Tissue inhibitors of metalloproteinases level and collagenase activity in gingival crevicular fluid: the relevance to periodontal diseases

OBJECTIVES: To provide an overall assessment of levels of tissue inhibitors of metalloproteinases (TIMPs), collagenase activities, and of immuno-reactivities for matrix metalloproteinases (MMP)-1 and -8 in gingival crevicular fluid (GCF) obtained from healthy subjects, and gingivitis and periodontitis patients, and to analyse the relationships between periodontal tissue destruction and the GCF components in periodontal diseases by principal component analysis. MATERIALS AND METHODS: GCF was sampled with sterile paper strips from 10 gingivitis and 11 periodontitis patients. Ten volunteers served as clinically healthy controls. TIMP-1 and -2 protein amounts in GCF were measured by ELISA, and active and APMA-activatable collagenase activities were determined by functional assays using image-analysis after SDS-PAGE. RESULTS: GCF TIMP-1 level and both active and latent collagenase activities were significantly higher in the diseased groups than in the healthy group. TIMP-2 was detectable in only 29% of all subjects (mean: 2.06 ng). Western blot analysis showed that MMP-8 was the major interstitial collagenase in the GCF of the diseased groups. Principal component analysis using clinical parameters and the GCF components has indicated components one to three account for 87% of total variation when evaluating the relevance of their measurements to periodontal diseases. CONCLUSIONS: We conducted the functional and immunological characterization of MMPs and TIMPs in the GCF of periodontally diseased patients. Principal component analysis indicated components one to three explaining 87% of total variation, and further suggested that higher collagenase activity (especially in active collagenase) would be an important marker in evaluating the pathogenesis of periodontitis. Consequently, these observations may have significant therapeutic and diagnostic implications.     

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.     

Levels of gingival crevicular metalloproteinases-8 and -9 in periodontitis

Matrix metalloproteinases (MMPs), the key enzymes responsible for matrix degradation, are derived from polymorphonuclear leukocytes during the early stages of periodontitis. The aim of this study was planned to determine the levels of GCF (gingival crevicular fluid) matrix metalloproteinase-8 (MMP-8) and metalloproteinase-9 (MMP-9) patients with periodontitis and in healthy controls. Levels of crevicular MMP-8 and -9 were determined by ELISA in subjects with healthy without any periodontal disease (n = 10) and periodontitis (n = 10). Significantly higher crevicular MMP-8 and -9 were observed in cases of periodontitis compared to healthy adults. Crevicular MMP-8 and -9 may serve as biomarkers of periodontal disease and aid in early detection of periodontitis.     

Levels of tissue inhibitor of metalloproteinases-1 and matrix metalloproteinases-1 and -8 in gingival crevicular fluid following treatment with enamel matrix derivative (EMDOGAIN)

The mechanism of enamel matrix derivative (EM D) action on the periodontal wound healing process is not well understood. However, earlier in vitro studies from our laboratory demonstrated that EMD stimulated the proliferation of both periodontal ligament and gingival fibroblast cells. Therefore, the purpose of this study was to further evaluate the effect of EMD on the early wound healing process by assessing the protein levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in gingival crevicular fluid (GCF). Sixteen patients, each of whom had one or two pairs of infrabony defects located contralaterally in the same arch, were included in this clinical trial. Thirty-six infrabony defects were randomly assigned treatment with flap surgery plus EMD or flap surgery plus placebo. At baseline and at 2, 4 and 12 week follow-up evaluation visits, GCF was sampled with paper strips. After determination of GCF volume, TIMP-1, MMP-1 and MMP-8 GCF levels were measured by an enzyme-linked immunosorbent assay. Intragroup analysis: At week 2 following surgery, when compared to baseline all parameters in each study group, except MMP-1, significantly increased (p<0.05). There were no significant differences between 4 or 12 weeks and baseline in either study group. Intergroup analysis: At 4 weeks after surgery, GCF volume and TIMP-1 levels showed a significant decrease (p<0.05) in the EMD group, when compared to the placebo group. MMP-1 levels at weeks 2, 4 and 12, and MMP-8 levels at weeks 4 and 12 were significantly lower (p < 0.05) in the EMD group compared to the placebo group. EMD compared to placebo treated sites demonstrated a more rapid return to baseline levels of TIMP-1, MMP-1 and MMP-8. These findings suggest that treatment with flap surgery and EMD, compared to flap surgery with placebo, accelerated healing at an earlier stage of wound healing following surgery.     

