J Clin Periodontol 2014; 41: 875–882 doi: 10.1111/jcpe.12290
The effect of periodontal therapy on cardiovascular risk markers: a 6-month randomized clinical trial
Luis Cau la, Ronaldo Andre Lira-Junior, Eduardo Muniz Barretto ~es Tinoco and Ricardo Guimara Fischer Department of Periodontology, School of Dentistry, Rio de Janeiro State University, Rio de Janeiro, Brazil
Ca ula AL, Lira-Junior R, Tinoco EMB, Fischer RG. The effect of periodontal therapy on cardiovascular risk markers: a 6-month randomized clinical trial. J Clin Periodontol 2014; 41: 875–882. doi: 10.1111/jcpe.12290.
Abstract Aim: To determine the influence of non-surgical mechanical periodontal treatment on inflammatory markers related to risk for cardiovascular disease. Material and Methods: A total of 64 patients with severe chronic periodontitis was randomly subjected to immediately periodontal treatment (test group, n = 32) or delayed periodontal treatment, without treatment during the study period (control group, n = 32). Clinical periodontal and laboratory examinations were performed at baseline (T0), 2 months (T2), and 6 months (T6) after the initial examinations (Control group) or completion of periodontal treatment (Test group). Results: After 2 months of periodontal treatment there was a significant reduction of erythrocyte sedimentation rate (ESR) and triglycerides (p = 0.002, p = 0.004, respectively) in the test group. Median values of C-reactive protein, ESR, total cholesterol, and triglycerides were reduced after 6 month of periodontal treatment in the test group (p < 0.001, p < 0.001, p < 0.001, and p = 0.015, respectively). Conclusions: The non-surgical periodontal treatment was effective in reducing the levels of systemic inflammation markers and improved the lipid profile in subjects with severe chronic periodontitis.
Elevated inflammatory activity was identified as a major responsible for the atherogenic process and endothelial dysfunction that may lead to cardiovascular disease (Ross 1999, Libby 2002, 2006). Periodontitis is a chronic Conflict of interest and source of funding statement The authors declare that there are no conflicts of interest in this study. No external funding, apart from the support of the authors’ institution, was available for this study.
inflammatory disease characterized by bacterial infection and teeth supporting tissues destruction, able to initiate and maintain high systemic levels of various cytokines related to inflammatory response (Page & Schroeder 1976, Tonetti & Van Dyke 2013). These cytokines, particularly tumour necrosis factor-a (TNF-a) and interleukin-6 (IL-6), trigger an increase in hepatic synthesis and rapid secretion of intravascular plasma proteins, including C-reactive protein (CRP) and fibrinogen (Ebersole & Cappelli 2000).
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Key words: cardiovascular disease; C-reactive protein; erythrocyte sedimentation rate; periodontitis; triglycerides Accepted for publication 7 July 2014
Several studies showed that periodontitis was linked with several of these markers of inflammation (TNF-a, IL-6, CRP, and fibrinogen) and many systemic changes, including dyslipidemias, which are known risk factors for cardiovascular disease (CVD) (Cutler et al. 1999, Loos et al. 2000, Noack et al. 2001, Buhlin et al. 2003, Craig et al. 2003, D’Aiuto et al. 2004, Joshipura et al. 2004, Vidal et al. 2009). Two metaanalysis indicated evidences that CRP and low-density lipoprotein cholesterol are associated with CVD
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events (Buckley et al. 2009, Ip et al. 2009). The effect of periodontal treatment on systemic markers of inflammation is still conflicting. Some studies showed that non-surgical periodontal treatment of chronic periodontitis did not influence serum levels of inflammatory markers (Ide et al. 2003, Yamazaki et al. 2005). In contrast, results of others studies described significant reductions in systemic markers of inflammation (Mattila et al. 2002, D’Aiuto et al. 2005, Vidal et al. 2009, 2013, Bokhari et al. 2012). Behle et al. (2009) observed that periodontal therapy resulted in an overall reduction of systemic inflammation, but there was considerable inter-patient variability in concentrations for most of the markers evaluated. The authors stated that the failure to identify significant differences in the levels of inflammatory markers after therapy may be partly attributed to a high biological heterogeneity. Recently, D’Aiuto et al. (2013) performed a systematic review about the effect of periodontal treatment on CVD biomarkers and outcomes. The authors concluded there is moderate evidence that does not support a positive effect of periodontal treatment on lipid profile and there is a moderate positive effect in reducing serum CRP levels after periodontal therapy. Considering the possible association between periodontitis and CVD, potential benefits of periodontal treatment to reduce cardiovascular risk and inconsistency about these beneficial influences, studies on the effect of periodontal therapy are still necessary. The aim of this research was to evaluate the influence of nonsurgical mechanical periodontal treatment on CRP serum level, erythrocyte sedimentation rate (ESR), and lipid profile in patients with severe chronic periodontitis. Material and Methods Study population
The study design was a randomized parallel clinical trial. The study population consisted of 66 patients with severe non-treated chronic periodontitis, referred to the clinic of periodontology from the Military Dental
