Estimation of tissue inhibitor of matrix metalloproteinase-1 levels in gingival crevicular fluid in periodontal health, disease and after treatment.

BACKGROUND: Initial research has shown a negative correlation between the severity of periodontal disease and tissue inhibitor of matrix metalloproteinases (TIMPs) concentrations in gingival crevicular fluid (GCF). However, there are no enough reports to correlate the TIMP-1 concentrations in GCF in periodontal health, disease and after treatment. Hence, the present study aim is to estimate the levels of TIMP-1 in GCF in periodontal health, disease and to evaluate the effect of periodontal therapy on TIMP-1 concentrations in GCF.
MATERIALS AND METHODS: Periodontal examination and collection of GCF by extracrevicular method was performed in 25 subjects selected randomly and categorized into three groups. Group I (Healthy, n = 10), Group II (chronic periodontitis (CP), n = 15) and Group III (After treatment group, n = 15). Scaling and root planning was performed and GCF was collected after 8 weeks of treatment. TIMP-1 levels were estimated in GCF samples by using enzyme linked immunosorbent assay.
RESULTS: TIMPs-1 was detected in all samples. Highest mean TIMP-1 concentrations in GCF were obtained for Group I, whereas the lowest concentrations were seen in Group II. This suggests that TIMP-1 levels in GCF decreases proportionally with the progression of periodontal disease and increases after treatment.
CONCLUSION: There is a substantial decrease in the concentrations of TIMP-1 in CP patients than in healthy and after treatment individuals. Since TIMP-1 levels in GCF are negatively correlated with periodontal disease, TIMP-1 may be considered as a biomarker in periodontal disease progression. However, controlled, longitudinal studies are needed to confirm this possibility.

INTRODUCTION

Chronic periodontitis (CP) is a disease characterized by an intense inflammatory infiltrate associated with irreversible loss of alveolar bone and/or connective tissue attachment in the periodontium, which ultimately results in loss of teeth. This tissue breakdown is thought to be the result of activation of host cells by inflammatory mediators, such as arachidonic acid metabolites, cytokines and enzymes. These factors in turn trigger the resorption of alveolar bone and generation of proteases that degrade extracellular matrix (ECM), resulting in tissue destruction.[1]

Matrix metalloproteinases (MMPs) are able to degrade most proteins of the ECM. MMPs are counteracted by tissue inhibitor of matrix metalloproteinases (TIMPs) which inhibit MMP activity, there by restricting ECM breakdown. The balance between MMPs and TIMPs play an important role in maintaining the integrity of healthy tissues. A disturbed balance of MMPs and TIMPs is found in various pathologic conditions such as rheumatoid arthritis, cancer and periodontitis. In healthy periodontal tissues, TIMP levels are generally higher than in inflamed periodontal tissue, in which MMP levels exceed TIMP levels.[2]

TIMPS form classical non-covalent bimolecular complexes with active forms of MMPs and in some instances with latent MMP precursors as well. TIMPs appear to regulate matrix degradation both by proteinase elimination and by blockage of autocatalytic MMP activation. TIMPs are widely distributed in tissues and fluids and are expressed by many cell types including fibroblasts, keratinocytes, monocytes/macrophages and endothelial cells.[3]

Currently four TIMPs (TIMP 1-4) are known to be expressed in vertebrates. TIMPs inhibit MMPs by forming 1: 1 stoichiometric enzyme-inhibitor complexes. TIMP-1, -2 and -4 are secreted while TIMP-3 is sequestered to the ECM. All currently known TIMP proteins contain 6 loops and have a junction between N- and C-terminal domains. TIMPs are produced in many tissues although not every tissue expresses all four TIMPs. In general most mesenchymal and epidermal cells are able to produce TIMPs. TIMPs are produced by many different cell types and are also found in all body fluids such as saliva, gingival crevicular fluid (GCF), serum and urine.[24]

