Evaluation of dietary supplementation of omega-3 polyunsaturated fatty acids as an adjunct to scaling and root planing on salivary interleukin-1β levels in patients with chronic periodontitis: A clinico-immunological study.

CONTEXT: Host modulation with omega-3 polyunsaturated fatty acids (ω-3 PUFAs) has anti-inflammatory and pro-resolution actions through replacing and inhibiting metabolism of arachidonic acid (AA). AIM: This study was aimed at evaluating clinical parameters and salivary levels of interleukin-1 beta (IL-1β) after scaling and root planing (SRP) in chronic periodontitis patients, with and without dietary supplementation of ω-3 PUFAs. SETTINGS AND
DESIGN: Forty otherwise healthy participants with severe chronic periodontitis were included. Full-mouth periodontal examination and scaling and root planing were done for test and control group (20 participants each).
MATERIALS AND METHODS: The control group received SRP alone while test group received daily dose of 700 mg ω-3 PUFA capsules for 3 months. All clinical parameters were checked at baseline, 1 month, and 3 months' duration, whereas levels of IL-1β were checked in saliva at baseline and 3 months after therapy. STATISTICAL ANALYSIS USED: T-test and repeated measure analysis of variance were used with Statistical Package for the Social Science Windows software.
RESULTS: Improvements in all clinical outcomes were similar in both groups over 3 months. Improvements in clinical parameters were not statistically significant on intergroup comparison, except for improvement in Bleeding Index and reduction in levels of IL-1β, which were statistically significant in test group as compared to control group (P < 0.05).
CONCLUSION: Even though adjunct therapy with ω-3 PUFAs can modulate cytokine levels and show proresolution properties, its importance on clinical outcomes may be controversial. Thus, this may be used as an adjunctive management of chronic periodontitis.

INTRODUCTION

Periodontal diseases are associated with specific pathogenic bacteria present within plaque biofilm, these bacteria cause extended inflammation which leads to a chronic condition.[1] Estimated 600 different bacteria can colonize in human mouth, with any person typically harboring 150–200 of these species. Virulence factors from these pathogenic species enable the bacteria to colonize and cause damage to the periodontal tissue by producing potent substances that trigger the host inflammatory response.[2] In addition to huge number of pathogenic bacterial species, recolonization after any type of oral hygiene intervention is also very rapid and thorough, making treatments such as scaling and root planning (SRP) insufficient.[3] Periodontal disease is not universally expressed in everyone with poor oral hygiene but manifests in a susceptible host.[4] In susceptible hosts, periodontal inflammation fails to resolve and chronic inflammation becomes periodontal pathology with systemic impact. Studies have also revealed that most tissue damage is caused by host response to infection and not by the infectious agent directly.[56]

The rationale of host modulation therapy is to maintain the equilibrium between destructive mediators and anti-inflammatory or protective mediators. A variety of drugs have been evaluated as host modulating agents including the nonsteroidal anti-inflammatory drugs, bisphosphonates, tetracyclines, enamel matrix proteins, growth factors, and bone morphogenic proteins.[7] One such agent for host modulation is omega-3 polyunsaturated fatty acids (ω-3 PUFAs).

Inflammed gingival tissue synthesizes larger amounts of prostaglandins compared to healthy gingiva.[8] Host modulation by ω-3 PUFAs is by reduction in arachidonic acid (AA)-derived eicosanoids and inflammatory cytokines and also by making the local environment more favorable to resolution of inflammation.[9]

ω-3 PUFAs act both directly, by replacing AA as an eicosanoid substrate and inhibiting AA metabolism and indirectly by altering the expression of inflammatory genes through effects on transcription factor activation. This action happens through a novel series of lipid mediators, which are resolvins, lipoxins, and protectins. Resolvins and protectins reduce neutrophil infiltration and increase nonphlogistic recruitment of monocytes and help in resolution of inflammation.[567]

Periodontitis is one of the major reasons for tooth loss in adults in India. Data from National Oral Health Survey, 2002–2003, states that the prevalence of chronic periodontitis in the 35–44-year age group was 89.6%, and in 64–75 years, it was 79.9%.[10] Since it is a huge public health concern, measures to overcome this are being explored extensively. Supplementation of ω-3 PUFAs can be a promising adjunct to conventional periodontal therapy to combat this problem. Thus, the aim of this study was to evaluate clinical parameters and immunological parameter like IL-1β levels from saliva, with or without dietary supplementation of ω-3 PUFAs as an adjunct to SRP in the treatment of chronic periodontitis and to compare these parameters before and after treatment.

