Impact of periodontal therapy on systemic markers of inflammation in patients with metabolic syndrome: A randomized clinical trial

To determine the impact of periodontal treatment on systemic markers of inflammation in patients with metabolic syndrome (MetS) and periodontitis.


| INTRODUCTION
Metabolic syndrome (MetS) is a cluster of medical conditions associated with the risk of developing atherosclerotic cardiovascular disease (CVD). 1 It is characterized by central obesity (defined by waist circumference [WC]), impaired glucose regulation, high blood pressure and dyslipidaemia (i.e. low high-density lipoprotein [HDL] cholesterol and/ or elevated triglycerides). 2 MetS is not a disease in itself, but it is a serious health condition and a major global public health concern that affects 10%-40% of adults in developed countries, in parallel with the rise in the prevalence of obesity. 3 In the United States, its prevalence has increased from 28% in 1988-1994 to 34% in 1994-2004, according to the National Health and Nutrition Examination Surveys. 4 Periodontitis is a highly prevalent chronic inflammatory disease characterized by the destruction of tooth-supporting tissues, eventually leading to tooth loss and significantly impairing the patient's quality of life. In fact, severe periodontitis is one of the most prevalent diseases worldwide, with an age-standardized prevalence of 9.8% and with 796 million cases. 5 Several epidemiological studies and systematic reviews have consistently shown an association between periodontitis and an increased risk of future atherosclerotic cardiovascular events, independent of traditional risk factors (e.g. smoking, diabetes, hypertension and hypercholesterolaemia). 6 This association has been explained by the systemic effect of this infectious disease, evidenced by a significant increase in high-sensitivity C-reactive protein (hs-CRP) and other biomarkers of systemic inflammation, which may contribute to the overall inflammatory burden and to an increased risk of atherogenesis and hyperglycaemia. 7,8 A recent systematic review with meta-analysis has shown evidence of a potential association between periodontitis and MetS. 9 This association may be explained by the shared chronic state of systemic inflammation in both conditions, resulting in oxidative stress and insulin resistance. 10,11 This bi-directional association may be explained by the increase of systemic inflammation as a consequence of periodontitis, which may act as a contributor to the development of MetS, 12 while the low-grade systemic inflammation characteristic of obesity and impaired glucose regulation may negatively affect the periodontal status. 13 The possible effect of periodontal treatment on systemic inflammation has been reported in several studies assessing the levels of inflammatory markers after treatment, showing significant reductions in interleukin (IL)-6 and hs-CRP. 14,15 However, this systemic impact has not been consistent and the variability in the reported outcomes has been attributed to inclusion of patients without clear measures of exposure, based on clinical or radiographic surrogates, rather than on the actual infectious burden, which may not reflect the degree of chronic inflammatory exposure. 16 Also, the selection of patients may not have included those with a relevant risk of CVD. 17 It was, therefore, the purpose of this parallel-arm, double-blind, randomized controlled clinical trial, to determine whether the treatment of periodontitis in patients with MetS could reduce the cardiometabolic risk, measured by means of (a) markers of systemic inflammation and prothrombotic states (hs-CRP, α-1 antitrypsin and fibrinogen levels), (b) proinflammatory cytokine profiles, and (c) markers of carbohydrate and lipid metabolism.

