Abstract
Background:
Supplementation with omega-3 polyunsaturated fatty acids (ω-3 PUFA) and low-dose aspirin (ASA) have been proposed as a host modulation regimen to control chronic inflammatory diseases. The aim of this study was to investigate the clinical and immunological impact of orally administered ω-3 PUFA and ASA as adjuncts to periodontal debridement for the treatment of periodontitis in patients type 2 diabetes.
Methods:
Seventy-five patients (n=25/group) were randomly assigned to receive placebo and periodontal debridement (CG), ω-3 PUFA + ASA (3g of fish oil/day + 100mg ASA/day for 2 months) after periodontal debridement (Test Group [TG]1), or ω-3 PUFA + ASA (3g of fish oil/day + 100mg ASA/day for 2 months) before periodontal debridement (TG2). Periodontal parameters and GCF were collected at baseline (t0), 3 months after periodontal debridement and ω-3 PUFA + ASA or placebo for TG1 and CG (t1), after ω-3 PUFA + ASA (before periodontal debridement) for TG2 (t1), and 6 months after periodontal debridement (all groups) (t2). GCF was analyzed for cytokine levels by multiplex ELISA.
Results:
Ten patients (40%) in TG1 and 9 patients (36%) in TG2 achieved the clinical endpoint for treatment (≤4 sites with PD ≥ 5mm), as opposed to 4 (16%) in CG. There was clinical attachment gain in moderate and deep pockets for TG1. IFN-γ and IL-8 levels decreased over time for both test groups. IL-6 levels were lower for TG1. HbA1c levels reduced for TG1.
Conclusion:
Adjunctive ω-3 and ASA after periodontal debridement provides clinical and immunological benefits to the treatment of periodontitis in patients with type 2 diabetes.
Keywords: Periodontitis, Diabetes, Omega-3 fatty acids, Aspirin, Immunomodulation, Inflammation
Summary sentence
Omega-3 and aspirin after periodontal debridement improve clinical and immunological outcomes in patients with periodontitis and type 2 diabetes.
INTRODUCTION
Periodontitis is an inflammatory condition associated with bacterial infection that is modified by multiple host response genes in combination with lifestyle and environmental factors and leads to the destruction of periodontal tissues1. The disease results from complex interactions between the biofilm and inflammatory immune response, and the latter accounts for almost 80% of the risk of periodontal tissue damage2,3. Diabetes is one of the major risk factors for periodontitis4. The level of hyperglycemia, rather than the diagnosis and etiology of diabetes, is associated with periodontitis and an increased risk of tooth loss4–6. In hyperglycemic individuals, impaired wound healing may increase susceptibility to periodontitis and compromise long-term periodontal stability. While the underlying mechanism of diabetes as a risk factor for periodontitis has been well-established, the response of patients with diabetes to different periodontal treatments has been less explored. The gold-standard treatment for periodontitis is subgingival debridement associated with an effective supragingival biofilm control7. Conventional treatment is quadrant scaling and root planing, but full-mouth approaches have been demonstrated to be equally effective8. Considering the hyperinflammatory profile of patients with diabetes, it has been hypothesized that adjunctive treatments that modulate the host-response could represent a good and effective treatment approach for these patients.
Omega-3 polyunsaturated fatty acids (ω-3 PUFA), including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have been investigated for their therapeutic actions in rheumatoid arthritis, ulcerative colitis, atherosclerosis, cardiovascular diseases, cancer, psoriasis and periodontitis9. Beneficial results were observed and attributed to the actions of specialized proresolving mediators (SPMs) biosynthesized from ω-3 PUFA (resolvins, protectins and maresins) and upregulation of endogenous SPMs (lipoxins)10–12. Studies also showed that aspirin (ASA) triggers the synthesis of more potent SPMs, called aspirin-triggered resolvins, aspirin-triggered protectins, and aspirin-triggered lipoxins that have a longer half-life in blood13–15. SPMs promote the resolution of inflammation by reducing neutrophil infiltration16, regulating cytokine/chemokine synthesis17, attenuating systemic C-reactive protein (CRP) and interleukin (IL)-118 production, decreasing the production of RANKL19, and regulating macrophage-secreted pro-inflammatory cytokines20.