MATERIALS AND METHODS

By purposive sampling, 50 patients were selected from our institution. They were prescreened from May 2014 to January 2015. Out of which, 4 were not eligible and 6 were not interested to participate in the study [Figure 1].

Figure 1

Study design. SRP – Scaling and root planing, IL-1b – Interleukin-1 beta

The inclusion criteria for selection of study participants were participants with severe form of chronic generalized periodontitis (which is, ≥30% of the sites showing ≥5 mm of clinical attachment loss).[11] Patients having at least 20 teeth and nonsmokers. Pregnant/lactating women, participants on any medication, participants who have undergone any periodontal treatment in the past 6 months, and participants requiring any emergency dental treatment were excluded from the study. The formula to calculate the sample size was N= (Z2 × p × [1-p])/d2, where N is sample size, Z is levels of confidence set at 1.96, p is prevalence which is 0.975, and d is precision which is 0.05. This made twenty participants per group, hence making it a total of forty participants, after adjusting for all inclusion and exclusion criteria for this study. The duration of follow-up was 3 months. The institutional ethical committee approved this trial and its protocols. Written informed consent was obtained from all individuals approved by the Institutional review board.

Participants were divided into two groups (20 patients each) in a parallel, examiner-masked design by a computer-generated list. Control group was assigned to receive only SRP, whereas test group received dietary supplementation with 0.960 g capsules (Seacod Active, Sanofi [Sanofi House, CTS No. 117-B, L and T Business Park, Saki Vihar Road, Powai, Mumbai - 400 072, Maharashtra, India]) daily following SRP. Each capsule contained net 700 mg fish oil (eicosapentaenoic acid [EPA] – 180 mg/docosahexaenoic acid [DHA] – 120 mg); these were given once a day for 3 months. Standardized oral hygiene instructions were given for supragingival plaque control to all participants.

The clinical parameters recorded at baseline, 1, and 3 months were plaque index by Silness and Loe,[12] gingival index by Loe and Silness,[13] gingival bleeding index by Ainamo and Bay,[14] and probing pocket depth and clinical attachment level (CAL) by Ramjford method [Figure 1].[15]

Whole unstimulated saliva samples were collected by expectoration in polypropylene tubes and then stored at −20°C. Levels of interleukin-1 beta (IL-1β) were determined in saliva at baseline and 3 months, using enzyme-linked immunosorbent assay (by Ray Bio [RayBiotech, Inc. 3607 Parkway Lane, Suite 100, Norcross GA 30092, USA]).

The recorded data were then subjected to descriptive statistics, paired samples and independent sample t-test, and repeated measure analysis of variance. The results were analysed using Statistical Package of Social Sciences (IBM corp. Released 2011 IBM SPSS statistics for windows, version 20.0. Armonk, NY, USA).

RESULTS

Out of 20 patients in control group, 13 were male and 7 were female. Test group comprised of 20 participants, out of which 12 were male and 8 were female. The mean age in control group was 43.5 ± 5.8 years, and in test group, it was 44 ± 6.44 years, hence showing similar age and gender distribution in both groups. Thirteen patients had vegetarian diet and rest were nonvegetarians. Most participants with nonvegetarian diet did not consume fish or fish products on a daily basis.

All the clinical parameters such as plaque index, gingival index, bleeding index, probing pocket depth, and CAL were checked at baseline, 1 and 3 months in this study. There was no significant difference at baseline in the clinical parameters between the groups. Plaque index, gingival index, probing depth, and CAL did not show significant improvement on intergroup comparison. The improvement in BI was statistically significant in test group at 3 months, when compared to control group with a P = 0.001 [Table 1].

