalamic-pituitary-adrenal axis, resulting in increased cortisol release. The present study evaluates the effect of two different hydrocortisone concentrations on mRNA expression of matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) in cultured, human gingival fibroblasts. METHODS: Gingival fibroblasts were stimulated with 10(-7) or 10(-9) M hydrocortisone for 24 hours; untreated cells served as controls. Alterations in the expression of MMP-1, -2, -3, -7, -11 and TIMP-1 and -2 were evaluated using real-time polymerase chain reaction and Western blotting. beta-actin mRNA expression was used as a reference to normalize gene expression. RESULTS: Although the higher hydrocortisone concentration upregulated MMP-1, -2, -7, -11, and TIMP-1 (P <0.05) expression, the lower concentration induced downregulation or diminished upregulation. The lower hydrocortisone concentration induced a 23-fold increase in MMP-3 gene expression, whereas the higher concentration induced less upregulation; however, protein expression was regulated similarly by both hydrocortisone concentrations. The effect of hydrocortisone on TIMP-2 expression was not significant (P >0.05). CONCLUSIONS: Hydrocortisone produced a dose-dependent regulation of MMP and TIMP expression. The higher hydrocortisone concentration significantly upregulated expression of MMP-1, -2, -7, and -11 and TIMP-1 in human gingival fibroblasts, which may constitute a mechanism underlying the increased periodontal breakdown associated with psychosocial stress status.     

Matrix metalloproteinases, tissue inhibitor of matrix metalloproteinase-1, and laminin-5 gamma2 chain immunolocalization in gingival tissue of endotoxin-induced periodontitis in rats: effects of low-dose doxycycline and alendronate

BACKGROUND: Matrix metalloproteinases (MMPs) play important roles in tissue destruction mechanisms of periodontitis. MMP-8 and -13 are the major collagenases that act in extracellular matrix degradation in periodontal tissues. MMP-14 is a membrane-type MMP, and laminin (Ln)-5 is a basal membrane component. The aim of the present study was to evaluate the effects of doxycycline and alendronate on gingival tissue expression of MMP-8, -13, and -14; tissue inhibitors of MMP (TIMP)-1; and Ln-5 gamma2 chain in experimental periodontitis induced by Escherichia coli endotoxin (LPS) in rats. METHODS: Experimental periodontitis was induced by repeated injection of LPS. Forty-four adult male Sprague-Dawley rats were divided into five study groups: saline control, LPS, LPS + doxycycline, LPS + alendronate, and LPS + doxycycline + alendronate. Doxycycline and alendronate were given as a single agent or as combination therapy during the 7 days of the experimental study period. On day 7, the rats were sacrificed, and the gingival tissues were analyzed immunohistochemically for expression of MMP-8, -13, and -14, Ln-5 gamma2 chain, and TIMP-1. Alveolar bone loss was evaluated morphometrically under a stereomicroscope. Data were tested statistically by Kruskal-Wallis and Mann-Whitney tests and Spearman correlation analysis. RESULTS: Alveolar bone loss was significantly higher in the LPS, doxycycline, alendronate, and combination groups than in the saline control group (all P <0.01). MMP-8 expression was significantly higher in the LPS group than in the saline control group (P = 0.001). Individual administration of doxycycline or alendronate significantly decreased the expression of MMP-8 compared to LPS (P = 0.01). Combined drug administration reduced MMP-14 significantly compared to doxycycline (P = 0.004). No significant differences in Ln-5 gamma2 chain expression were found between the study groups (P >0.05). MMP-14 significantly correlated with the Ln-5 gamma2 chain in the LPS + alendronate group (P = 0.04) and with the amount of alveolar bone loss in the LPS + doxycycline + alendronate group (P = 0.03). CONCLUSIONS: Our findings suggest that alendronate and/or doxycycline may inhibit MMP-8 expression significantly; particularly, their combined administration may provide beneficial effects in periodontal treatment. Moreover, individual administration of alendronate and doxycycline results in significant increases in TIMP-1 expression in gingiva. However, these effects of combined low-dose doxycycline and alendronate on MMPs and TIMP should be verified by clinical human trials before these agents are used in dental practice.     