In a placebo-controlled randomized clinical trial (RCT), clinical benefits of adjunctive oral administration of ω-3 PUFA + ASA, including reduced mean probing depth (PD) and clinical attachment (CA) gain, and decreased RANKL and MMP-8 salivary levels were shown in individuals with generalized severe chronic periodontitis who used ω-3 and ASA for 6 months21. Other clinical studies with fewer patients and shorter follow-up periods also evaluated the use of ω-3 PUFA alone22–25, but less evident favorable results were observed when compared to the combined use of ω-3 PUFA and ASA. The aim of this study was therefore to investigate periodontal and immunological effects of daily supplementation with 3g fish oil with ω-3 and 100mg ASA for 2 months as an adjunctive therapy to periodontal debridement for the treatment of periodontitis in patients with type 2 diabetes. We hypothesized that the combined treatment protocol would lead to improved periodontal and immunological outcomes when compared to periodontal debridement alone after 6 months of follow-up.
MATERIALS AND METHODS
Experimental Design
This was a placebo-controlled, double-blind, randomized clinical study to evaluate the superiority of two dosing regimens of ω-3 PUFA + ASA vs. control. The study was designed in accordance with the CONSORT 2010 Statement and the SPIRIT 2013 Statement. This study was approved by the human subjects ethics board of UNESP (CAAE: 51626115.5.0000.0077) and was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2013. The study protocol was registered in ClinicalTrials.gov (NCT02800252).
Study Population and Inclusion and Exclusion Criteria
Patients with type 2 diabetes and moderate to severe generalized chronic periodontitis 26 were selected from the population referred to the Department of Periodontology at the Institute of Science and Technology, São Paulo State University (Unesp). Detailed dental and medical records were obtained. Patients who fulfilled the inclusion criteria were invited to participate in the study. Inclusion criteria were: Stages III and IV, Grades B and C periodontitis with at least 6 sites with PD and CAL ≥5 mm and bleeding on probing (BoP)27,28; ≥15 teeth; aged ≥35; diagnosis of type 2 diabetes for ≥5 years, under treatment for diabetes with oral hypoglycemic agents and/or insulin, with glycated hemoglobin (HbA1c) levels ≥6.5% to ≤11%. Exclusion criteria were: need for prophylactic antimicrobial coverage; scaling and root planing in the previous 6 months; antimicrobial therapy in the previous 6 months; systemic conditions (other than diabetes) that could affect the progression of periodontitis; long-term use of medication that could interfere with periodontal response; pregnancy or lactation; smoking; allergy to fish/seafood or ASA. Written informed consent was provided by each volunteer after a thorough explanation of the nature, risks, and benefits of the clinical investigations.
Interventions, Randomization, Allocation Concealment, and Treatment Protocol
The study coordinator (MPS) created a computer-generated blocking sequence (3, 6, 9) to randomly allocate the selected patients to one of the following groups: (i) periodontal debridement and placebo (CG), (ii) 3g fish oil (900mg of ω-3 PUFA) and 100mg ASA daily for 2 months after periodontal debridement (TG1), or (iii) 3g fish oil (900mg of ω-3 PUFA) and 100mg ASA daily for 2 months before periodontal debridement (TG2). This sequence was placed in sealed opaque envelopes, each one containing a treatment modality, ensuring allocation concealment.
All patients received instructions about the relationship between periodontitis and type 2 diabetes, oral hygiene instructions, supragingival biofilm and calculus removal, extraction of hopeless teeth, dental decay removal and provisional restoration, and removal of overhanging restorations. All patients received periodontal debridement in one session. After local anesthesia, patients received subgingival debridement using ultrasonic device with subgingival inserts* and manual curettes†. All pockets received meticulous scaling and root planing (SRP), and the endpoint for this procedure was smoothness of the scaled roots. This procedure was performed by one experienced and trained periodontist (NCCS). Patients in the CG received placebo capsules for 2 months after periodontal debridement. Patients in the TG1 received 3g fish oil with ω-3 PUFA‡ and 100mg ASA§ daily for 2 months after periodontal debridement. Patients in the TG2 received 3g of fish oil with ω-3 PUFA plus 100mg ASA daily for 2 months before periodontal debridement. All patients were blinded for the treatment assignment. An investigator (NMRBA) assigned participants to the interventions according to the content of the envelopes.