Collagenolytic fragments and active gelatinase complexes in periodontitis

Background: Periodontal tissues remodel rapidly, which enables quick adaptation to mechanical changes. Matrix metalloproteinases (MMPs) are involved in these remodeling processes under control of tissue inhibitor of metalloproteinases (TIMPs). In periodontitis, overactivity of MMPs results in pathologic tissue degradation. The aim of this study was to analyze MMPs and TIMPs in healthy and diseased gingiva, periodontal ligament (PDL), and gingival crevicular fluid (GCF). Methods: Samples of gingiva, PDL, and GCF were obtained from healthy controls (gingiva: n = 18; PDL: n = 15; GCF: n = 8) and subjects with periodontitis (gingiva: n = 11; PDL: n = 18; GCF: n = 12). MMPs and TIMPs were analyzed by gelatin-, collagen-, and reverse zymography and by Western blotting. Total MMP activity was analyzed using a fluorogenic substrate. Results: TIMP-1 and -2, active and pro-MMP-2 and -9, and active MMP-1 and -8 were present in all samples. Large amounts of active MMP-2 complexes and collagenolytic fragments were also found. Their levels were higher in PDL and GCF from subjects with periodontitis. In general, TIMP levels were lower in diseased periodontal tissues. Especially diseased GCF contained more MMPs. Surprisingly, some MMPs were more abundant in healthy gingiva and PDL than in diseased tissue. Conclusions: Unexpected variations in MMP and TIMP levels in gingiva, PDL, and GCF may result from differences in subject characteristics and disease activity. The levels of active MMP-2 complexes and collagenolytic fragments are higher in the periodontium of subjects with periodontitis and might contribute significantly to periodontal destruction.     

龈沟液中基质金属蛋白酶8及基质金属蛋白酶组织抑制物1水平与牙周病的关系

目的 探讨龈沟液中基质金属蛋白酶8(matrix metalloproteinases-8,MMP-8)及金属蛋白酶组织抑制物1(tissue inhibitors of metalloproteinases,TIMP-1)的水平变化与牙周病的关系,以期为临床提供参考.方法 试验人群共48例,分为牙周健康组16例、慢性牙龈炎组18例及慢性牙周炎组14例,采用酶联免疫吸附测定法检测各组人群龈沟液中MMP-8和TIMP-1水平,并计算MMP-8与TIMP-1的比值.结果 牙周健康组、慢性牙龈炎组及慢性牙周炎组中MMP-8的平均质量浓度分别为:(0.62±0.09)、(0.68 ±0.02)及(0.78±0.05) mg/L,3组之间差异有统计学意义(P＜0.01),并且两两比较差异均有统计学意义(P＜0.05);牙周健康组、慢性牙龈炎组及慢性牙周炎组龈沟液中MMP-8与TIMP-1的比值分别为:0.16 ±0.06、0.23±0.09及0.32 ±0.15,3组相比差异有统计学意义(P＜0.05).结论 龈沟液中MMP-8的质量浓度及MMP-8/TIMP-1比值的变化均可以作为判断牙周炎症水平有意义的临床指标.     

MMP-13 and TIMP-1 determinations in progressive chronic periodontitis

Matrix metalloproteinase (MMP)-13 is a collagenase involved in extracellular matrix degradation either by its direct degradative effects or by processing bioactive substrates. The aim of this study was to determine the levels of MMP-13 and tissue inhibitor of metalloproteinase (TIMP)-1 in gingival crevicular fluid (GCF) and gingival biopsies obtained from active and inactive sites during chronic periodontitis progression. MATERIALS AND METHODS: This was a longitudinal study in which chronic periodontitis patients with moderate to severe disease were included and followed until they developed progression determined by the tolerance method. GCF samples were obtained from periodontitis, active, inactive and healthy sites and additional gingival biopsies were taken from active and inactive sites. MMP-13 and TIMP-1 determinations were carried out by immunodot blots and immunowestern blots. RESULTS: In progressive periodontitis, MMP-13 and TIMP-1 remained unchanged between active and inactive sites, but as the TIMP-1 relative levels increased together with MMP-13 elevation in inactive samples, an inverse correlation was observed in active sites. Besides, MMP-13 was undetectable in healthy controls. CONCLUSION: Chronic periodontitis is characterized by increased MMP-13 expression. During disease progression, active sites tended to decrease TIMP-1 levels in association with MMP-13 elevation.     