The associations between gingival crevice fluid matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1 and periodontitis in human immunodeficiency virus-positive patients

BACKGROUND AND OBJECTIVE: The study aimed to determine whether matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in gingival crevice fluid could serve as prognostic factors for the progression of periodontitis in human immunodeficiency virus (HIV) -positive patients. Activated inflammatory cells produce inflammatory mediators, which stimulate the production of MMPs and their inhibitors. It is likely that the compromised immune system contributes to the pathogenesis of periodontitis in HIV-positive patients. METHODS: Clinical measurements including gingival index, plaque index, bleeding index, probing depth, attachment loss, and gingival crevice fluid samples were taken from two healthy sites (including sites with gingival recession, gingival index = 0; probing depth < or = 3 mm; attachment loss < or = 2 mm), three gingivitis sites (gingival index > 0; probing depth < or = 3 mm; attachment loss = 0) and three periodontitis sites (gingival index > 0; probing depth > or = 5 mm; attachment loss > or = 3 mm) of each of the 35 patients at baseline visits and 6-month visits by means of paper strips. Gingival crevice fluid levels of MMP-9 and TIMP-1 were determined by sandwich enzyme-linked immunosorbent assays. RESULTS: The mean amounts of MMP-9 and TIMP-1 in the gingivitis and periodontitis sites sites were significantly higher than in the healthy sites (P < 0.0001). The progressing site was defined as a site that had 2 mm or more attachment loss during the 6-month study period. Gingival crevice fluid levels of MMP-9 were significantly correlated with probing depth, attachment loss, TIMP-1, age, smoking pack years, and viral load values at baseline and 6-month visits (0.0001 < P < 0.001). TIMP-1 levels were only correlated with CD4, viral load, attachment loss, and MMP-9 (0.001 < P < 0.01). Repeated measures analysis of 11 active sites vs. 269 inactive sites indicated that MMP-9 and TIMP-1 levels were significantly higher in active sites than in inactive sites (P < 0.0001). These data indicate that sites with high ginigval crevice fluid levels of MMP-9 and TIMP-1 in HIV-positive patients are at significantly greater risk for progression of periodontitis.     

Influence of phase I periodontal therapy on levels of matrix metalloproteinase 1 and tissue inhibitor of metalloproteinase 1

Background

Matrix metalloproteinase-1 (MMP-1) is a member of a family of enzymes that can degrade most extracellular matrix macromolecules. Extracellularly, MMPs are controlled by tissue inhibitors of metalloproteinases (TIMPs) and by mechanisms of pro-MMP activation. Levels of MMPs and TIMPs change during healing, inflammation, and normal tissue turnover. Herein we aimed to evaluate the levels of MMP-1 and TIMP-1 in gingival crevicular fluid (GCF) from periodontally healthy patients (control group) and chronic periodontitis patients before and after phase 1 therapy.

Methods

In this study we examined 30 patients who had chronic periodontitis with probing depth sites ⩾5 mm and a clinical attachment level (CAL) ⩾5 mm. We included 30 periodontally healthy patients as a control. Clinical measurements such as plaque (PI) and gingival (GI) indices, papillary bleeding index (PBI), probing depths (PD), and CAL were recorded both before treatment (BT) and after phase I periodontal treatment (AT). Assays for MMP-1 and TIMP-1 were performed with an enzyme-linked immunosorbent assay (ELISA) method.

Results

All clinical parameters were significantly reduced at the post-therapy visit. MMP-1 levels were significantly higher in patients BT than the controls; however, the patients AT were not statistically different than the controls. TIMP-1 levels in patients BT were significantly lower than in the controls and significantly lower than patients AT. We observed a significant positive correlation between GCF volume and MMP-1 levels. Furthermore, TIMP-1 levels were significantly negatively correlated with both GCF volume and all clinical parameters.

Conclusions

We observed that as the extent of periodontal destruction increases, MMP-1 concentration increases and TIMP-1 concentration decreases in GCF. When chronic periodontitis patients were treated by scaling and root planing (SRP), the average MMP-1 concentrations decreased and TIMP-1 concentrations increased in GCF.     