Clinic, Rio de Janeiro State Fire Department (CBMERJ). All patients gave written informed consent. The study had been reviewed and approved by the Ethics Committee in Research of Pedro Ernesto University Hospital (HUPE) of the Rio de Janeiro State University (UERJ). Each subject had at least 15 remaining teeth without untreated periapical lesions, and with at least two or more inter-proximal sites with clinical attachment loss (CAL) ≥ 6 mm, not in the same tooth, and the presence of probing pocket depth (PPD) ≥ 5 mm in one or more interproximal sites, excluding third molars (Page & Eke 2007). Exclusion criteria were: (1) patients who require antibiotic prophylaxis, (2) patients with immune or inflammatory conditions (arthritis, gastrointestinal disorder, bronchitis), (3) patients who were pregnant or breastfeeding, (4) patients received periodontal treatment prior to 6 months before the start of the study, and (5) patients using, or who received prior treatment (6 months) with medicines such as antibiotics, anti-inflammatory drugs, steroids, immunosuppressants, anticoagulants, and regulators of cholesterol. Sample size
Changes in CRP levels after 6 month of periodontal treatment were established as primary outcome. Based on a = 0.05, mean difference of 0.5 mg/l (Paraskevas et al. 2008) and standard deviation of 0.9, we should enrol 28 to achieve 80% of power. Assuming 20% of drop-out, 33 patients per group were enrolled. Medical history and physical exams
The medical history of each patient was obtained through interviews and all participants underwent physical examinations. The information collected included: (1) sex, (2) age, (3) ethnicity, (4) educational level, (5) presence of systemic diseases, (6) habits of smoking, and (7) body mass index (BMI). Periodontal examination
Patients underwent a comprehensive periodontal examination including
PPD, CAL, bleeding on probing (BOP), and plaque index. The measurements were determinate at six sites per tooth (mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual), excluding third molars. The clinical examination was performed by a single calibrated examiner using a manual probe (UNC-15, Hu-Friedy Manufacturing Company, Inc., Chicago, IL, USA). To measure the degree of intra-examiner reproducibility, a duplicate examination of periodontal data was done, for which five individuals not participating in the study were examined according to the methodology used in the study and were re-examined within 2 h after the first evaluation. The kappa coefficient was 0.78. The periodontal PPD and CAL were stratified into three groups each: (a) % of sites with PPD ≤ 3 mm, (b) % of sites with PPD from 4 to 5 mm, (c) % of sites with PPD ≥ 6 mm, (d) % of sites with CAL ≤ 3 mm, (e) % of sites with CAL from 4 to 5 mm, (f) % of sites with CAL ≥ 6 mm. BOP was registered as the number of bleeding sites times 100 divided by the number of examined sites. Plaque index was stratified into two groups each: (a) no visible plaque, (b) presence of visible plaque. Laboratorial exams
Venous blood samples (10 ml) were collected in the morning of the periodontal examination, after a 12-h fasting period, in the laboratory of the CBMERJ Hospital. The levels of CRP were determined in serum on an IMMULITEâ Automated Analyzer using the commercially available high sensitivity immulite chemiluminescent enzyme immunometric assay (Immuliteâ, Diagnostic Products Corp., Los Angeles, CA, USA). The ESR was determined by Westergren method, manual system with the reading of the 1st. hour. Lipid profile was determined by an automated method, using the clinical analyzer apparatus Wiener lab. Metrolab 2300 Plus Random Access (UV-Vis Metrolab SA, Bernal, Buenos Aires, Argentina). The laboratory staff was blinded to the allocation group.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Periodontal therapy reduces CVD risk markers Experimental protocol
Statistical analysis
Patients were randomly allocated to one of two groups: Test Group (received periodontal treatment immediately, n = 32) or control group (delayed periodontal treatment, without treatment during the study period, n = 32). The control group received periodontal treatment similar to the Test Group, but started after a period of 6 months. The mean (SD) number of days from baseline to 2 months follow-up was 60.2 (11), range between 57–67 days, while the corresponding values from baseline to 6 months were 183 (2.9), range 177–190 days. To avoid the chance of having more patients in one group than another, a randomization in blocks of four was performed using opaque envelopes placed in the IDs of the treatment group (Test group and Control group). The envelopes were sealed, shuffled, and then numbered in sequential order from 1 to 4. Each new patient entering the study an envelope was opened. This procedure was repeated for each group of four patients. Thus, the numbers in the two groups would never differ by more than half the length of the block. Periodontal treatment consisted of four visits, scheduled weekly, with no restriction on duration. All treatment was performed by a single operator. Periodontal treatment consisted of instruction and monitoring of oral hygiene, supragingival and subgingival scaling, and root planning. It was performed manual and ultrasonic instruments under local anaesthesia. For manual instrumentation, Gracey curettes (Hu-Friedy Manufacturing Company, Inc., Chicago, IL, USA) and ultrasonic instrumentation (Sonic Jet, Dental Medical Equipment Gnatus Ltda. Ribeirao Preto, Brazil) were used under local anaesthesia. Extraction of hopeless teeth and remaining roots was performed in the same clinic session. Clinical periodontal and laboratory examinations were performed pre-treatment (T0) and at 2 and 6 months (T2 and T6, respectively) after initial examination (control group) or completion of periodontal treatment (Test Group). The flow chart of the study is presented in Fig. 1.
