Supragingival biofilm control and systemic inflammation in patients with type 2 diabetes mellitus

The objective of this study was to evaluate the effect of strict supragingival biofilm control on serum inflammatory markers and on periodontal clinical parameters in type 2 diabetes mellitus (T2DM) patients with chronic severe periodontitis. Twenty-four individuals with T2DM and periodontitis were randomly allocated to two treatment groups. The supragingival therapy group (ST, n = 12) received supragingival scaling, whereas the intensive therapy group (IT, n = 12) underwent supraand subgingival scaling, as well as root planing. Patients from both groups received professional oral hygiene instructions every month. Data regarding visible plaque index (VPI), gingival bleeding index (GBI), bleeding on probing (BOP), probing pocket depth (PPD), clinical attachment level (CAL), serum levels of interleukin (IL)-6, IL-17A, IL-8, tumor necrosis factor α (TNF-α), monocyte chemoattractant protein (MCP)-1 enzyme-linked immunosorbent assay (ELISA), and glycated hemoglobin (HbA1c) levels were obtained at baseline and at 6 months post-therapy. Both therapies resulted in the improvement of almost all clinical periodontal parameters (p < 0.05). There were no differences in TNF-α, IL-8, IL-17A and HbA1c levels in either group (p > 0.05), between the two periods. However, MCP-1 levels were significantly reduced in both the ST (p = 0.034) and the IT (p = 0.016) groups, whereas the serum IL-6 levels were significantly reduced only in the IT group (p = 0.001). Strict control of supragingival biofilm has a limited effect on systemic inflammatory markers, and a moderate effect on periodontal clinical parameters.


Introduction
Periodontitis is a destructive form of periodontal disease (PD), affecting approximately 50% of all adults and more than 60% of over 65-year-olds.Severe periodontitis is known to affect approximately 10%-15% of the population. 1,2Periodontal destruction is mediated by the host's immune-inflammatory response 3 elicited by pathogendominated biofilms.This response leads to the production of specific antibodies and inflammatory mediators within the local tissues, as well as a systemically elicited response. 4claration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript.
Cytokines participate actively in the initiation and maintenance of immune responses to oral bacteria. 5n imbalance between pro-and anti-inflammatory cytokines leads to the destruction of periodontal tissue and to clinical signs of periodontal disease. 6he local production of cytokines in response to periodontal bacteria is related to serum concentrations of pro-inflammatory cytokines. 7Non-surgical periodontal therapy promotes the reduction of bacterial challenge, 8,9,10 and may also affect the inflammatory burden. 11iabetes and PD are highly prevalent in the population, and share a common etiopathogenesis. 12ype 2 diabetes mellitus (T2DM) is a recognized risk factor for PD, affecting its prevalence, incidence, and severity; 13,14 the relationship between the two disorders is bidirectional 12 .PD may represent an additional factor contributing to the total inflammatory burden in individuals with T2DM.Furthermore, periodontal treatment has been shown to improve metabolic control in T2DM. 15,16he role of periodontal treatment in serum inflammatory markers is somewhat conflicting, possibly because of differences in periodontal therapy protocols (time of follow-up and use of local/systemic antimicrobials).Some studies have shown considerable changes in the serum levels of IL-6 and TNF-α, 17,18 whereas others have disclosed no significant changes in TNF-α, 19 IL-6, 20 and IL-8. 21o the best of our knowledge, there is no information regarding the effect of strict supragingival biofilm control on inflammatory burden.Thus, the purpose of this study was to test the null hypothesis that supragingival scaling does not have beneficial effects on the serum levels of inflammatory markers or on periodontal clinical parameters in patients with chronic generalized periodontitis and T2DM, compared with the effects of intensive periodontal therapy (supra-and subgingival scaling, as well as root planing).

