Ikramuddin Aukhil,â Clay Walker,â¡ and Luciana Macchion Shaddoxâ . Background: Matrix metalloproteinases (MMPs) are a family of host-derived proteinases ...
J Periodontol • December 2013
Periodontal Treatment Reduces Matrix Metalloproteinase Levels in Localized Aggressive Periodontitis Patricia Furtado Gonc xalves,*† Hong Huang,† Suzanna McAninley,† Barnett Alfant,‡ Peter Harrison,† † Ikramuddin Aukhil, Clay Walker,‡ and Luciana Macchion Shaddox†
Background: Matrix metalloproteinases (MMPs) are a family of host-derived proteinases reported to mediate multiple functions associated with periodontal breakdown and inflammation. High MMP levels in African-American children with localized aggressive periodontitis (LAgP) have been reported previously by the present authors. However, little is known about MMP reductions in gingival crevicular fluid (GCF) after therapy. This study aims to evaluate MMP levels in the GCF after treatment of LAgP and to correlate these levels with clinical response. Methods: GCF samples were collected from 29 AfricanAmerican individuals diagnosed with LAgP. GCF was collected from one diseased site (probing depth [PD] >4 mm, bleeding on probing [BOP], and clinical attachment level ‡2 mm) and one healthy site (PD £3 mm, no BOP) from each individual at baseline and 3 and 6 months after periodontal treatment, which consisted of full-mouth scaling and root planing (SRP) and systemic antibiotics. The volume of GCF was controlled using a calibrated gingival fluid meter, and levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-12, and MMP-13 were assessed using fluorometric kits. Results: MMP-1, MMP-8, MMP-9, MMP-12, and MMP-13 levels were reduced significantly up to 6 months, comparable to healthy sites at the same point. Significant correlations were noted between MMP-2, MMP-3, MMP-8, MMP-9, MMP-12, and MMP-13 levels and percentage of sites with PD >4 mm. MMP-3, MMP-12, and MMP-13 levels also correlated with mean PD of affected sites. Conclusion: Treatment of LAgP with SRP and systemic antibiotics was effective in reducing local levels of specific MMPs in African-American individuals, which correlated positively with some clinical parameters. J Periodontol 2013;84:1801-1808. KEY WORDS Adolescent; aggressive periodontitis; anti-infective agents; child; metalloproteinases; root planing. * Department of Dentistry, Federal University of Jequitinhonha and Mucuri Valleys, Diamantina, Minas Gerais, Brazil. † Department of Periodontology, College of Dentistry, University of Florida, Gainesville, FL. ‡ Department of Oral Biology, College of Dentistry, University of Florida.
A
ggressive periodontitis (AgP) is a relatively rare inflammatory condition, mostly seen in young individuals, characterized by severe and rapid breakdown of the periodontium.1 It affects individuals with a non-contributory medical history and presents a familial aggregation pattern.2-4 When AgP is localized (LAgP), this disease affects first molars and incisors.2 As a result of the specific bacteria that are commonly found subgingivally in these individuals,5,6 a marked immune response is mounted by the host,7,8 resulting in periodontal breakdown. Connective tissue breakdown is essentially controlled by matrix metalloproteinases (MMPs), a family of zincand calcium-dependent proteolytic enzymes that are secreted by immune cells, including polymorphonuclear leukocytes and fibroblasts,9,10 in response to inflammatory stimuli.11,12 MMPs target extracellular matrix components, such as collagen, elastin, fibronectin, laminin, and entactin, among others.13 They are classified into 28 distinct but structurally related enzymes and are commonly divided into six groups: 1) collagenases (MMP-1, MMP-8, and MMP-13); 2) gelatinases (MMP-2 and MMP-9); 3) stromelysins (MMP-3); 4) matrilysins; 5) membrane-type MMPs; and 6) others, such as the macrophage metalloelastase (MMP-12). MMPs also doi: 10.1902/jop.2013.130002
1801
Matrix Metalloproteinase Levels in Aggressive Periodontitis