The data were processed and analysed using Statistical Package for the Social Sciences (IBM SPSS 19.0, SPSS Inc., Chicago, IL, USA) version 19. Descriptive statistical analysis included medians and first and third quartiles of quantitative variables and frequency and percentage for qualitative variables. The significance of differences of each parameter between the test and control groups, and among T0, T2, and T6 periods was analysed by the nonparametric Wilcoxon test (Wilcoxon signed rank test with continuity correction). For categorical variables, we used chi-square test (Pearson’s Chi-squared test with Yates’ continuity correction). Spearman’s rank correlation was calculated for differences in clinical and haematological variables. Significance level was determined at p < 0.05. Results
From a total of 66 participants invited to participate in the study, two withdrew before starting the treatment phase. Thus, 64 participants completed the study and were included in the analysis. There were no significant differences in participant characteristics between groups at baseline (T0). The sample
characterization, which presents the frequencies, percentages, averages, and standard deviations of the demographic and medical characteristics, is presented in Table 1. The patients did not change their medications or their smoking habit during the study. Periodontal conditions were similar on baseline in the test and control groups (Table 2). However, there was a significant difference in all periodontal parameters between the test and control groups at 2 and 6 months after treatment (T2 and T6). When comparing the median values of periodontal parameters between T0/T2 and T0/T6 in the test group, we observed statistically significant improvement in almost all values, except the percentage of sites with CAL 4–5 mm, which showed no significant difference (p = 0.342 and p = 0.466 between T0/T2 and T0/T6). However, when comparing median values of periodontal parameters in the control group, we observed statistically significant worsening of almost all values, except the percentage of sites with CAL ≥ 6 mm (p = 0.776) between T0/T2, and the percentage of sites with CAL ≥ 6 mm (p = 0.47) and percentage of sites with PPD ≥ 6 mm (p = 0.49) between T0/ T6 (Table 2). In the period between
Assessment for eligibility (n = 66) Enrollment •
Exclusion (n =2) Withdrew (n = 2)
Randomization (n = 64)
Allocation Test group (n = 32)
Control group (n = 32) Follow-up
Lost to follow-up (n = 0)
Lost to follow-up (n = 0) Analysis
Analyzed (n = 32)
Fig. 1. Flow chart of the study.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
877
Analyzed (n = 32)
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Table 1. Medical and demographic characteristics of the test and control groups
Discussion
Variable
Test (n = 32)
Control (n = 32)
p
Age (years) BMI (kg/m2) Ethnicity [n (%)] White Pardo (brown-skinned, mixed-race) Black Gender [n (%)] Men Women Education Level [n (%)] Fundamental Medium Superior Smoking [n (%)] Smokers Ex-smokers Non-smokers Diabetes [n (%)] Arterial hypertension [n (%)]
44.4 (38; 50) 26.5 (25; 28.2)
44.0 (38.5; 51.5) 26.4 (24.1; 29.2)
0.89 0.75
13 (40.6%) 12 (37.5%) 7 (21.9%)
15 (46.9%) 10 (31.2%) 7 (21.9%)
0.85
20 (62.5%) 12 (37.5%)
19 (59.4%) 13 (40.6%)
1.00
15 (46.9%) 16 (50.0%) 1 (3.1%)
9 (28.1%) 21 (65.6%) 2 (6.2%)
0.20
4 13 15 5 7
6 6 20 7 3
0.16
(12.5%) (40.6%) (46.9%) (15.6%) (21.9%)
(18.8%) (18.8%) (62.5%) (21.9%) (9.4%)
0.37 0.15
Results are expressed as median (first quartile; third quartile) and frequency (percentage).