Sample Size Calculation
This study was designed to compare the impact of 3 different approaches on the number of periodontal pockets with PD ≥ 5mm. Considering a difference of at least 4 pockets with PD ≥ 5mm between each test group and the control group, and standard deviation of 5 pockets, 25 patients would be necessary to provide power of 80% with a significance level of 0.0529.
Clinical Analyses
Periodontal measurements were performed by one calibrated examiner (CFA) who was blinded for treatment allocation. The examiner participated in a calibration exercise in which PD and CAL of 10 patients were measured twice within a 24-hour interval. These measurements were subjected to an intraclass correction test. The agreement for the variables was 92%.
Clinical evaluations were performed at baseline (t0) and at 3 (t1) and 6 months (t2) after debridement for CG and TG1. For TG2, measurements were performed before ω-3 and ASA therapy (t0), after ω-3 and ASA therapy/before debridement (t1), and 6 months after debridement (t2). The evaluated parameters were: 1. PD; 2.CAL; 3. Gingival recession (GR); 4. Bleeding on probing (BoP); 5. Supragingival biofilm accumulation (PI)30. All clinical measures were assessed using a manual probe**. Supragingival biofilm/calculus removal and oral hygiene instructions were performed monthly, as necessary. Peripheral blood was collected to evaluate HbA1c levels at t0 and t2. Anthropometric measures including weight and height were recorded. Body mass index (BMI) was calculated as the weight divided by the square of height (kg/m2).
Immunological Assays
To analyze the cytokine levels in the GCF, samples were collected from 2 sites with PD ≥5mm in a pooled sample from a subset of 45 randomly selected subjects (n=15/group). Each site was isolated and supragingival biofilm was removed. GCF was collected with PerioPaper strips†† that were introduced in the pockets until slight resistance was felt and kept there for 30s. Samples were placed in sterile microtubes and kept frozen until Multiplex assay31.
The inflammatory markers of interferon (IFN)-γ, IL-1β, IL-6, IL-8, IL-4, IL-10, tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP-1α), and monocyte chemotactic protein 1 (MCP-1) were analyzed. Multiplexed sandwich immunoassays, based on flowmetric Luminex xMAP technology, were conducted at the Forsyth Institute (Cambridge, MA, USA). Each panel for biomarker analysis was performed according to manufacturer’s protocols. GCF samples were eluted in 100μL L-AB buffer, vortexed for 30min, 600rpm. PerioPaper strips were transferred to spin column using sterile forceps, the spin baskets were placed in the original sample tube and the solution was centrifuged for 10min at 4ºC, 10,000rpm. The spin baskets and PerioPaper strips were discarded. The samples were kept at 4ºC until the analysis was carried out. The reagents were prepared according to the manufacturer’s protocol. The plates were washed with 200μL of wash buffer and shook for 10min at room temperature, and the wash buffer was decanted from the plates. Then, 25μL of standards, controls and L-AB buffer (blanks) were added to the respective wells, and 25μL of L-AB buffer were added to all wells, including sample wells. After that, 25μL of the samples were added to their respective wells and 25μL of beads were added to all wells. The plates were incubated overnight at 4ºC on a shaker at 600rpm, and washed twice with 200μL of wash buffer. Following this, 25μL of detection antibody were added to all wells and incubated for 1h at room temperature on a shaker at 600rpm. Then, 25μL of streptavidin-phycoerythrin were added to all wells and incubated for 30min at room temperature on a shaker at 600rpm. The plates were washed twice with 200μL of wash buffer. Sheath fluid (150μL) was added to all wells. Assays were carried out on a Luminex 100 Bio-plex Platform, and data were read with Bio-Plex Manager 6.1 ‡‡ with 100μL of sample volume and 50 beads per region.