play an important role in physiologic events, such as morphogenesis, development, and tissue repair.14,15 Normally, MMP activity is tightly regulated to prevent unnecessary tissue breakdown.16 Several studies have shown that MMP levels are higher in patients with chronic periodontitis (CP) compared with healthy patients and patients with gingivitis,17-19 but little information is available about the levels of various MMPs associated with AgP. In a previous study,20 the present authors found that MMP levels were elevated in diseased sites relative to non-diseased sites in patients with LAgP, in siblings, and in unrelated controls. In addition, MMPs associated with the diseased sites in patients with LAgP were generally elevated compared with an adult cohort with CP.20 Considering that the control of LAgP and the maintenance of treatment outcomes represent a major challenge for clinicians, monitoring MMP levels after treatment may be an important noninvasive tool to guide clinicians toward early chairside and point-of-care treatment directions. Thus, the objective of this study is to evaluate MMP levels in the gingival crevicular fluid (GCF) after treatment of patients diagnosed with LAgP and to correlate these levels with their clinical response. MATERIALS AND METHODS Study Population Twenty-nine systemically healthy African-Americans, (11 males and 18 females, aged five to 21 years; mean age: 14.86 +/- 3.65 years) were recruited from August 2009 to August 2012 to participate in this study, which is part of a larger longitudinal clinical trial of LAgP in children (ClinicalTrials.gov Identifier NCT01330719). Only children and young individuals were included because LAgP is known to be an early-onset type of periodontal disease. These patients were recruited in Leon County Health Department, Tallahassee, Florida and in Duval County Health Department, Jacksonville, Florida. Patients or their legal authorized representatives were asked a series of questions to address health conditions and inclusion/exclusion criteria. A complete dental and medical history was taken for each individual. Inclusion criteria were as follows: African-American patients diagnosed with LAgP, defined by ‡2 teeth (incisor or first molar and no more than two other teeth other than first molars and incisors) presenting probing depth (PD) ‡5 mm with bleeding on probing (BOP), clinical attachment level (CAL) ‡2 mm, and radiographic bone loss.1,2 Exclusion factors were: 1) having any history of systemic disease that could interfere with the clinical characteristics, incidence, or pro1802
Volume 84 • Number 12
gression of periodontal disease; 2) having taken antibiotics within the previous 3 months; 3) allergy to penicillin; 4) use of medications that could influence the characteristics of periodontal disease; and 5) smokers or pregnant/lactating females. The study protocol was explained, and written informed consent was obtained from all individuals or their legal authorized representatives as required in the study protocol, approved by the University of Florida Institutional Review Board. Sample Collection GCF was collected from one diseased site (PD >4 mm, BOP, and CAL ‡2 mm) and one healthy site (PD £3 mm with no BOP) from each individual with LAgP at baseline and 3 and 6 months after treatment using a paper strip.§ The same sites were sampled at each time point. Each GCF collection site was isolated with cotton gauze and gently air dried, and supragingival plaque was removed. The paper strip was then inserted 1 to 2 mm into the sulcus for 10 seconds. The volume of fluid collected was measured using a calibrated gingival fluid meteri and placed in a microcentrifuge tube and kept on ice. Samples were stored at -80C until processed. Clinical Measurements and Treatment Protocol Periodontal clinical parameters were recorded at baseline and 3 and 6 months after treatment, including the following: 1) PD; 2) CAL; 3) BOP; and 4) visible plaque. CAL was calculated by PD + gingival margin position (GM). GM was assigned a negative value when located coronal to the cemento-enamel junction. All measurements were performed with the use of a periodontal probe¶ at six sites per tooth and were recorded using computer software.# After completion of baseline measurements, individuals received full-mouth debridement with an ultrasonic device,** followed by scaling and root planing (SRP) and oral hygiene instructions, along with oral care products.†† Local anesthesia was provided as required. Immediately after treatment, all individuals were prescribed 500 mg amoxicillin and 250 mg metronidazole three times daily for 7 days. The antibiotics dosage was adjusted for children weighing 4 mm
%BOP
%Plaque
Mean PD Sites (mm)
Mean PD All Sites (mm)
Mean CAL (mm)
PD Site (mm)
14.86 – 3.65
11 males, 18 females
17.81 – 15.19
17.67 – 13.11
38.04 – 21.79
5.22 – 0.75
2.25 – 0.82
2.92 – 1.30
6.00 – 1.69
Values are given as mean – SD. %PD >4 mm = mean percentage of sites with PD >4 mm; Mean PD Sites = mean PD of sites with PD >4 mm; Mean PD All Sites = mean PD of all sites; Mean CAL = mean CAL of affected sites; PD Site = mean PD from diseased sites sampled for GCF.