T0 and T2, five control patients had discomfort and pain symptoms and were undergoing dental extraction (lost a total of six teeth with mobility grade 3). There was no tooth loss between T2 and T6 in two groups. Haematological data are presented in Table 3. At the pre-treatment (T0), there was no significant difference between groups for CRP level, ESR, and lipid profile. However, there was a significant difference after treatment between the test and control groups for the median values of CRP and ESR at T2 [0.19 (CI 95% 0.1–0.32) difference, p = 0.001 and 3.5 (1.1–6.2) difference, p = 0.003, respectively] and for median values of CRP, ESR, and total cholesterol, in T6 [0.7 (0.51–0.83) difference, p < 0.001; 7.0 (5.8–10.4) difference, p < 0.001; and 10.0 (4.7–38.6) difference, p = 0.021, respectively]. When comparing median values of haematological data in the test group, we observed statistically significant improvement between T0/T2, of the values of ESR and triglycerides [2.5 (1.2–5.0) difference, p = 0.002 and 23.0 (2.0–75.8) difference, p = 0.004, respectively]. Reduction in the median levels of CRP, ESR, total cholesterol, and triglycerides, between T0/ T6 was observed in the test group [0.46 (0.21–0.73) difference, p < 0.001; 5.0 (4.0–7.9) difference, p < 0.001; 20.5 (10.9–30.4) difference, p < 0.001; and 23.5 (3.1–72.7) difference, p = 0.015, respectively]. The
LDL cholesterol showed a trend towards reduction [3.5 ( 0.2–12.8) difference, p = 0.06]. When comparing median values of haematological data in the control group between T0/T2 and T0/T6, we observed a statistically significant increase in the values of CRP [0.14 (0.06–0.2) difference, p = 0.003 and 0.24 (0.1–0.5) difference, p < 0.001, respectively] and ESR [1.0 (0.2–4.1) difference, p = 0.007 and 2.0 (1.4–6.1) difference, p = 0.001, respectively]. There was no significant correlation between PPD and BOP reductions and haematological changes at T2 in the test group. However, there was a significant correlation between CAL decrease and a decline in triglycerides at T2 in the test group (q = 0.395, p < 0.05). At T6, there were significant correlations between PPD reduction and a decrease in ESR (q = 0.502, p = 0.003) and between CAL improvement and CRP decrease in the test group (q = 0.371, p < 0.05). No significant correlation was observed between PPD, CAL, and BOP alterations and haematological changes at T2 and T6 in the control group. Correlations among CRP, TC, HDL, and LDL at baseline (T0), 2 (T2) and 6 months (T6) after periodontal treatment are described in Table 4. There was a statistically significant correlation between TC and LDL at T0, T2, and T6 and between TC and triglycerides at T0 and T2.
The aim of this study was to evaluate the effect of non-surgical periodontal treatment on markers of cardiovascular disease (CRP, ESR, and lipid profile). The results of non-surgical treatment are in accordance with the classical studies (Badersten et al. 1981, 1984, 1987). We found that periodontal treatment was effective in improving the periodontal parameters and reducing CRP level, ESR, total cholesterol, and triglycerides after 6 months. Inflammation is important in all phases of CVD, including initiation of atherogenic process, as well as the acute rupture of atherosclerotic plaque (Ridker 2003). Low-grade inflammation is a marker for subsequent CVD (Ridker et al. 1998) and C-reactive protein is an acute-phase protein that has been shown to be a simple, reliable, and reproducible marker of underlying systemic inflammation and a strong predictor of myocardial infarction and stroke (Willerson & Ridker 2004). ESR is an indicator of red cell aggregation and thus of blood viscosity, and it is a simple and inexpensive laboratory test. ESR is an independent, longterm predictor of coronary heart disease (Andresdottir et al. 2003). Lipid profile is also associated with coronary heart disease (Sarwar et al. 2007). Elevated levels of CRP have been observed in periodontal patients (Paraskevas et al. 2008). The elevation of CRP level is considered a risk factor for atherosclerosis and cardiovascular disease (Buckley et al. 2009). In the present study, no significant changes in CRP levels were observed within 2 months, however, a significant reduction was observed in CRP after 6 months of treatment (p < 0.0001). These results are similar to those found by D’Aiuto et al. (2004). Mattila et al. (2002) have also found that treating periodontitis decreases CRP level. The authors concluded that it possibly reduces CVD risk. Ide et al. (2003) and Yamazaki et al. (2005) did not find significant effect of periodontal therapy on CRP level. They evaluated CRP levels, respectively, after 6 weeks and 3 months following completion of treatment which
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
32.5 (24; 42)
17 (7; 31)
62 (50; 76.5)
29 (18; 39.5) 7 (4; 15.5)
42.5 (26.5; 62) 41.5 (24.5; 55.5)
16 (7.5; 25.5)
64 (49.5; 78)
24 (17; 36.5)
7 (4; 15.5)
55 (38; 68) 45 (31; 62.5)
24 (20.5; 26.5) 50 (25.5; 61.5)
Control
29.5 (21; 36.5)
23.5 (20.5; 26) 54.0 (34.5; 67.5)
Test
T0
0.06 0.15
0.60
0.30
0.73
0.94
0.25
0.53 0.35
pa
9 (0; 20) 5 (0; 23)
0 (0; 1.5)
11.5 (6.5; 18.5)
86.5 (78.5; 93.5)
4.5 (1; 11)
30 (11.5; 41)
22.5 (18.5; 26) 65.5 (46.5; 85.5)
Test
41.5 (29.5; 68.5) 40.5 (28.5; 55)
7 (4; 15)
31.5 (18; 42.5)
60 (40.5; 74)
17 (7; 33.5)
35 (25.5; 42.5)
24 (20.5; 26.5) 48 (25; 58.5)
Control
T2
The effect of periodontal therapy on cardiovascular risk markers: a 6-month randomized clinical trial
Luis Cau la, Ronaldo Andre Lira-Junior, Eduardo Muniz Barretto ~es Tinoco and Ricardo Guimara Fischer Department of Periodontology, School of Dentistry, Rio de Janeiro State University, Rio de Janeiro, Brazil
Ca ula AL, Lira-Junior R, Tinoco EMB, Fischer RG. The effect of periodontal therapy on cardiovascular risk markers: a 6-month randomized clinical trial. J Clin Periodontol 2014; 41: 875–882. doi: 10.1111/jcpe.12290.