TIMP-1 molecule has a wedge like shape that fits into the active site cleft of an MMP, as would a substrate molecule. The conserved cystine in N-terminal domain in TIMP-1, will subsequently chelate the active zinc site and expel the water molecule, thereby inactivating the MMP protein.[25]

Although it is now clear that TIMPs are multifunctional proteins that possess both mainstream Metalloproteinase (MP) inhibitory activities and divergent other functions, their attributes may be exploited in search for novel therapies. The current trend of seeking to readdress the MP: TIMP balance to block or reverse disease progression involves either inhibition of MMP activity by small molecule drugs or increasing the local concentration of TIMPs by recombinant protein administration or gene transfer.[6]

The present study is aimed to estimate the levels of TIMP-1 in GCF of periodontal health, disease and to evaluate the effect of periodontal therapy on TIMP-1 concentration in GCF.

MATERIALS AND METHODS

This study was conducted in the division of periodontics, C.K.S Teja Institute of Dental Sciences and Research. The study population consisted of 25 subjects who attended our out-patient section, patients were included if they are in between 20 and 50 years of age group and had not received any periodontal treatment in previous 6 months and were excluded if suffering from any systemic diseases including diabetes mellitus, hypertension, psoriasis, tumors, having smoking habit, Sjogren's syndrome, delayed hypersensitivity, Cardiac valvular diseases, subjects who have received anti-inflammatory drugs, antibiotics and neo vascularization inhibitors in the previous 6 months and Rheumatoid arthritis. Ethical clearance for the study was obtained from the ethical committee of the institution. The patients were explained regarding the study procedure and written informed consent was obtained from those who agreed to participate voluntarily in this study.

The subjects selected randomly were categorized into three groups. Group-1 consisted of 10 subjects with clinically healthy periodontium and with no evidence of disease. Group-II consisted of 15 CP subjects with the clinical signs of inflammation, probing pocket depth (PPD) ≥5 mm and clinical attachment loss (CAL) ≥2 mm, with radiographic evidence of bone loss. Group-III consisted of 15 subjects of Group-II treated by scaling and root planning (SRP) only.

Gingival index (GI) scores, PPD and CAL measurements were recorded for all patients. Test site for GCF sample collection was selected based on the highest scored sites in the oral cavity i.e., the site showing greatest amount of attachment loss (in CP cases) and the same test site for after treatment group was selected. GCF collection was done in the next appointment to avoid contamination of the sample.

GCF sampling and phase I therapy

After making the subjects sit comfortably in an upright position on the dental chair, the selected test site was air dried and isolated with cotton rolls. Without touching the marginal gingiva, supragingival plaque was removed to avoid contamination and blocking of the micro capillary pipette. GCF was collected by placing 1-3 μ1 calibrated volumetric micro capillary pipettes obtained from Sigma Aldrich Chemical Company, USA (Catalog No.p0549). By placing the tip of the pipette extracrevicularly (unstimulated) for 5-20 min, a standardized volume of 3 μl GCF was collected using the calibration on the micropipette from each test site. The test sites, which did not express standard volume (3 μ1) of GCF and micropipette contaminated with blood and saliva, were excluded. Samples of GCF were collected at the initial visit in Group II patients. Periodontal treatment (SRP) was performed for periodontitis patients at the same appointment after GCF collection. After 8 weeks, GCF was collected from the same site of these subjects were considered as Group III. For this 8 week period subjects were called at 1-week interval and plaque control measures were performed. The GCF collected was immediately transferred to aliquots and stored at −70°C until the time of the assay.

GCF enzyme linked immunosorbent assay analysis for TIMP-1

This assay employs the quantitative sandwich enzyme immunoassay technique with catalog number DTM100 from R and D systems. A polyclonal antibody specific for TIMP-1 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any TIMP-1 present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for TIMP-1 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the concentrations of total TIMP-1 (pro-and/or active) bound in the initial step. After the color development is stopped then the intensity of the color is measured.