Methodology Study population
Patients selected for the study were recruited between February 2011 and December 2013 in complete accordance with the Helsinki Declaration of 1975, as revised in 2000.Ethical approval was received by the Ethics Committee of the Universidade de São Paulo -SP, São Paulo, Brazil (FR 284313-127/2009 protocol).All patients with T2DM were diagnosed according to the World Health Organization classification, 22 and remained under the supervision of an endocrinologist.The participants were treated with a dietary intervention and/or oral hypoglycemic agents.
The inclusion criteria were as follows: individuals ≥ 35 years of age, confirmed diagnosis of T2DM for a period of over 3 years, generalized severe chronic periodontitis (number of probing pocket depth [PPD] sites ≥ 30%, clinical attachment level [CAL] > 4 mm, and bleeding on probing), and ≥ 15 teeth.Pregnant women, smokers, patients with body mass index (BMI) > 35 kg/m 2 , or those who had received periodontal therapy, systemic antibiotic, or oral antiseptic therapy 6 months prior to the study were excluded.
Sample size calculation was undertaken by assuming a reduction of 2 mm in mean pocket depth, with 0.6 mm standard deviation in the IT group, and 1 mm mean pocket depth reduction, with 0.6 mm standard deviation in the ST group (90% statistical power and 5% significance level).The required sample size for each group was determined as 11, and 12 patients were recruited to account for potential dropouts and missing data.

Periodontal Clinical Data
The presence of supragingival biofilm was recorded as visible plaque index (VPI), 23 whereas marginal gingival bleeding was recorded as gingival bleeding index (GBI). 23Bleeding on probing (BOP), PPD, and CAL were also evaluated.The North Carolina manual probe was used in this study, (Hu-Friedy ® , Chicago, USA); VPI, GBI, and BOP were recorded as (0) absent, or (1) present.Clinical examinations were performed by two blinded and calibrated examiners (H.P.C.A. and A.M. F.) on six sites per tooth (excluding the third molars), at baseline and at 6 months after periodontal therapy.Reproducibility during the study was assessed in 10% of the participants, with intraclass correlation coefficients ranging from 0.90 to 0.94 for periodontal PPD, and 0.85 to 0.89 for CAL.

Experimental Design and Periodontal Therapy
This study comprised 24 individuals randomly assigned by a computer random number generator (Excel), and allocated by sequentially numbered sealed opaque envelopes.All individuals were given oral hygiene instructions.The supragingival therapy group (ST, n = 12) received supragingival scaling using an ultrasonic device and periodontal curettes (Hu-Friedy ® , Chicago, USA).A Single appointment lasted ~ 60 minutes.The intensive therapy group (IT, n = 12) received supra-and subgingival scaling and root planing, (in sites with PPD ≥ 4 mm) using an ultrasonic device and periodontal curettes.The procedures for the IT group were performed under local anesthesia (3% prilocaine with felypressin), in two appointments lasting ~ 120 minutes each.Supportive therapy for biofilm control consisted of professional instructions on oral hygiene given to each patient every month.Periodontal therapy was carried out by an experienced periodontist (G.H.G.).No changes in medication or diet were made during the study period.

Inflammatory Markers and HbA1c Measurements
Samples of approximately 5 mL of blood were collected by venipuncture and placed in untreated Vacutainer blood collection tubes (Becton Dickinson Co., São Paulo, Brazil).An experienced nurse technician (J.P.R.) performed the blood collections.Glycated hemoglobin (HbA1c) was analyzed by high-performance liquid chromatography (DiaSTAT Hemoglobi n A1c Analyzer System, BioRad Laboratories, Hercules, USA), at the clinical laboratory located in the Hospital Universitário -HU (University Hospital), at the USP.The serum was obtained by centrifugation at 2,000 g for 10 minutes, aliquoted, and stored at -80 °C until further analysis.

Statistical analysis
Data from the ELISA were analyzed using Prism software (Prism 5 Project, Graphpad Software Inc., La Jolla, USA).Shapiro-Wilk normality test was used and the data did not follow Gaussian distribution.The data were analyzed by non-parametric statistical methods.Periodontal clinical data were analyzed using SPSS software (SPSS for Windows, version 17.0, SPSS Inc., Chicago, USA), considering the individual as a study unit.Intergroup comparison was determined using the Mann-Whitney test.The intragroup comparison was determined using the Wilcoxon signed rank test.The tests were based on the median values with variability measures (25% and 75% quartiles).The results were considered statistically significant at p < 0.05.The GBI, VPI, and BOP data were obtained by calculating percentages of positive sites per patient, and thereafter, median values were calculated for the groups.For PPD and CAL, measured in millimeters, the median value per patient was obtained first, followed by the median value for the group.