Statistical analysis
The primary outcome variable of this study was the number of pockets with PD ≥ 5mm29. Mean and standard deviations were calculated for each parameter. Normal distribution was tested by Shapiro-Wilk. Demographic data were assessed using one-way ANOVA. Gender differences between groups were assessed using the Chi-square test. The differences among full-mouth variables were assessed using a Generalized Estimated Equations Model, after adjusting for baseline. The difference of PD reduction, %BoP and %PI, CA gain, and mean HbA1c were analyzed using a General Lineal Model. The number and percentage of patients who achieved or did not achieve the clinical endpoint for periodontal treatment at t2 were assessed by Chi-square test. The data corresponding to the concentration of each cytokine were analyzed by Friedman’s test. Correlation analyses between clinical and immunological data were assessed by Pearson Correlation test. The post-hoc test applied for each pairwise comparison was Bonferroni. Data were analyzed using the intent-to-treat concept with the last observation carried forward32. All data analyses were performed using IBM SPSS and RStudio Statistics. The significance level applied was 5%.
RESULTS
The study was conducted between December 2015 and June 2018. A total of 76 volunteers were recruited at baseline. One patient decided not to go through the treatment. Thus, 75 patients received periodontal treatment. At the 3-month follow-up, all patients were reassessed. At 6-month follow-up, 2 patients were lost (1 in TG2 and 1 in CG). As the intention-to-treat statistical method was applies, 25 patients were analyzed at 6 months (Figure 1). None of the patients reported adverse events or presented complications due to long-term use of low-dose ASA.
FIGURE 1.
CONSORT flowchart of the study.
Demographic and baseline data are represented in Table 1. The percentage of female patients did not differ between groups (p>0.05). Mean age (years) was 54.9 ± 9.7 for CG, 55.6 ± 8.3 for TG1, and 54.4 ± 10.2 for TG2, with no statistical difference between groups (p>0.05). Mean number of teeth was 21.5 ± 3.4 for CG, 20.8 ± 4.6 for TG1, and 22.2 ± 3.9 for TG2 (p>0.05). In all groups, BMI for most patients was above overweight at t0. Mean BMI (kg/m2) was 31.5 ± 3.5 for CG, 30.8 ± 4.6 for TG1, and 29.1 ± 6.2 for TG2 (p>0.05).
Table 1 –
Demographic and baseline data (mean±SD) for CG, TG1, and TG2.
| Treatment groups | ||||
|---|---|---|---|---|
| Parameters | CG (n=25) | TG1 (n=25) | TG2 (n=25) | p value |
| Female (%) | 64 | 64 | 48 | 0.60 |
| Age (years) | 54.9 ± 9.7 | 55.6 ± 8.3 | 54.4 ± 10.2 | 0.98 |
| Number of teeth (n) | 21.5 ± 3.4 | 20.8 ± 4.6 | 22.2 ± 3.9 | 0.48 |
| BMI (kg/m2) | 31.5 ± 3.5 | 30.8 ± 4.6 | 29.1 ± 6.2 | 0.22 |
BMI, body mass index; CG, control group; TG1, test group 1; TG2, test group 2.
Statistically significant difference for the parameter “Female” – Chi-square test; p < 0.05
Statistically significant difference – one-way ANOVA; p < 0.05.
Periodontal parameters
Periodontal full-mouth parameters, such as mean PD, mean CAL, %BoP and %PI are represented in Table 2. At t0, no statistically significant differences were observed between groups for these parameters (p>0.05). At t1, intragroup comparisons for mean PD presented statistically significant differences for CG and TG1, which was not observed for TG2. After 6 months of periodontal debridement, intragroup comparisons showed that all groups presented statistically significant differences for mean PD. Intergroup comparisons did not present significant differences for mean PD and for PD reduction (ΔPD) from t0 to t2 (p>0.05). For mean CAL, intragroup comparisons presented significant changes for TG1 at t1, and for both test groups at t2. No significant differences were detected for CG over time. Intergroup comparisons did not present significant differences for mean CAL and for clinical attachment (CA) gain (ΔCA) from t0 to t2 (p>0.05). For BoP, intragroup comparisons revealed statistically significant differences for CG and TG1 at t1 and t2. No significant differences were detected for TG2 at t1. Intergroup comparisons showed statistically significant differences at t1, revealing significant differences between CG and TG1 and between TG1 and TG2. When BoP reduction (ΔBoP) was compared, statistically significant differences were detected for TG2 (p=0.02). For PI, intragroup comparisons showed significant differences for all groups at t1 and for both test groups at t2. Intergroup comparisons did not detect significant differences at anytime points (p>0.05). When PI reduction (ΔPI) was compared between groups, significant differences were detected for TG1 (p=0.02). Moderate (5-6mm) and deep (≥7mm) periodontal pockets were analyzed for ΔPD and ΔCA (Table 2). For ΔPD, no significant differences were detected between groups for moderate and deep pockets. For ΔCA, TG1 presented significantly greater CA gain for moderate (p=0.04) and deep (p<0.00) pockets.