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.     

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.     

Biomarkers of periodontitis in oral fluids

Matrix metalloproteinases (MMPs), the key enzymes responsible for matrix degradation, are derived from polymorphonuclear leukocytes during the early stages of periodontitis. The present study determined the levels of GCF matrix metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) and salivary MMP-8 in patients with gingivitis and periodontitis and in healthy controls. Levels of crevicular MMP-2, MMP-9 and salivary MMP-8 were determined by ELISA in subjects with healthy gingiva (n = 15), gingivitis (n = 18) and periodontitis (n = 20). Significantly higher salivary MMP-8 and crevicular MMP-9 were observed in cases of periodontitis compared to gingivitis and healthy adults. On the other hand, crevicular MMP-2 levels in periodontitis subjects were lower than those in gingivitis and healthy subjects. The three MMP levels were highly correlated to probing depth, and bleeding on probing. Salivary MMP-8, crevicular MMP- 2 and 9 may serve as biomarkers of periodontal disease and aid in early detection of periodontitis or gingivitis.     

Expression of HMGB1 and HMGN2 in gingival tissues, GCF and PICF of periodontitis patients and peri-implantitis

Background and objective

High mobility group chromosomal protein B1 (HMGB1) and N2 (HMGN2), two members of high mobility group (HMG) family, play important role in inflammation. The purpose of this study was to investigate the expression of HMGB1 and HMGN2 in periodontistis.

Materials and methods

The expression of HMGB1 and HMGN2 mRNA in gingival tissues and gingival crevicular fluid (GCF) in chronic periodontitis (CP), generalised aggressive periodontitis (G-AgP) patients and healthy subjects was detected by real-time PCR. The protein level of HMGB1 and HMGN2 in peri-implant crevicular fluid (PICF), peri-implant crevicular fluid of peri-implantitis (PI-PICF) and normal patients was determined by Western blotting. Furthermore, IL-1β, IL-6, IL-8, TNF-α and HMGB1 levels in GCF, PI-PICF and healthy-PICF samples from different groups were determined by ELISA.

Results

HMGN2 expression was increased in inflamed gingival tissues and GCF from CP and G-ApG groups compared to control group. HMGB1 expression was the highest in the gingival tissues and GCF from CP patients and was accompanied by increased concentrations of IL-1β, IL-6, IL-8 proinflammaory cytokines.

Conclusion

To our knowledge, this is the first study reporting that the expression of HMGB1 and HMGN2 was increased in the gingival tissues and GCF in CP and G-AgP and the PICF in PICF. Our data suggest that HMGB1 may be a potential target for the therapy of periodontitis and PI.     

Longitudinal analysis of metalloproteinases, tissue inhibitors of metalloproteinases and clinical parameters in gingival crevicular fluid from periodontitis-affected patients

BACKGROUND: The aim of this work was to improve the assessment of the periodontal disease status through measurements of extracellular matrix metalloproteinases (MMPs) and their tissular inhibitors (TIMPs) in the gingival crevicular fluid from patients diagnosed with chronic periodontitis. METHODS: Gingival crevicular fluid samples from patients (n = 13) were taken from 60 sites initially, and from 51 and 41 sites, respectively, 3 and 6 months after scaling and root planing. Gingival crevicular fluid samples were also taken from healthy subjects (n = 11, 24 sites). The presence of MMP-9 and MMP-8 was assessed by zymography and immunowestern blotting, respectively. The actual MMP activity (gelatinase and collagenase) was measured using the fluorogenic substrate assay. TIMP-1 and -2 levels were measured by immunodot blot. RESULTS: The fluorogenic substrate assay determinations showed higher MMP activity in sites with probing depth > or = 4 mm, with significant reduction post-treatment. Gelatinase activity followed by zymography consisted mainly of MMP-9. A different pattern of MMP-8 in control and patient sites was found. Controls only showed species of a partially active form (69 kDa), whereas patient sites showed a high frequency of the active form (56 kDa), and in some cases the latent form (85 kDa) was also observed. The active form reduced its frequency in sites with probing depth > or = 4 mm. TIMP-1 and -2 levels in patients were significantly lower than in controls, and after treatment the recovery of TIMP-1 level similar to control was observed. CONCLUSION: Significant correlations between the severity of the periodontal disease and the actual MMP activity, the active form of MMP-8 and the low level of both TIMP-1 and TIMP-2 were found.     