Statistical analysis

All the data were analyzed using a software program SPSS 17.1, IBM, Chicago, IL. To test the hypothesis of equality of means among the three groups non-parametric Kruskal-Wallis test was carried on, Further multiple comparison using Mann-Whitney U-test was carried on to find out which pair or pairs differ significantly. Group III (after treatment) and Group II were compared using Wilcoxon signed Rank Test. Spearman's rank correlation test was carried out to observe for any correlation between the GCF MMP-3 concentration and clinical parameters.

RESULTS

The mean concentration of TIMP-1 concentration in Group I was 8.623 ng/ml with standard deviation (SD) 0.648, in Group II was 1.658 ng/ml with SD 0.592 and in Group III 6.492 ng/ml with SD 0.472. The mean TIMP-1 concentrations in GCF were observed to be highest in Group I and lowest in Group II. The mean TIMP-1 concentration in Group III fell between the highest and lowest values. The mean GI was significantly higher in Group II when compared with Group I and Group III, which was statistically significant. The mean PPD was significantly higher in Group II when compared with Group I and Group III, which was statistically significant. The mean CAL was significantly higher in Group II when compared with Group I and Group III which was statistically significant [Table 1].

Table 1

Clinical characteristics and TIMP-1 levels in Group I, II and III (mean±SD)

To test the hypothesis of equality of means among the three groups non parametric Kruskal-Wallis test was carried on, the results of which are tabulated in Table 2. The mean ranks obtained for Groups I-III are 35.60, 8.52 and 23.00 for GCF with P = 0.00. Therefore, the hypothesis of equality of means is rejected at 5% level of significance (P < 0.05), which indicates that the means differ significantly.

Table 2

Results of Kruskal-Wallis test comparing mean TIMP-1 concentration in GCF among groups

Further multiple comparisons using Mann-Whitney U-test were carried on to find out which pair or pairs differ significantly. When Groups I and II, II and III and I and III were compared, the differences were statistically significant with mean ranks between Group I and Group II i.e., 20.70 and 8.00, between Group II and Group III i.e., 8.00 and 23.50 and between Group I and Group III i.e., 20.50 and 8.00 with P = 0.000 as shown in Table 3.

Table 3

Mann-Whitney U-test for pair wise comparison of TIMP-1 concentrations

When Group III (after treatment) and Group II were compared using Wilcoxon signed Rank Test, the difference in the concentration of TIMP-1 was statistically significant (P < 0.05), indicating that, after SRP, TIMP-1 levels increased considerably from 1.65 ng/ml to 6.49 ng/ml as shown in Table 4.

Table 4

Results of Wilcoxon signed rank test to compare TIMP-1 concentration in GCF before and after treatment

Spearman's rank correlation test was done to observe for any correlation between the GCF TIMP-1 concentration and clinical parameters i.e., GI, plaque index (PI), PPD and CAL. The results of the test showed a negative correlation between levels of TIMP-1 in GCF and clinical parameters [Table 5].

Table 5

Results of Spearman correlation test between TIMP-1 and clinical parameters

DISCUSSION

Periodontitis is characterized by the loss of connective tissue attachment between the root and the supporting alveolar bone. As degradation of the periodontal connective tissues is a critical component of periodontitis, there appears to be therapeutic value in regulating the activity of enzymes involved in this pathogenic process. One group of enzymes thought to be important in this degradative process is the MMP family. MMPs are able to degrade most proteins of the ECM. MMPs are counteracted by TIMPs which inhibit MMP activity there by restricting ECM breakdown.[27] In the present study, un stimulated GCF collection was done using micro capillary pipettes and TIMP-1 concentrations were analyzed by ELISA. GCF was collected using micro capillary pipettes to avoid non-specific attachment of the analyte to filter paper fibers, which would have falsely reduced levels of detectable cytokines, leading to underestimation of the correlation between TIMP-1 levels and disease severity.[8]