Table 2 –
Periodontal parameters (mean±SD) for CG, TG1, and TG2 at t0, t1, and t2.
| Treatment groups | |||||
|---|---|---|---|---|---|
| Parameters | Time points | CG (n=25) | TG1 (n=25) | TG2 (n=25) | p value |
| Mean PD (mm) | t0 | 3.3 ± 0.5a | 3.3 ± 0.6a | 3.2 ± 0.5a | >0.05 |
| t1 | 2.9 ± 0.4bA | 2.8 ± 0.4bA | 3.1 ± 0.6aB | 0.00 | |
| t2 | 2.9 ± 0.4b | 2.9 ± 0.4b | 2.9 ± 0.4b | >0.05 | |
| Δt0-t2 | 0.4 ± 0.3A | 0.4 ± 0.3A | 0.4 ± 0.3A | 0.40 | |
| Mean CAL (mm) | t0 | 3.8 ± 0.6aA | 3.9 ± 0.8aA | 3.8 ± 0.8aA | >0.05 |
| t1 | 3.5 ± 0.7aA | 3.5 ± 0.8bA | 3.8 ± 1.0aA | >0.05 | |
| t2 | 3.6 ± 0.6aA | 3.4 ± 0.8bA | 3.5 ± 0.8bA | >0.05 | |
| Δt0-t2 | 0.2 ± 0.4A | 0.5 ± 0.6A | 0.3 ± 0.3A | 0.14 | |
| BoP (%) | t0 | 48.9 ± 20.0aA | 46.1 ± 20.2aA | 44.7 ± 18.3aA | >0.05 |
| t1 | 32.5 ± 15.7bA | 30.4 ± 16.1bA | 37.7 ± 13.0aB | 0.04 | |
| t2 | 34.7 ± 15.9bA | 28.6 ± 14.8bA | 24.4 ± 9.8bA | >0.05 | |
| Δt0-t2 | 14.1 ± 17,9A | 17.5 ± 14.9A | 20.3 ± 12.5B | 0.02 | |
| PI (%) | t0 | 58.1 ± 22,0aA | 52.6 ± 16.0aA | 55.1 ± 13.1aA | >0.05 |
| t1 | 39.2 ± 19,8bA | 37.3 ± 17.9bA | 39.2 ± 11.9bA | >0.05 | |
| t2 | 44.1 ± 16,6aA | 32.1 ± 14.7bA | 42.7 ± 11.2bA | >0.05 | |
| Δt0-t2 | 14.1 ± 25.2A | 20.6 ± 21.1B | 12.3 ± 14.9A | 0.02 | |
| PD reduction in pockets with PD 5-6 (mm) | Δt0-t2 | 1.1 ± 0.2A | 1.2 ± 0.2A | 1.3 ± 0.2A | >0.05 |
| PD reduction in pockets with PD ≥7 (mm) | Δt0-t2 | 2.0 ± 0.2A | 1.9 ± 0.2A | 2.0 ± 0.2A | >0.05 |
| CA gain in pockets with PD 5-6 (mm) | Δt0-t2 | 0.8 ± 0.2A | 1.3 ± 0.2B | 0.8 ± 0.2A | 0.04 |
| CA gain in pockets with PD ≥7 (mm) | Δt0-t2 | 1.6 ± 0.3A | 2.6 ± 0.3B | 0.8 ± 0.3A | 0.00 |
BoP, bleeding on probing; CA, clinical attachment, CG, control group; PD, probing depth; PI, plaque index; TG1, test group 1; TG2, test group 2.