Osteocalcin,deoxypyridinoline and interleukin-1beta in peri-implant crevicular fluid of patients with peri-implantitis

The aim of the present study was to analyze the levels of osteocalcin, deoxypyridinoline (Dpd) and interleukin-1beta as markers of bone metabolism in peri-implant crevicular fluid (PICF) from peri-implantitis patients. PICF was sampled from a total of 34 endosseous titanium implants from 16 patients; nine females (mean age 52.8, range 40-62 years) and seven males (mean age 56.0, range 36-66 years). The implants had been in place for a period of 9-112 months (mean; 35.8 months) since the loading. These sites were categorized as six peri-implantitis, eight peri-implant mucositis and 20 healthy implant. PICF volume from peri-implantitis sites was significantly higher than mucositis and healthy implant sites (P < 0.01). Osteocalcin levels in PICF from mucositis sites were significantly higher than healthy implants (P < 0.05), whereas peri-implantitis sites were not significantly different from either mucositis or healthy implant sites. Dpd could not be detected in any of the samples examined. IL-1beta levels in PICF from peri-implantitis sites were significantly higher than levels from peri-implant mucositis (P < 0.05) and healthy implant sites (P < 0.01). In conclusion, osteocalcin in PICF may reflect increased local bone turnover around implants. Further, IL-1beta should be a useful marker for peri-implant inflammation.     

牙周非手术治疗对龈沟液MMP-8及其抑制剂TIMP-1水平的影响

目的: 比较慢性牙周炎患者非手术治疗前、后龈沟液MMP-8、TIMP-1水平的变化。方法: 选择慢性中重度牙周炎30例, 治疗前、后临床检查牙龈出血指数(SBI)、菌斑指数(PLI)、探诊深度(PD), 采集龈沟液, 采用双抗体夹心ABC-ELISA法检测MMP-8、TIMP-1的含量。采用SSPS 19.0软件包对数据进行配对样本t检验和多元相关分析。结果: 治疗后MMP-8、TIMP-1水平显著下降, MMP-8/TIMP-1 比率治疗前、后无显著差异。多元相关分析各变量间相关性, 发现MMP-8和龈沟液重量呈正相关关系。结论: 牙周非手术治疗能显著降低龈沟液MMP-8、TIMP-1水平, 从而减轻牙周组织损伤。     

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-1 and -8 in gingival crevicular fluid during orthodontic tooth movement: a pilot study during 1 month of follow-up after fixed appliance activation

The role of matrix metalloproteinases (MMPs) in response to mechanical forces in orthodontic tooth movement has only been partially clarified. In the present in vivo pilot study, the presence, levels, and degree of activation of MMP-1 and -8 were measured daily for 1 month in gingival crevicular fluid (GCF) of patients treated with orthodontic fixed appliances. GCF samples were collected from five orthodontic patients and three controls from one upper or lower central incisor or from one upper canine before fixed appliance activation and every 24 hours for 1 month thereafter. The molecular forms and activation degrees of MMP-1 and -8 in GCF were analysed by Western blotting, and MMP-8 levels determined by immunofluorometric assay (IFMA).IFMA revealed, during the study period, on average 12-fold higher levels (56 +/- 50 versus 4.6 +/- 4 microg/l) of MMP-8 in orthodontic GCF than in control GCF. The MMP-8 levels in orthodontic GCF were lower than those detected in gingivitis and periodontitis GCF, but significantly higher than in control GCF. IFMA analysis was confirmed by Western blot analysis showing elevated MMP-8 levels from orthodontic GCF relative to control GCF. Forty-one per cent of total MMP-8 immunoreactivities were high-molecular weight complexes (>100 kDa), 32 per cent in the 75 kDa pro-polymorphonuclear (PMN)-MMP-8 form, 14 per cent in the 60 kDa active-PMN-MMP-8 form, and 13 per cent in the 55 kDa fibroblast-type pro-MMP-8 form. In the GCF of orthodontic patients no MMP-1 immunoreactivities were detected. MMP-8 and -1 levels in the control GCF were low and not detectable.These results demonstrate that in vivo in human GCF, elevation and partial activation of multiple species of PMN- and fibroblast-type MMP-8 reflect periodontal remodelling during orthodontic tooth movement.     