In the present study the mean concentrations of TIMP-1 in GCF were found to be lower in healthy individuals compared with CP patients with P < 0.001, these results are in accordance with Biyikoğlu et al.[9] and Ingman et al.[10] who also demonstrated lower TIMP-1 levels in CP patients than in gingivitis patients. Hernαndez et al.[11] and Tüter et al.[67] in their longitudinal studies demonstrated a decreased tendency in TIMP-1 levels in active sites of progressive periodontitis patients. In the present study when Groups I and II, II and III and I and III were compared, the differences were statistically significant with mean ranks between Group I and Group II i.e., 20.7 and 8.00, between Group II and Group III i.e., 8.00 and 23.50 and between Group I and Group III i.e., 20.50 and 8.00 with P < 0.01. This clearly suggests that TIMP-1 concentrations in GCF decrease progressively from health to periodontitis.

In the present study, CP subjects were treated by non-surgical periodontal therapy (SRP) and strict oral hygiene measures were instituted. The mean TIMP-1 concentrations in GCF in CP group increased from 1.65 ng/ml to an after treatment levels of 6.49 ng/ml, which were statistically significant with P < 0.001. The results are in accordance with Pozo et al.,[12] in his study Pozo et al. observed significantly reduced values of TIMP-1 in periodontitis patients than in after treatment values. Contrary to our study, Marcaccini et al.[13] found no significant differences among TIMP-1 values between healthy, CP and after treatment groups.

Tüter et al.[67] evaluated the effect of SRP on GCF levels of TIMP-1 in a group of patients with advanced periodontal diseases. As per the results of Tüter et al.[7] the mean GCF TIMP-1 concentrations were found to be less in periodontitis group compared to after treatment group with P < 0.01.

The results in the present study showed a positive correlation between levels of GCF TIMP-1 concentrations and clinical parameters with ‘r’ value of 0.664 for GI, 0.540 for PI, 0.780 for PPD, 0.821 for CAL in Group II and 0.750 for GI, 0.734 for PI, 843 for PPD, 0.23 for CAL in Group III. These results are in accordance with Tüter et al.[7]

According to Tüter et al.[67] the negative correlations among all clinical parameters and TIMP-1 levels in GCF were significant with ‘r’ value of − 0.397 (P < 0.01) for GI, −0.401 (P < 0.01) for PI, −0.425 (P < 0.01) for PPD and − 0.485 (P < 0.001) for CAL.

Soell et al.[14] in their study have reported higher levels of TIMP-1 and TIMP-2 in the GCF of healthy versus periodontitis patients, as observed in the present study. They provided evidence that tissue destruction results from an imbalance of MMPs over tissue inhibitors.

In contrast to the present results, Alpagot et al.[15] conducted a study to determine the GCF levels of TIMP-1 with smoking and age as prognostic factors for the development of attachment loss in established periodontitis sites. They found increased levels of TIMP-1 in progressing periodontitis sites than in non-progressing periodontitis sites. Gulnur Emingil et al.[16] conducted a study to determine the levels of TIMP-1 in GCF samples of patients with generalized aggressive periodontitis, CP gingivitis and healthy subjects. They found increased TIMP-1 levels in GCF of generalized aggressive periodontitis compared to CP indicating that this molecule can participate in the regulation of progression of periodontal diseases. On the other hand, Maeso et al.[17] used ELISA technique and found increased TIMP-1 levels after SRP in periodontitis patients, suggesting a breakdown of the balance between the amount of MMPs and their inhibitor, which were consistent with our findings.

CONCLUSION

Within the limits of our study, it can be postulated that mean concentrations of TIMP-1 in GCF are lower in CP patients than in healthy and in after treatment individuals. In the present study, CP subjects treated by non-surgical periodontal therapy (SRP), showed increased mean GCF TIMP-1 concentrations. However, further longitudinal studies are needed to evaluate the concentrations of TIMP-1 in the periodontal disease tissues and GCF will be beneficial in clarifying the role in the pathogenesis of periodontitis and to validate TIMP-1 as a biomarker of periodontal disease progression.