Abstract Aim: To determine the influence of non-surgical mechanical periodontal treatment on inflammatory markers related to risk for cardiovascular disease. Material and Methods: A total of 64 patients with severe chronic periodontitis was randomly subjected to immediately periodontal treatment (test group, n = 32) or delayed periodontal treatment, without treatment during the study period (control group, n = 32). Clinical periodontal and laboratory examinations were performed at baseline (T0), 2 months (T2), and 6 months (T6) after the initial examinations (Control group) or completion of periodontal treatment (Test group). Results: After 2 months of periodontal treatment there was a significant reduction of erythrocyte sedimentation rate (ESR) and triglycerides (p = 0.002, p = 0.004, respectively) in the test group. Median values of C-reactive protein, ESR, total cholesterol, and triglycerides were reduced after 6 month of periodontal treatment in the test group (p < 0.001, p < 0.001, p < 0.001, and p = 0.015, respectively). Conclusions: The non-surgical periodontal treatment was effective in reducing the levels of systemic inflammation markers and improved the lipid profile in subjects with severe chronic periodontitis.
Elevated inflammatory activity was identified as a major responsible for the atherogenic process and endothelial dysfunction that may lead to cardiovascular disease (Ross 1999, Libby 2002, 2006). Periodontitis is a chronic Conflict of interest and source of funding statement The authors declare that there are no conflicts of interest in this study. No external funding, apart from the support of the authors’ institution, was available for this study.
inflammatory disease characterized by bacterial infection and teeth supporting tissues destruction, able to initiate and maintain high systemic levels of various cytokines related to inflammatory response (Page & Schroeder 1976, Tonetti & Van Dyke 2013). These cytokines, particularly tumour necrosis factor-a (TNF-a) and interleukin-6 (IL-6), trigger an increase in hepatic synthesis and rapid secretion of intravascular plasma proteins, including C-reactive protein (CRP) and fibrinogen (Ebersole & Cappelli 2000).
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Key words: cardiovascular disease; C-reactive protein; erythrocyte sedimentation rate; periodontitis; triglycerides Accepted for publication 7 July 2014
Several studies showed that periodontitis was linked with several of these markers of inflammation (TNF-a, IL-6, CRP, and fibrinogen) and many systemic changes, including dyslipidemias, which are known risk factors for cardiovascular disease (CVD) (Cutler et al. 1999, Loos et al. 2000, Noack et al. 2001, Buhlin et al. 2003, Craig et al. 2003, D’Aiuto et al. 2004, Joshipura et al. 2004, Vidal et al. 2009). Two metaanalysis indicated evidences that CRP and low-density lipoprotein cholesterol are associated with CVD
875
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Ca ula et al.
events (Buckley et al. 2009, Ip et al. 2009). The effect of periodontal treatment on systemic markers of inflammation is still conflicting. Some studies showed that non-surgical periodontal treatment of chronic periodontitis did not influence serum levels of inflammatory markers (Ide et al. 2003, Yamazaki et al. 2005). In contrast, results of others studies described significant reductions in systemic markers of inflammation (Mattila et al. 2002, D’Aiuto et al. 2005, Vidal et al. 2009, 2013, Bokhari et al. 2012). Behle et al. (2009) observed that periodontal therapy resulted in an overall reduction of systemic inflammation, but there was considerable inter-patient variability in concentrations for most of the markers evaluated. The authors stated that the failure to identify significant differences in the levels of inflammatory markers after therapy may be partly attributed to a high biological heterogeneity. Recently, D’Aiuto et al. (2013) performed a systematic review about the effect of periodontal treatment on CVD biomarkers and outcomes. The authors concluded there is moderate evidence that does not support a positive effect of periodontal treatment on lipid profile and there is a moderate positive effect in reducing serum CRP levels after periodontal therapy. Considering the possible association between periodontitis and CVD, potential benefits of periodontal treatment to reduce cardiovascular risk and inconsistency about these beneficial influences, studies on the effect of periodontal therapy are still necessary. The aim of this research was to evaluate the influence of nonsurgical mechanical periodontal treatment on CRP serum level, erythrocyte sedimentation rate (ESR), and lipid profile in patients with severe chronic periodontitis. Material and Methods Study population
The study design was a randomized parallel clinical trial. The study population consisted of 66 patients with severe non-treated chronic periodontitis, referred to the clinic of periodontology from the Military Dental