| Study design and participants
This study was designed as a parallel-group, double-blind, single-centre, randomized clinical trial (RCT) with a 6-month follow-up. Patients were recruited among those diagnosed with MetS and included in an active programme of cardiovascular risk prevention at the Fuenla-  18 in 2009 based on the presence of at least three of the following risk factors: • Elevated WC (≥94 cm in men, ≥80 cm in women).
-Presence of at least 16 teeth.
-Diagnosis of stage III-IV generalized periodontitis, according to the criteria of the 2018 European Federation of Periodontology-American Academy of Periodontology classification. 19 These are the most severe forms of periodontitis, showing an attachment loss of ≥5 mm with radiographic bone loss extending to the middle or apical third of the root.
-Presence of at least eight sites with a probing pocket depth (PPD) of ≥6 mm and four sites with a clinical attachment level (CAL) of ≥5 mm, distributed in at least two different quadrants, 20 with the objective of recruiting participants with severe periodontal destruction (defined by CAL) and a high level of inflammation (defined by PPD).
Participants were excluded by the presence of: -Uncontrolled systemic diseases other than diabetes or hypertension (e.g. chronic kidney disease, chronic lung disease, acute pulmonary disease during the previous 3 months, history of stroke, myocardial infarction, angor pectoris or revascularization during the previous 6 months, neoplasms).
-History of any surgical treatment in the previous 3 months.
-Alcoholism or psychiatric disorders.
-History of systemic antibiotic usage over the previous 3 months.
-Non-surgical periodontal treatment during the previous 6 months, or surgical periodontal treatment over the previous 12 months.
All the patients recruited according to these criteria and willing to participate in the study received verbal and written information. Upon signing the approved consent form, they were referred for periodontal treatment to the Postgraduate Clinic of Periodontology at the Faculty of Odontology (University Complutense of Madrid, Spain).

| Study interventions
All patients received standardized oral hygiene instructions (consisting of the use of a manual toothbrush and specific inter-dental brushes) and, when indicated, extraction of hopeless teeth. Then patients were randomized to one of the two following groups: • An intensive periodontal treatment (IPT) test group, which con- Both treatment groups received, after the first treatment session, an antiseptic mouth rinse, containing 0.12% chlorhexidine and 0.05% cetylpyridinium chloride (Perio-Aid 0.12%; Dentaid, Barcelona, Spain), to be used twice daily for 14 days. Oral hygiene instructions were reinforced in both groups at the 3-and 6-month visits, but no additional periodontal treatment was provided during the study, unless a subject showed periodontitis progression, defined as the presence of interproximal attachment loss of ≥3 mm in at least two teeth. 22 These patients were withdrawn from the study and immediately received the appropriate periodontal treatment.

| Outcomes
The primary outcome was the evaluation of the differences between the test group and the control group in the mean hs-CRP serum levels at 6 months after the periodontal intervention. As secondary outcomes, the mean levels at 6 months of α-1 antitrypsin, fibrinogen, inflammatory markers (IL-1β, IL-6, IL-8 and TNF-α), HbA1c, FPG, insulin, lipid fractions and blood pressure were compared.

| Statistical methods
The sample size calculation was based on detecting a mean difference between groups of 2 mg/L in serum hs-CRP levels with an estimated standard deviation (SD) of 2.5 mg/L. To detect statistically significant differences (α = 0.05) with 80% power, a minimum of 25 subjects per group were needed, which including an expected 20% dropout rate, resulting in a final sample of 30 participants per group.
Data were reported as mean and SD unless otherwise specified (e.g. n [%]). All analyses were performed using the intention-to-treat population and the last observation carried forward approach for missing values. Analyses were repeated for all primary and secondary outcomes using post hoc missing value analyses. In addition, perprotocol analyses were conducted for all outcomes; the estimates derived from these analyses were reported for the primary outcome (hs-CRP) and any secondary or post hoc outcomes if they differed from estimates obtained in the intention-to-treat population.
The primary and secondary outcomes were modelled using multi-  (Table S1).
The cardiometabolic risk profiles at baseline (Table 1)

| Clinical and microbiological periodontal outcomes
Periodontal clinical variables were similar at baseline in both groups (Table 2, Figure S1). The mean number of teeth at baseline in the IPT and MPT groups was 22.6 (SD = 3.   Creatinine, mg/dL 0.9 (0.