ultrasonic device and additional therapy (SRP) for residual pockets >4 mm, as required. Antibiotics were only prescribed once at initial therapy. Professional plaque removal and reinforcement of home-care procedures were also performed. GCF Processing and MMP Enzymatic Assay The GCF absorbed to the strip was eluted with 50 µL MMP buffer (50 mM Tris-HCL, 0.2 M NaCL, and 5 mM CaCl2 [pH 7.5]), followed by centrifugation to minimize retention of proteins on the filter paper. The activities of MMP-1, MMP-2, MMP3, MMP-8, MMP-9, MMP-12, and MMP-13 were assessed using commercially available fluorometric MMP kits specific for each MMP,‡‡ in accordance with the instructions of the manufacturer. The detection limits of these kits are given by the manufacturer as 0.1 to 1.0 ng depending on the particular assay kit. Briefly, each specific MMP substrate (50 µL) was diluted in 5 mL assay buffer (provided with the kit), and 50 µL diluted substrate was added to the desired well of a black fluorometric 96-well microtiter plate.§§ Two microliters of the sample were added to each well and mixed by gently shaking for 15 minutes in the dark and then incubated for an additional 45 minutes in the dark. Each of the seven MMP substrates was arranged on each plate in rows, and the samples were applied in columns, with the first two wells of each row serving as controls for background fluorescence. Fluorescence intensity was measured at 490 nm excitation and 520 nm emission using a fluorometric reader and associated software.ii The data, initially expressed as relative fluorescence units, were converted to nanograms based on standard curves (r2 >0.99) generated with the purified recombinant MMP¶¶ and its specific substrate. This value was then adjusted to concentration (nanograms per microliters) based on the volume of the GCF collected, according to a calibration curve. MMPs were assayed without activating the proenzyme MMP form, and the active forms of seven MMPs (MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-12, and MMP-13), which are more
closely related to progressive periodontal disease, were chosen.11 Statistical Analyses Statistical analysis was performed using statistics software.## Analysis of variance (ANOVA) on ranks (Kruskal-Wallis test) was applied among all time points using concentration (nanograms per microliters) of each MMP and clinical parameters. Dunn multiple comparisons test was used to determine differences among time points. Wilcoxon matched-pairs signed-rank test was used for comparisons between baseline diseased and healthy sites within LAgP patients. In addition, Spearman correlations were run between MMP levels and clinical parameters. Statistical significance was considered to be P £0.05. RESULTS A summary of the demographics and clinical aspects of the individuals included in the study is presented in Table 1. Four participants had mixed dentition, and disease was present on primary teeth. Participants reported full compliance with the antibiotic regimen, and no side effects were reported. Missed time points are indicated in Figure 1. Periodontal treatment resulted in improvement of clinical parameters (Fig. 1), specifically in the percentage of sites with PD >4 mm, mean PD of affected sites (PD >4 mm with attachment loss and radiographic bone loss), mean PD from specific sampled sites, BOP, and mean CAL (one-way ANOVA, P 4 mm correlated significantly with MMP-2, MMP-3, MMP8, MMP-9, MMP-12, and MMP-13. Significant correlations were also noted between MMP-3, MMP-12, and MMP-13 with mean PD of affected sites. Considering correlations among different MMPs, it was noted that MMPs strongly and positively correlated with each other (P 0.80) with each of the two gelatinases. Figure 1.
A through G) Clinical parameters at baseline (BL) and after treatment. PD >4 mm = mean percentage of sites with PD >4 mm; Mean PD sites = mean PD of sites with PD >4 mm; Mean PD all sites = mean PD of all sites; Mean CAL = mean CAL of affected sites; PD site = mean PD from diseased sites sampled for GCF. Values given at baseline (n = 29) and at 3 (n = 16) and 6 months (n = 22) after treatment. Values in boxes are represented as mean (horizontal bars) – SD (whiskers). *Statistically significant differences (one-way ANOVA by Dunn multiple comparisons, P