Results
All patients selected for analysis in the present study completed 6 months of the clinical trial.The ST group was comprised of 56.3% women (mean age, 54.4 ± 5.8 years; BMI, 25.6 ± 4.4), whereas the IT group was comprised of 52.0 % women (mean age, 52.0 ± 3.3 years; BMI, 26.9 ± 3.8).Both groups were similar in age, gender, and BMI at baseline (p > 0.05).
No significant differences in periodontal parameters were observed between the two groups at baseline (Table 1).Both therapies resulted in statistically significant improvements in most clinical parameters after 6 months of follow-up, except for CAL in the ST group.However, the effects on BOP and PPD were more pronounced in the IT group, with a significant gain in CAL observed in this group only (Table 1).Changes in periodontal clinical parameters are represented in Table 2.The effectiveness of periodontal treatment on PPD (4-6 mm; ≥ 7 mm) and CAL (4-6 mm; ≥ 7 mm) was observed in both groups.Changes in PPD (4-6 mm) (p = 0.001) and CAL (4-6 mm) (p = 0.001) were found to be significantly different between the two groups in this study (Table 2).
The levels of inflammatory markers and HbA1c, at baseline and at the end of the experimental period, are shown in Figure 1.Both groups of patients showed similar levels of serum inflammatory markers at baseline (p > 0.05).No significant differences for serum levels of IL-8, IL-17A and TNF-α were recorded between the two groups (p > 0.05), 6 months after periodontal therapy.
However, there was a statistically significant reduction in IL-6 serum levels in the IT group, after 6 months of therapy (p < 0.001), but not in the ST group (p = 0.33).Furthermore, MCP-1 levels were reduced in both groups (ST, p = 0.03; IT, p = 0.01).There were no significant differences in serum HbA1c levels between the groups at baseline and at 6 months post-therapy (p > 0.05) (Figure 1).

Discussion
The results of the current study demonstrate that supragingival biofilm control reduces the local symptoms of inflammation, with a limited systemic effect on serum biomarkers in T2DM individuals.This is in concurrence with the findings from previous clinical studies that have reported similar effects of periodontal therapy on IL-6 levels. 17,24To our knowledge, there are no previously published studies available regarding the effect of strict supragingival biofilm control on serum levels of IL-6, IL-8, IL-17, TNF-α, and MCP-1 in T2DM patients.Our follow-up data showed that both therapies were comparable in promoting clinical improvement of the periodontal parameters; however, the best effect was seen in the IT group.
Minimal periodontal treatment in the form of supragingival scaling, prophylaxis, and oral hygiene instructions was given to the patients in the ST group.Although not considered the ideal form of therapy, it promoted clinical improvement of most of the periodontal parameters in this study.It has been known for decades that professional supragingival biofilm control has an effect on the subgingival microbiota, and, consequently, on periodontal clinical parameters. 25This effect is attributed to the reduced inflammation of adjacent periodontal tissues, leading to a restriction in the availability of nutrients necessary for bacterial multiplication.It is important to highlight that previous studies have not been performed on diabetic individuals.Moreover, in the present study, no microbiological or local cytokine level analyses were performed, and thereby are recommended for further studies on T2DM individuals.
In order to determine more specific differences between the two groups, periodontal sites were divided   The mechanisms by which periodontitis influences T2DM have not been precisely defined.Increasing evidence suggests that severe chronic periodontitis induces subclinical bacteremia, leading to a low-grade systemic inflammation that may affect metabolic control. 26,27Some of the inflammatory markers linked to T2DM complications and insulin resistance are IL-6, IL-12, MCP-1, and IL-8.Moreover, IL-6 has also been associated with vascular complications, 28 and atherogenesis may be regulated by chemotactic factors, such as MCP-1, which has been linked to both insulin resistance and hyperglycemia.Our study demonstrated that both therapies were able to reduce serum MCP-1 levels, as also achieved by some recently developed drugs. 29This reduction may be partly explained by the partial improvement of periodontal parameters and local inflammation.On the other hand, this partial periodontal improvement was not able to reduce IL-6 serum levels in the ST group.

Conclusions
Supragingival therapy has a modest effect on periodontal clinical parameters, and a limited effect on systemic inflammatory markers, compared with the effect of intensive periodontal therapy.Therefore, intensive periodontal therapy should be prioritized to control the inflammatory burden on T2DM individuals.

Table 1 .
Medians values (75 th /25 th percentiles) for clinical periodontal parameters at baseline and 6 months post-therapy for the supragingival therapy (ST; n = 12) and the intensive periodontal therapy (IT; n = 12) groups.
IT group, indicating the importance of root debridement in treating diseased sites.We could speculate that the reduction of inflammation in the adjacent periodontal tissue was the major cause for the changes in PPD and CAL measurements.