Clinic, Rio de Janeiro State Fire Department (CBMERJ). All patients gave written informed consent. The study had been reviewed and approved by the Ethics Committee in Research of Pedro Ernesto University Hospital (HUPE) of the Rio de Janeiro State University (UERJ). Each subject had at least 15 remaining teeth without untreated periapical lesions, and with at least two or more inter-proximal sites with clinical attachment loss (CAL) ≥ 6 mm, not in the same tooth, and the presence of probing pocket depth (PPD) ≥ 5 mm in one or more interproximal sites, excluding third molars (Page & Eke 2007). Exclusion criteria were: (1) patients who require antibiotic prophylaxis, (2) patients with immune or inflammatory conditions (arthritis, gastrointestinal disorder, bronchitis), (3) patients who were pregnant or breastfeeding, (4) patients received periodontal treatment prior to 6 months before the start of the study, and (5) patients using, or who received prior treatment (6 months) with medicines such as antibiotics, anti-inflammatory drugs, steroids, immunosuppressants, anticoagulants, and regulators of cholesterol. Sample size
Changes in CRP levels after 6 month of periodontal treatment were established as primary outcome. Based on a = 0.05, mean difference of 0.5 mg/l (Paraskevas et al. 2008) and standard deviation of 0.9, we should enrol 28 to achieve 80% of power. Assuming 20% of drop-out, 33 patients per group were enrolled. Medical history and physical exams
The medical history of each patient was obtained through interviews and all participants underwent physical examinations. The information collected included: (1) sex, (2) age, (3) ethnicity, (4) educational level, (5) presence of systemic diseases, (6) habits of smoking, and (7) body mass index (BMI). Periodontal examination
Patients underwent a comprehensive periodontal examination including
PPD, CAL, bleeding on probing (BOP), and plaque index. The measurements were determinate at six sites per tooth (mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual), excluding third molars. The clinical examination was performed by a single calibrated examiner using a manual probe (UNC-15, Hu-Friedy Manufacturing Company, Inc., Chicago, IL, USA). To measure the degree of intra-examiner reproducibility, a duplicate examination of periodontal data was done, for which five individuals not participating in the study were examined according to the methodology used in the study and were re-examined within 2 h after the first evaluation. The kappa coefficient was 0.78. The periodontal PPD and CAL were stratified into three groups each: (a) % of sites with PPD ≤ 3 mm, (b) % of sites with PPD from 4 to 5 mm, (c) % of sites with PPD ≥ 6 mm, (d) % of sites with CAL ≤ 3 mm, (e) % of sites with CAL from 4 to 5 mm, (f) % of sites with CAL ≥ 6 mm. BOP was registered as the number of bleeding sites times 100 divided by the number of examined sites. Plaque index was stratified into two groups each: (a) no visible plaque, (b) presence of visible plaque. Laboratorial exams
Venous blood samples (10 ml) were collected in the morning of the periodontal examination, after a 12-h fasting period, in the laboratory of the CBMERJ Hospital. The levels of CRP were determined in serum on an IMMULITEâ Automated Analyzer using the commercially available high sensitivity immulite chemiluminescent enzyme immunometric assay (Immuliteâ, Diagnostic Products Corp., Los Angeles, CA, USA). The ESR was determined by Westergren method, manual system with the reading of the 1st. hour. Lipid profile was determined by an automated method, using the clinical analyzer apparatus Wiener lab. Metrolab 2300 Plus Random Access (UV-Vis Metrolab SA, Bernal, Buenos Aires, Argentina). The laboratory staff was blinded to the allocation group.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Periodontal therapy reduces CVD risk markers Experimental protocol
Statistical analysis
Patients were randomly allocated to one of two groups: Test Group (received periodontal treatment immediately, n = 32) or control group (delayed periodontal treatment, without treatment during the study period, n = 32). The control group received periodontal treatment similar to the Test Group, but started after a period of 6 months. The mean (SD) number of days from baseline to 2 months follow-up was 60.2 (11), range between 57–67 days, while the corresponding values from baseline to 6 months were 183 (2.9), range 177–190 days. To avoid the chance of having more patients in one group than another, a randomization in blocks of four was performed using opaque envelopes placed in the IDs of the treatment group (Test group and Control group). The envelopes were sealed, shuffled, and then numbered in sequential order from 1 to 4. Each new patient entering the study an envelope was opened. This procedure was repeated for each group of four patients. Thus, the numbers in the two groups would never differ by more than half the length of the block. Periodontal treatment consisted of four visits, scheduled weekly, with no restriction on duration. All treatment was performed by a single operator. Periodontal treatment consisted of instruction and monitoring of oral hygiene, supragingival and subgingival scaling, and root planning. It was performed manual and ultrasonic instruments under local anaesthesia. For manual instrumentation, Gracey curettes (Hu-Friedy Manufacturing Company, Inc., Chicago, IL, USA) and ultrasonic instrumentation (Sonic Jet, Dental Medical Equipment Gnatus Ltda. Ribeirao Preto, Brazil) were used under local anaesthesia. Extraction of hopeless teeth and remaining roots was performed in the same clinic session. Clinical periodontal and laboratory examinations were performed pre-treatment (T0) and at 2 and 6 months (T2 and T6, respectively) after initial examination (control group) or completion of periodontal treatment (Test Group). The flow chart of the study is presented in Fig. 1.