Significant differences between time points and groups were analyzed using Generalized Estimating Equations (GEE) after adjusting for baseline. Significant differences between t0 e t2 (Δ) were assessed using ANCOVA. Different lowercase letters represent differences over time, and different uppercase letters represent differences between groups (p<0.05).
The number and percentage of patients who achieved or did not achieve the clinical endpoint for treatment (≤4 pockets with PD ≥5mm)29,33 was compared between groups. Four patients (16%) in CG, 10 patients (40%) in TG1 and 9 patients (36%) in TG2 achieved the clinical endpoint for periodontal treatment. Statistically significant differences were detected for TG1 (p<0.00) and TG2 when compared to CG (p<0.00) (Table 3).
Table 3 –
Number and percentage of patients who achieve or did not achieve the clinical endpoint for treatment (≤4 sites with PD ≥5mm) (Feres et al., 2012; Borges et al., 2017) and number of sites with PD ≥5mm (mean±SD).
| Treatment groups | |||||
|---|---|---|---|---|---|
| Parameters | Time points | CG (n=25) | TG1 (n=25) | TG2 (n=25) | p value |
| Yes | t2 | 4 (16%)A | 10 (40%)B | 9 (36%)B | 0.00 |
| No | t2 | 21 (84%) | 15 (60%) | 16 (64%) | |
| Sites with PD ≥ 5mm | t0 | 22.5 ± 9.2aA | 20.2 ± 12.2aA | 20.3 ± 18.7aA | > 0.05 |
| t1 | 11.4 ± 8.5bA | 9.4 ± 8.6bA | 19.1 ± 17.2aA | > 0.05 | |
| t2 | 11.1 ± 10.0bA | 9.1 ± 7.8bA | 9.6 ± 9.9bA | > 0.05 | |
| Δt0-t2 | 10.6 ± 1.2A | 11.6 ± 1.2A | 11.0 ± 1.2A | > 0.05 | |
CG, control group; PD, probing depth; TG1, test group 1; TG2, test group 2.
Significant differences between groups for the parameter “achieved the clinical endpoint” was assessed using Chi-square test; p < 0.05. Significant differences between time points and groups for the parameter “sites with PD ≥ 5mm” was assessed using Generalized Estimating Equations (GEE) after adjusting for baseline. Significant differences between t0 e t2 (Δ) were assessed using ANCOVA. Different lowercase letters represent differences over time, and different uppercase letters represent differences between groups (p<0.05).
Glycemia
Figure 2 represents HbA1c levels for all study groups. Intragroup comparisons were performed to verify if there were statistically significant changes in HbA1c levels after periodontal treatment. For CG, mean HbA1c was 8.16% at t0 and 8.10% at t2 (p>0.05). For TG1, mean HbA1c was 8.14% at t0 and 7.63% at t2, with statistically significant differences (p=0.038). For TG2, mean HbA1c was 8.08% at t0 and 7.97% at t2 (p>0.05).
FIGURE 2.
Glycated hemoglobin (HbA1c) levels at baseline (t0) and 6 months (t2). * Intragroup statistically significant difference -- General Lineal Model; p < 0.05.
Immunological parameters
Samples were collected from two periodontal pockets with PD ≥ 5mm at t0, t1, and t2 and analyzed by Multiplex ELISA assay. Cytokine concentration levels were compared over time and between groups (Figure 3). Intragroup comparisons for the concentration levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 were performed. At t1, IL-1β showed significant changes for TG1. At t2, all groups presented statistically significant changes for IL-1β levels. TNF-α levels did not present significant differences over time for any of the study groups. Concentration levels of IL-6 presented reductions at t1 and t2 for TG1. No significant differences were detected for CG and TG2. For the analysis of chemokines, we observed reduction in the concentration levels of IL-8 at t1 and t2 for TG1, and at t2 for TG2, with no significant differences for CG. Concentration levels of MIP-1α presented reduction at t2 for TG2, and did not present significant differences for CG and TG1. No statistically significant differences were detected for concentration levels of MCP-1 for all the study groups. When we analyzed cytokines from the Th family, we observed a reduction in concentration levels of IFN-γ at t1 for TG1, and at t2 for TG2. Statistically significant differences were not detected for CG. Anti-inflammatory cytokines IL-4 and IL-10 did not present changes for intra- and intergroup comparisons.