Assessment of a novel screening test for neutrophil collagenase activity in the diagnosis of periodontal diseases

BACKGROUND: Increased levels of active neutrophil collagenase (MMP-8) in the gingival crevicular fluid (GCF) are associated with progressive periodontitis. The measurement of this enzyme in GCF could facilitate diagnosis. However, assays with sufficient sensitivity to detect collagenase in whole-mouth GCF currently use radiolabeled substrates and require several days to complete. To provide more rapid analyses of collagenase activity that are better adapted to clinical studies, we developed and validated a novel assay (soluble biotinylated-collagen assay: SBA) based on chemiluminescent detection of biotinylated collagen digestion products. METHODS: The concordance of the novel SBA assay with a radioactive collagen substrate assay was assessed by parallel analyses of enzyme from 35 neutrophil preparations and from 41 samples of GCF from periodontitis patients, followed by Pearson correlation analysis. To test whether the assay appropriately measured MMP-8 activity, enzyme activity was assessed after incubation with specific collagenase blockers. We examined the diagnostic utility of the SBA in cross-sectional and longitudinal analyses of 125 patients with adult periodontitis, 5 patients with early-onset periodontitis, 1 edentulous patient, and in 32 control patients without periodontitis. RESULTS: The assay detected <56 pg collagen degraded/hour/microl sample, which is comparable to the most sensitive radioactive assay. The total assay time was 22 hours and reproducibility on replicate measurements was high (r = 0.96). In direct comparisons of MMP-8 activity in GCF with enzyme from peripheral blood neutrophils using the SBA and radioactive assays, there was a high correlation (r = 0.97). As expected, EDTA and TIMP-1 and -2, known inhibitors of MMP-8, completely blocked enzyme activity with this assay. Cross-sectional and longitudinal analyses of GCF showed that MMP-8 activity was >18-fold higher in severe periodontitis than in stable periodontitis and decreased to <25% of pretreatment levels following therapy. Based on measurements of collagenase activity in different disease groups, we estimated a value of 80 nano units as a threshold for severe periodontitis. CONCLUSIONS: These results indicate that active MMP-8 is detected in GCF by a novel assay that is specific, simple, rapid, and reproducible and which may facilitate diagnostic discrimination between stable and progressive lesions.     

Modulation of host matrix metalloproteinases by bacterial virulence factors relevant in human periodontal diseases

OBJECTIVE: Bacterial pathogens involved in periodontal diseases exert a part of their destructive effect by triggering and inducing host cells to elevate their secretion of matrix metalloproteinases (MMPs). Pathogen-secreted phospholipase (PLC) is one bacterial product that may trigger this host response. The roles of exogenous PLC leading to the release, secretion and expression of MMPs by peripheral blood neutrophils (PMNs), cultured epithelial cells of human gingiva and porcine periodontal ligament were investigated. Also the activities of PLC in the diseased and healthy gingival sulcular fluid (crevicular fluid, GCF) and molecular forms of gelatin-ases present in dental plaque were investigated. MATERIALS AND METHODS GCF, salivary and dental plaque samples were analyzed for PLC and proteinase activities. The abilities of PLC to induce PMNs and oral epithelial cells to release and express their MMPs were examined by specific functional, immunological and molecular biology means. RESULTS: PMN-derived MMPs were found to predominate in periodontitis GCF and plaque, and PLC activities were higher in GCF of adult periodontitis patients than in healthy controls. Purified bacterial PLC (1 mU ml^-I) efficiently induced PMN degranulation. PLC also induced MMP expression in the cultured epithelial cells. The strongest response was seen in MMP-9 and less in MMP-2. The induction was dose-dependent in the range of 0.I-1.0 U ml^-1 PI-PLC, and quiescent cultures were more responsive than proliferating ones. PLC induction of MMPs was polar, with increased levels of MMP-9 in the apical region and increased MMP-2 levels secreted in the basal direction. Northern analysis showed a strong increase in mRNA levels of MMP-9 and a smaller increase for MMP-2 and MMP-I. In the second part of the study we investigated the molecular forms of the released MMPs during periodontitis. In bacterial plaque of periodontitis patients the MMP-9 were found to be converted into lower molecular weight forms. Isolated proteinase from Porphyromonas gingivalis (ATCC 33277) was able to convert human proMMPs to their active forms. CONCLUSION: Bacterial PLC may induce degranulation of PMN MMPs and increase MMP expression in oral epithelial cells. The released proteases can be converted into active form by the proteases of plaque bacteria. Thereby, the pathogenic oral bacteria may indirectly participate in the destruction of periodontal tissues.     