Tissue inhibitor of metalloproteinases-1, matrix metalloproteinases-1 and -8, and collagenase activity levels in peri-implant crevicular fluid after implantation

The integrity of connective tissues surrounding dental implants may be influenced by a balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The purpose of this study was to provide an overall assessment of TIMP-1, MMP-1 and -8 levels as well as collagenase activities during the wound healing process after implantation and in peri-implantitis lesions. Peri-implant crevicular fluid (PICF) was sampled with sterile paper strips from 10 osseointegrated implants of 6 subjects. Ten implants from 6 patients affected with peri-implantitis were also assessed. Gingival crevicular fluid (GCF) from 11 periodontitis-affected patients and 10 healthy volunteers served as controls. TIMP-1 and MMP-1 and -8 protein levels in the PICF were measured by ELISA, and active and APMA-activatable collagenase activities were determined by functional assays using image-analysis after SDS-PAGE. The experiment showed a significant increase in the TIMP-1 level at 1 week after implantation as compared with that in GCF from healthy periodontium. Four weeks after implantation it had reached the same level as that in the GCF of healthy subjects. The data has also disclosed a higher post-implantation collagenase activity level at 1 week than at weeks 2, 4, and 12. This may be due to the increase in MMP-1 and -8. Furthermore, peri-implantitis and periodontitis were shown to be similar inflammatory lesions in respect to MMP-1 and -8 and collagenase activities, even though the TIMP-1/MMP-1 + MMP-8 ratio was significantly lower in peri-implantitis than in periodontitis. In conclusion, the overproduction of TIMP-1 in the wound area after implantation could, to some extent, inhibit excessive tissue destruction and degradation of the neo-matrix in wound repair due to MMPs.     

MMP-7 and TIMP-1, New Targets in Predicting Poor Wound Healing in Apical Periodontitis

Introduction

Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) are strongly associated with tissue destruction because of inflammation. In this study, we investigated the expression of MMPs and TIMPs messenger RNA and protein levels in apical periodontitis lesions.

Methods

Tissue samples from patients presenting clinical signs of chronic apical abscess (CAA) or asymptomatic apical periodontitis (AAP) were collected postoperatively and used for gene expression analysis of MMP-2, -3, -7, -9, -14, -16, and -25; TIMP-1; and TIMP-2 in real-time polymerase chain reaction. Immunohistochemistry was also performed to detect the expression of MMP-7 and TIMP-1 proteins. Lastly, U-937 cells were induced to terminal differentiation into macrophages, infected with purified Escherichia coli lipopolysaccharide, and assessed for the expression of MMP-7 and TIMP-1 using immunocytochemistry and confocal microscopy.

Results

Significantly higher messenger RNA levels were found for all genes in AAP and CAA samples when compared with healthy control samples (P < .001). AAP cases exhibited significantly higher TIMP-1 when compared with CAA cases, whereas CAA cases showed higher MMP-2, MMP-7, and MMP-9 messenger RNA levels (P < .05). We also detected positive the expression of MMP-7 and TIMP-1 proteins in the tissue samples. The expression of both MMP-7 and TIMP-1 were increased in lipopolysaccharide-stimulated cells compared with nonstimulated cells and appear to colocalize in the Golgi apparatus.

Conclusions

MMPs appear to have an influential role in CAA cases in which ongoing tissue destruction is observed. TIMPs are preferentially associated with AAP, perhaps as a subsequent defense mechanism against excessive destruction. Taken together, our findings implicate MMP and TIMP molecules in the dynamics of inflammatory periapical lesion development.     