FIGURE 3.
Concentration levels of cytokines in gingival crevicular fluid. * Intragroup statistically significant differences -- Friedman test; p < 0.05.
Correlation Analysis
We analyzed correlations between cytokine levels at t2 and the periodontal parameters ΔPD and ΔCA from the sites where GCF samples were collected. Whereas both test groups presented significant negative correlations among cytokines and clinical parameters, CG presented a different pattern of significant positive correlations among these variables (see Supplementary Figure 1 in online Journal of Periodontology).
DISCUSSION
Host modulation therapy with oral administration of ω-3 PUFA and ASA promoted clinical and immunological benefits for the treatment of periodontitis in patients with type 2 diabetes. Among the patients who took ω-3 PUFA and ASA, 40% from TG1 and 36% from TG2 achieved the clinical endpoint for treatment, whereas 16% achieved this result in CG. TG1 presented CA gain in moderate and deep pockets when compared to the other groups. When compared to CG, TG1 presented significant difference for ΔPI, and TG2 presented significant difference for ΔBoP.
The clinical benefits of the adjunct use of ω-3 and ASA have been observed in previous randomized studies. El-Sharkawy et al. performed a study in which patients with severe chronic periodontitis were treated with scaling and root planing alone or with ω-3 and ASA (n=40/group) for 6 months. The test group presented greater PD reduction and CA gain than the control group21. Elwakeel and Hazaa applied a similar protocol for patients with chronic periodontitis and type 2 diabetes (n=20/group) and observed favorable preliminary results for the test group34. However, in a study in which a lower dose of ω-3 PUFA was used, without ASA administration, no statistically significant differences were observed for clinical variables when test and control groups were compared (n=15/group)35. Although we applied a reduced supplementation/medication protocol (2 months), we observed significant differences favoring test groups.
We proposed a new protocol for the use of ω-3 and ASA, with changes in the time of administration. After initial dental therapy, patients from TG2 received ω-3 and ASA for 2 months before periodontal debridement. This protocol aimed to assess clinical and immunological variables in the use of ω-3 PUFA and ASA alone, with no subgingival instrumentation. The secondary objective of this modality was to compare results of the 3 groups at 6 months. When we compared clinical parameters among groups at t1, there was a statistically significant difference in mean PD, mean CAL, and BoP. These findings suggest that subgingival biofilm removal was critical for clinical changes related to periodontal inflammation in the short-term. However, when the groups were compared at 6 months after periodontal treatment, TG2 presented significant reduction in mean CAL over time and a greater reduction in BoP (ΔBop) than CG and TG1. We did not detect significant differences in cytokine levels between t0 and t1 for TG2. However, after 6 months of follow-up TG2 presented similar results to TG1 for IL-1β and IL-8, and presented reduction for IFN-γ and MIP-1α. These results suggest that the use of ω-3 PUFA and ASA alone did not promote clinical and immunological changes in the periodontium. However, once subgingival biofilm was removed, additional benefits of the host modulation therapy were significant.
Hemoglobin A1c (HbA1c) is a gold-standard parameter for glycemic control and clinical management of patients with diabetes in medicine, as it represents the serum glucose levels during the 120-day life of red blood cells. Whilst chronic inflammation impairs glycemic control, high levels of HbA1c negatively affect systemic inflammation, as observed in diabetic individuals with periodontitis36. Evidence suggests that periodontal therapy has an impact of −0.36% change in HbA1c levels, that is comparable to the effect of adjunct medication to metformin for metabolic control in patients with diabetes37. Although favorable results were observed in this systematic review, the authors reported that there was limited confidence in this conclusion due to high risk of bias, small sample sizes and heterogeneity in periodontal treatment in the evaluated investigations. In our study we observed statistically significant reduction of −0.51% in mean HbA1c levels (from 8.14 to 7.63%) between t0 to t2 for TG1, while CG presented a reduction of −0.06% and TG2, −0.11%. This result suggests that the combination of periodontal debridement followed by ω-3 and ASA might improve glycemic control and contribute to the management of type 2 diabetes.