| Impact of the interventions on systemic inflammatory status
The primary outcome, mean hs-CRP concentration, was reduced after  (Table 3). If assigned to the IPT group versus the MPT group, the odds ratio for a patient to move from a hs-CRP value of ≥3 to <3 mg/L was 5.4 (95% CI [1.0; 31.6]; P = .040). At 6 months, the percentage of patients in the IPT group experiencing a reduction in hs-CRP levels was 68.8%, while this percentage was 29.0% in the MPT group (P < .001). Among the variables included in the multilevel linear regression, only belonging to the IPT group (P = .004), baseline hs-CRP levels (P < .001) and being a smoker (P = .014) significantly and independently predicted the variance of hs-CRP decline over 6 months.
In the secondary outcomes, significant reductions in IL-1β and TNF-α at 3 months were observed in the IPT group compared with the MPT group (Table 3). However, no differences between the groups were observed for these biomarkers at 6 months, or for the other inflammatory markers (α-1 antitrypsin, fibrinogen, WBC count, IL-6 and IL-8) at any time point after therapy. No differences in BMI or WC between patients in the IPT or MPT groups were observed throughout the study.

| DISCUSSION
The present study has shown that effective periodontal treatment in Despite periodontitis being defined as a chronic multifactorial inflammatory disease associated with dysbiotic plaque biofilms, 19 most of the previous reports evaluating the impact of periodontal treatment on cardiovascular risk have relied on surrogate markers of exposure that were either clinical (probing depths) or radiographic (bone loss), but they did not assess exposure by measuring the bacterial burden or the presence of putative pathogens. 16,27,31 In the present investigation we have identified high counts of anaerobic bacteria and high proportions and counts of P. gingivalis, a keystone periodontal pathogen strongly associated with periodontitis, in all T A B L E 3 Inflammatory biomarkers and clinical variables 3 and 6 months after periodontal treatment, by study group patients at baseline. The tested intervention significantly reduced both the counts of anaerobic bacteria and P. gingivalis. Although previous cross-sectional studies have shown an independent significant association between presence of a significant subgingival bacterial burden and levels of periodontal pathogens with surrogate measurements of atherosclerotic risk as intima-media thickness or hs-CRP, 32 this is the first RCT to show that effective periodontal treatment significantly reduced these microbiological exposure measurements and that this microbiological impact was associated with significant reductions in hs-CRP.
Other significant improvements associated with the effective periodontal treatment were the significant reduction (0.3%) in HbA1c at 3 months, and a significant reduction in arterial blood pressure, thus suggesting that this effect was not limited to the improvement in systemic inflammation, but also impacted vascular function and metabolic control. In fact, periodontal inflammation has been associated with both insulin resistance and endothelial dysfunction. 28,31,33 The fact that the significance in HbA1c reduction in the treatment group was lost at 6 months may be caused by the lack of repeated periodontal interventions during the course of the study. Effective periodontal maintenance care has been shown in other studies to maintain a positive effect in glycaemic control. 20,29 Similarly, recent studies have shown a significant association between periodontitis and hypertension and have suggested that periodontal treatment could reduce arterial blood pressure. 34 In this RCT, we have reported an adjusted statistically significant reduction in both systolic and diastolic blood pressure (7.8 and 7.3 mmHg, respectively), 3 months after effective periodontal treatment. These findings may be meaningful, because a 10 mmHg reduction in systolic blood pressure or a 5 mmHg reduction in diastolic blood pressure has been associated with a 25%-30% reduction of cardiovascular events, 35 and half of hypertensive adults remain poorly controlled despite effective medications. 36 The current clinical trial, however, may have relevant limitations.
One is that the tested intervention consisted of a combination of subgingival debridement and the adjunctive administration of azithromycin. This antibiotic has shown significant reductions in a global inflammatory score consisting of hs-CRP, IL-1β, IL-6 and TNF-α at 6 months, but not on CRP alone, 37 and hence the cardiovascular effect shown in this study could be attributed to the antibiotic rather than the periodontal therapy. However, the adjunctive use of systemic antimicrobials to subgingival instrumentation is clearly justified by the periodontitis severity in the selected sample, as this modality of therapy has shown a significant added effect on clinical and microbiological outcomes compared with subgingival debridement alone in severe periodontitis patients, 38 as well as in RCTs evaluating the impact of intensive periodontal therapy in cardiovascular risk outcomes. 29,31 Another possible limitation could be the selection of the main outcome variable (hs-CRP) as a surrogate for cardiovascular risk, as its predictive value may be limited, 39