The data were processed and analysed using Statistical Package for the Social Sciences (IBM SPSS 19.0, SPSS Inc., Chicago, IL, USA) version 19. Descriptive statistical analysis included medians and first and third quartiles of quantitative variables and frequency and percentage for qualitative variables. The significance of differences of each parameter between the test and control groups, and among T0, T2, and T6 periods was analysed by the nonparametric Wilcoxon test (Wilcoxon signed rank test with continuity correction). For categorical variables, we used chi-square test (Pearson’s Chi-squared test with Yates’ continuity correction). Spearman’s rank correlation was calculated for differences in clinical and haematological variables. Significance level was determined at p < 0.05. Results
From a total of 66 participants invited to participate in the study, two withdrew before starting the treatment phase. Thus, 64 participants completed the study and were included in the analysis. There were no significant differences in participant characteristics between groups at baseline (T0). The sample
characterization, which presents the frequencies, percentages, averages, and standard deviations of the demographic and medical characteristics, is presented in Table 1. The patients did not change their medications or their smoking habit during the study. Periodontal conditions were similar on baseline in the test and control groups (Table 2). However, there was a significant difference in all periodontal parameters between the test and control groups at 2 and 6 months after treatment (T2 and T6). When comparing the median values of periodontal parameters between T0/T2 and T0/T6 in the test group, we observed statistically significant improvement in almost all values, except the percentage of sites with CAL 4–5 mm, which showed no significant difference (p = 0.342 and p = 0.466 between T0/T2 and T0/T6). However, when comparing median values of periodontal parameters in the control group, we observed statistically significant worsening of almost all values, except the percentage of sites with CAL ≥ 6 mm (p = 0.776) between T0/T2, and the percentage of sites with CAL ≥ 6 mm (p = 0.47) and percentage of sites with PPD ≥ 6 mm (p = 0.49) between T0/ T6 (Table 2). In the period between
Assessment for eligibility (n = 66) Enrollment •
Exclusion (n =2) Withdrew (n = 2)
Randomization (n = 64)
Allocation Test group (n = 32)
Control group (n = 32) Follow-up
Lost to follow-up (n = 0)
Lost to follow-up (n = 0) Analysis
Analyzed (n = 32)
Fig. 1. Flow chart of the study.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
877
Analyzed (n = 32)
878
Ca ula et al.
Table 1. Medical and demographic characteristics of the test and control groups
Discussion
Variable
Test (n = 32)
Control (n = 32)
p
Age (years) BMI (kg/m2) Ethnicity [n (%)] White Pardo (brown-skinned, mixed-race) Black Gender [n (%)] Men Women Education Level [n (%)] Fundamental Medium Superior Smoking [n (%)] Smokers Ex-smokers Non-smokers Diabetes [n (%)] Arterial hypertension [n (%)]
44.4 (38; 50) 26.5 (25; 28.2)
44.0 (38.5; 51.5) 26.4 (24.1; 29.2)
0.89 0.75
13 (40.6%) 12 (37.5%) 7 (21.9%)
15 (46.9%) 10 (31.2%) 7 (21.9%)
0.85
20 (62.5%) 12 (37.5%)
19 (59.4%) 13 (40.6%)
1.00
15 (46.9%) 16 (50.0%) 1 (3.1%)
9 (28.1%) 21 (65.6%) 2 (6.2%)
0.20
4 13 15 5 7
6 6 20 7 3
0.16
(12.5%) (40.6%) (46.9%) (15.6%) (21.9%)
(18.8%) (18.8%) (62.5%) (21.9%) (9.4%)
0.37 0.15
Results are expressed as median (first quartile; third quartile) and frequency (percentage).