The present investigation differs from previous studies with regard to the severity of periodontal disease. In our study, most patients were diagnosed with Stage III periodontitis at baseline, whereas in previous studies patients presented a more advanced stage of the disease. This characteristic is relevant since little clinical improvement was observed in the CG. Although all groups presented intragroup reduction in mean PD, periodontal debridement alone was not effective in reducing mean CAL over time for CG. In addition, this approach was less effective than adjunctive ω-3 PUFA and ASA for the percentage of patients who achieved the clinical endpoint for treatment. These findings suggest that periodontal treatment in patients with Stage III periodontitis and diabetes might represent a clinical challenge and that new therapies should be investigated for patients with these characteristics.
The concentration levels of the pro-inflammatory cytokine IL-1β significantly decreased over time for all 3 groups, with TG1 presenting early (t1) changes. From these results, we observe that periodontal debridement alone is as effective as adjunct ω-3 PUFA and ASA therapy in reducing IL-1β levels after 6 months. This finding agrees with one meta-analysis concerning GCF cytokine levels in periodontitis, where the authors concluded that non-surgical therapy reduced IL-1β concentration levels in normoglycemic patients38, and one previous clinical study patients with diabetes and periodontitis, that reported significant reductions of IL-1β in GCF over time39. Differently from IL-1β, TNF-α, another pro-inflammatory cytokine, did not present significant changes for any of the evaluated therapies. The limited number of studies evaluating levels of TNF-α, especially in diabetes, precludes a consistent conclusion about the effect of non-surgical therapy on concentration levels of this cytokine38. One of our main findings was the significant reduction of IL-6 levels in TG1 after 3 and 6 months of therapy when compared to baseline. IL-6 is an important pro-inflammatory marker that plays a central role in host defense mechanisms due to responses against infection, inflammation, and tissue injury, and high plasma concentration levels of IL-6 is a risk marker for diabetes and its complications40.
Correlation analysis demonstrated that, in general, cytokine levels inversely correlated with periodontal parameters when adjunctive ω-3 PUFA and ASA therapy was used, as opposed to the positive correlations detected in the placebo group. These results suggest that the immunomodulation promoted by ω-3 and ASA had an impact on reducing concentration levels of cytokines in GCF, which resulted in ΔPD and ΔCA in periodontal pockets. To the best of our knowledge, this is the first study to correlate clinical and GCF cytokine levels after different periodontal therapies in patients with type 2 diabetes.
The main limitation of this study is lack of systemic evaluations, such as serum inflammatory markers, that may be relevant secondary variables to better elucidate the systemic effects of ω-3 PUFA and ASA in patients with periodontitis and diabetes. The main strength of the present study is the evaluation different times of administration of adjunctive ω-3 PUFA and ASA. Comparing 3 different groups demonstrated the importance of periodontal debridement in reducing mean PD over time, and its limitation in providing significant changes in mean CAL when used alone.
The results of this randomized clinical study suggest that daily supplementation with ω-3 PUFA and low-dose ASA for 2 months as an adjunct to periodontal debridement promotes clinical and immunological benefits for patients with type 2 diabetes, especially when the supplementation/protocol starts after periodontal debridement.
Supplementary Material
ACKNOWLEDGMENTS
The authors would like to thank Ms. Danielle Stephens for excellent technical assistance. This study was funded by São Paulo Research Foundation (Fapesp) (Grant 2016/02234-7 and Grant 2017/21136-9) and the National Institute of Dental and Craniofacial Research (NIH) (USPHS Grant DE025020).
Footnotes
Cavitron Select, Dentsply, York, PA, USA
Gracey curettes, Hu-Friedy, Chicago, IL, USA
Catarinense Pharma, Joinville, SC, Brazil
Bayer, São Paulo, SP, Brazil
University of North Carolina Probe PCPUNC-BR 15, Hu-Friedy, Chicago, IL, USA
Oraflow, New York, NY, USA
Bio-Rad Laboratories Inc., Hercules, CA, USA
Publisher's Disclaimer: This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record.
CONFLICTS OF INTEREST
The authors declare no conflict of interest.
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