The effects of selective COX-2 inhibitor/celecoxib and omega-3 fatty acid on matrix metalloproteinases, TIMP-1, and laminin-5gamma2-chain immunolocalization in experimental periodontitis

BACKGROUND: Matrix metalloproteinases (MMPs) play important roles in tissue-destruction mechanisms-associated periodontitis. MMP-8 and -13 are the predominant collagenases that are important in the extracellular matrix degradation in periodontal tissues. MMP-14 is a membrane-type MMP, whereas laminin-5 indicates basal membrane modification and epithelial induction. The purpose of the present study was to evaluate the effects of celecoxib and omega-3 fatty acid administration on the gingival tissue expression of MMP-8, -13, and -14, tissue inhibitor of MMP (TIMP)-1, and laminin (Ln)-5gamma2-chain in rat experimental periodontitis induced by Escherichia coli endotoxin (lipopolysaccharide [LPS]). METHODS: Experimental periodontitis was induced in rats by repeated LPS injection. Fifty-one adult male Sprague-Dawley rats were divided into six study groups: saline control, LPS, LPS + celecoxib, LPS + therapeutic omega-3 (TO3), prophylactic omega-3 + LPS + omega-3 (P+TO3), and LPS + celecoxib + omega-3 fatty acid. Celecoxib and omega-3 fatty acid were given as a single agent or as combination therapy for 14 days. On day 15, all rats were sacrificed, and gingival tissues were analyzed immunohistochemically for the expression of MMP-8, -13, and -14, TIMP-1, and Ln-5gamma2-chain. Alveolar bone loss was evaluated morphometrically under a stereomicroscope. Data were tested statistically by Kruskal-Wallis and Mann-Whitney tests and Spearman correlation analysis. RESULTS: Alveolar bone loss was significantly higher in all study groups compared to the saline control group (all P <0.01). MMP-8 expression was significantly higher in the LPS group than in the saline group (P = 0.001). Very low expression of MMP-8 was found in the celecoxib, P+TO3, and combination groups. TO3 increased TIMP-1 expression significantly compared to the LPS group (P <0.05). Individual celecoxib and P+TO3 administration increased MMP-14 significantly compared to saline control and LPS groups (P <0.05). No significant differences were found among the study groups with regard to Ln-5gamma2-chain and MMP-13 expressions (P >0.05). CONCLUSIONS: Selective cyclooxygenase-2 inhibitor, prophylactic omega-3 fatty acid, and a combination of these two agents can inhibit gingival tissue MMP-8 expression. Moreover, the individual administration of therapeutic omega-3 may increase gingival TIMP-1 expression in contrast to no effect on MMP-8, -13, and -14 expressions in experimental periodontitis. These experimental findings in a rat model of LPS-induced periodontitis need to be verified by clinical human studies.     

Matrix metalloproteinases and their inhibitors in gingival crevicular fluid and saliva of periodontitis patients

Abstract Matrix metalloproteinases (MMPs) and serine proteinases seem to be related to tissue destruction in periodontitis. The presence of MMPs in gingival crevicular fluid (GCF) and saliva, however, has not been studied comprehensively with the enzyme-linked immunosorbent assay (ELISA)-technique, We therefore examined the levels of MMP-1,-3.-8 and -9, and their endogenous inhibitor, tissue inhibitor of matrix metalloproteinases (TIMP-1). in GCF and saliva of patients with adult periodontitiss (AP) and localized juvenile periodontitis (LJP). Elevated levels of MMP-1 were detected in LJP GCF compared to AP and control GCF. Elevated levels of TIMP-1 were also detected in LJP GCF in comparison to AP and control GCF Higher MMP-8 levels were detected in AP GCF compared to LJP and control GCF. The relative low levels of MMP-3 were present in all studied GCF samples. Elevated levels of MMP-8 were further detected in saliva of AP compared to LJP and the controls. Both MMP-1 and TIMP-1 were detected in all studied saliva samples, but not significant differences were detected between the studied groups. Our ELISA-results confirm that (i) PMN MMP-8 and MMP-9 are the main collagenase and gelatinase in AP GCF, whereas GCF collagenase in LJP seems to be of the MMP-1-type; (ii) only low levels of TIMP-1. endogenous MMP-inhibitor. are present in AP GCF. which emphasises the importance of doxycycline as a possible adjunctive drug in the treatment of AP patients; (iii) tests based on specific antibodies against PMN MMPs. especially MMP-8, might serve as a reliable method of measuring and monitoring enzyme levels in GCF from different periodontitis patients.