Longitudinal evaluation of GCF MMP-3 and TIMP-1 levels as prognostic factors for progression of periodontitis

BACKGROUND: To determine whether matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in gingival crevicular fluid (GCF) could serve as prognostic factors for the progression of periodontitis, we monitored GCF MMP-3 and TIMP-1 and periodontal status of selected sites in 40 medically healthy subjects over a 6-month period. METHOD: Clinical measurements including gingival index (GI), plaque index, bleeding on probing, suppuration, probing depth (PD), attachment loss (AL), and GCF samples were taken from 2 healthy sites (including sites with gingival recession, GI=0 PD < or =3 mm; AL < or =2 mm) and 2 periodontitis sites (GI > or =1; PD > or =5 mm; AL > or =3 mm) of each patient at baseline, 3-month and 6-month visits by means of sterile paper strips. GCF levels of MMP-3 and TIMP-1 were determined by sandwich ELISA assays. RESULTS: The mean amounts of MMP-3 and TIMP-1 in diseased sites were significantly higher than in healthy sites (p<0.0001). Significantly higher GCF levels of MMP-3 and TIMP-1 were found at progressing sites than in nonprogressing periodontitis sites (0.001 or =2 mm loss of attachment during 6- month study period. GCF levels of MMP-3 were highly correlated with clinical measurements taken at baseline, 3-month and 6-month visits (p<0.001). TIMP-1 levels were only moderately correlated with probing depth and attachment level (p<0.01). Step-wise multiple regression analysis was performed to construct models for the prediction of probing depth and attachment loss increases. The most parsimonious regression models which had the best R2 values included the following variables and accounted for the indicated % of variability. The regression model for the prediction of probing depth increase included MMP-3, smoking pack-years, TIMP-1 and accounted for 53% of the variability. The best model for the prediction of attachment loss increase included MMP-3, smoking pack-years, age, TIMP-1 and explained 59% of the variability. CONCLUSION: These data indicate that sites with high GCF levels of MMP-3 and TIMP-1 are at significantly greater risk for progression of periodontitis.     

Early wound healing following one-stage dental implant placement with and without antibiotic prophylaxis: a pilot study

BACKGROUND: One-stage implant placement has clinically acceptable treatment outcomes. Among other advantages, it may allow investigation of early wound healing. The purpose of this pilot study was to determine whether peri-implant crevicular fluid (PICF) can be used to detect early changes around implants placed with one-stage surgical protocol following 1 week of healing. METHODS: Twenty subjects (11 males and nine females; aged 22 to 72 years; two smokers) were included. Exclusion criteria were allergies to amoxicillin and systemic conditions that may affect healing. Subjects had a healthy periodontium and needed a single implant; eight received antibiotic prophylaxis, and 12 served as controls. Clinical healing was evaluated with plaque and gingival indices (PI and GI, respectively). Gingival crevicular fluid (GCF) from the surgical site was obtained prior to the surgery, whereas PICF was collected at the 1-week visit. Enzyme-linked immunosorbent assay was used to determine GCF/PICF interleukin (IL)-1beta and -8 concentrations. Peripheral blood and GCF antibiotic levels were measured by high-performance liquid chromatography. RESULTS: Postoperative PI and GI were slightly increased. Total GCF and PICF volumes did not show a significant difference between appointments. There was an increase in PICF IL-1beta and -8 levels at 1 week postoperatively. Mean amoxicillin serum concentration was 5.1 +/- 2 microg/ml at 1 to 4 hours following the initial dose, whereas GCF amoxicillin levels were below the limit of detection. Antibiotic prophylaxis had a modest effect on clinical indices (PI and GI) and no appreciable effect on biomarkers. CONCLUSIONS: PICF content can be studied as early as 1 week following one-stage implant placement. The results raise doubts regarding the clinical usefulness of amoxicillin prophylaxis.