T0 and T2, five control patients had discomfort and pain symptoms and were undergoing dental extraction (lost a total of six teeth with mobility grade 3). There was no tooth loss between T2 and T6 in two groups. Haematological data are presented in Table 3. At the pre-treatment (T0), there was no significant difference between groups for CRP level, ESR, and lipid profile. However, there was a significant difference after treatment between the test and control groups for the median values of CRP and ESR at T2 [0.19 (CI 95% 0.1–0.32) difference, p = 0.001 and 3.5 (1.1–6.2) difference, p = 0.003, respectively] and for median values of CRP, ESR, and total cholesterol, in T6 [0.7 (0.51–0.83) difference, p < 0.001; 7.0 (5.8–10.4) difference, p < 0.001; and 10.0 (4.7–38.6) difference, p = 0.021, respectively]. When comparing median values of haematological data in the test group, we observed statistically significant improvement between T0/T2, of the values of ESR and triglycerides [2.5 (1.2–5.0) difference, p = 0.002 and 23.0 (2.0–75.8) difference, p = 0.004, respectively]. Reduction in the median levels of CRP, ESR, total cholesterol, and triglycerides, between T0/ T6 was observed in the test group [0.46 (0.21–0.73) difference, p < 0.001; 5.0 (4.0–7.9) difference, p < 0.001; 20.5 (10.9–30.4) difference, p < 0.001; and 23.5 (3.1–72.7) difference, p = 0.015, respectively]. The
LDL cholesterol showed a trend towards reduction [3.5 ( 0.2–12.8) difference, p = 0.06]. When comparing median values of haematological data in the control group between T0/T2 and T0/T6, we observed a statistically significant increase in the values of CRP [0.14 (0.06–0.2) difference, p = 0.003 and 0.24 (0.1–0.5) difference, p < 0.001, respectively] and ESR [1.0 (0.2–4.1) difference, p = 0.007 and 2.0 (1.4–6.1) difference, p = 0.001, respectively]. There was no significant correlation between PPD and BOP reductions and haematological changes at T2 in the test group. However, there was a significant correlation between CAL decrease and a decline in triglycerides at T2 in the test group (q = 0.395, p < 0.05). At T6, there were significant correlations between PPD reduction and a decrease in ESR (q = 0.502, p = 0.003) and between CAL improvement and CRP decrease in the test group (q = 0.371, p < 0.05). No significant correlation was observed between PPD, CAL, and BOP alterations and haematological changes at T2 and T6 in the control group. Correlations among CRP, TC, HDL, and LDL at baseline (T0), 2 (T2) and 6 months (T6) after periodontal treatment are described in Table 4. There was a statistically significant correlation between TC and LDL at T0, T2, and T6 and between TC and triglycerides at T0 and T2.
The aim of this study was to evaluate the effect of non-surgical periodontal treatment on markers of cardiovascular disease (CRP, ESR, and lipid profile). The results of non-surgical treatment are in accordance with the classical studies (Badersten et al. 1981, 1984, 1987). We found that periodontal treatment was effective in improving the periodontal parameters and reducing CRP level, ESR, total cholesterol, and triglycerides after 6 months. Inflammation is important in all phases of CVD, including initiation of atherogenic process, as well as the acute rupture of atherosclerotic plaque (Ridker 2003). Low-grade inflammation is a marker for subsequent CVD (Ridker et al. 1998) and C-reactive protein is an acute-phase protein that has been shown to be a simple, reliable, and reproducible marker of underlying systemic inflammation and a strong predictor of myocardial infarction and stroke (Willerson & Ridker 2004). ESR is an indicator of red cell aggregation and thus of blood viscosity, and it is a simple and inexpensive laboratory test. ESR is an independent, longterm predictor of coronary heart disease (Andresdottir et al. 2003). Lipid profile is also associated with coronary heart disease (Sarwar et al. 2007). Elevated levels of CRP have been observed in periodontal patients (Paraskevas et al. 2008). The elevation of CRP level is considered a risk factor for atherosclerosis and cardiovascular disease (Buckley et al. 2009). In the present study, no significant changes in CRP levels were observed within 2 months, however, a significant reduction was observed in CRP after 6 months of treatment (p < 0.0001). These results are similar to those found by D’Aiuto et al. (2004). Mattila et al. (2002) have also found that treating periodontitis decreases CRP level. The authors concluded that it possibly reduces CVD risk. Ide et al. (2003) and Yamazaki et al. (2005) did not find significant effect of periodontal therapy on CRP level. They evaluated CRP levels, respectively, after 6 weeks and 3 months following completion of treatment which
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
32.5 (24; 42)
17 (7; 31)
62 (50; 76.5)
29 (18; 39.5) 7 (4; 15.5)
42.5 (26.5; 62) 41.5 (24.5; 55.5)
16 (7.5; 25.5)
64 (49.5; 78)
24 (17; 36.5)
7 (4; 15.5)
55 (38; 68) 45 (31; 62.5)
24 (20.5; 26.5) 50 (25.5; 61.5)
Control
29.5 (21; 36.5)
23.5 (20.5; 26) 54.0 (34.5; 67.5)
Test
T0
0.06 0.15
0.60
0.30
0.73
0.94
0.25
0.53 0.35
pa
9 (0; 20) 5 (0; 23)
0 (0; 1.5)
11.5 (6.5; 18.5)
86.5 (78.5; 93.5)
4.5 (1; 11)
30 (11.5; 41)
22.5 (18.5; 26) 65.5 (46.5; 85.5)
Test
41.5 (29.5; 68.5) 40.5 (28.5; 55)
7 (4; 15)
31.5 (18; 42.5)
60 (40.5; 74)
17 (7; 33.5)
35 (25.5; 42.5)
24 (20.5; 26.5) 48 (25; 58.5)
Control
T2