Table 1

Results for the effect of treatment on clinical parameters and salivary levels of interleukin-1β over time

The reduction in IL-1β was statistically significant in both groups over 3 months and in test group at 3 months, when compared to control group with a P = 0.001 [Table 1]. None of the patients had reported any change in general health during the study.

DISCUSSION

Treatments for chronic periodontitis are unmaintainable as it has multifactorial etiology and because the bacteria can recolonize rapidly and completely. Thus, this issue can be addressed by adjunctive therapy along with conventional periodontal therapy to some extent in the maintenance of treatment outcomes. The present study was conducted to evaluate adjunctive therapy with dietary supplementation of ω-3 PUFA in patients with generalized chronic periodontitis after SRP, and to compare this with the group that received only SRP. This was a randomized controlled, examiner-masked, interventional study conducted over 3 months' duration.

On evaluation of clinical outcomes in the study, we found that improvement in Bleeding Index was statistically significant on intergroup comparison. Overall, clinical analysis on intergroup comparison was not significant. At the end of 3 months, correlation with none of the clinical parameters was found to be significant with the levels of IL-1β [Table 1].

This is partly in accordance with study done by Martinez et al. They treated 15 patients with nonsurgical periodontal therapy along with 900 mg/day ω-3 PUFAs in divided doses for one year. They reported decrease in AA to EPA ratio. They also reported no effect on clinical outcomes.[16] Similarly, Rosenstein et al. and Campan et al. also did not report significant improvement in clinical outcomes.[1718] In contrast to this, in a study conducted by Deore et al., 60 otherwise healthy participants were treated with SRP; test group was given ω-3 PUFAs supplementation 300 mg/day for 3 months. Levels of CRP were checked in serum in both groups. They found statistically significant improvement in clinical parameters, but there was no change in levels of CRP between the test and control groups.[19]

There are two earlier studies by Elkhouli and Sharkawy et al.; reporting improvement in clinical outcomes, but both studies combined the ω-3 PUFA with low doses of aspirin. El-Sharkawy et al. found reduction in inflammatory biomarkers such as RANKL and MMP-8 in saliva.[20] Elkhouli reported reduction in inflammatory biomarkers IL-1β and IL-10 in gingival crevicular fluid.[21] The main difference between the present study and studies that showed a positive effect of ω-3 PUFAs is the low dose of aspirin. When ω-3 PUFA supplements are taken with aspirin, aspirin-triggered lipoxin, and resolvins are produced. These compounds are more potent and they are not only anti-inflammatory but also drive the pathway of inflammation toward resolution.[22] However, host modulation therapy with ω-3 PUFA and low-dose aspirin will change the direction of this study from dietary supplementation to a more complex situation including medication. This combination of drugs was convoluted to justify for otherwise healthy individuals, as even low-dose aspirin, over long time can hold risk of undue bleeding episodes. Furthermore, it is a topic of dispute, whether to give aspirin to participants who are not at risk of cardiovascular diseases or where any such indication is not present. Thus, low-dose aspirin was not given in the present study.

Varying doses of ω-3 PUFA supplementation have been used in other clinical trials, among which Martinez et al have used 3 capsules of 300 mg each per day for 12 months. Each capsule contained EPA 180 mg and DHA 120 mg.[16] Deore et al. had used one capsule/day with the same composition, being 300 mg of ω-3 PUFA with EPA 180 mg and DHA 120 mg for 3 months.[19]

Elkhouli used three capsules of 1 g of ω-3 PUFAs containing 300 mg of DHA and 150 mg of EPA with 75 mg of aspirin for 6 months. Whereas, Sharkawy et al. used 3 g fish oil and 81 mg aspirin daily. Each capsule contained 900 mg of fish oil (EPA/DHA 30%) and 100 mg wheat-germ oil for 6 months.[21] The American Heart Association has suggested that a dose of 0.5–1.8 g/day of EPA + DHA is generally regarded as safe in healthy people.[23] Hence, dosage of 700 mg/day was taken in this study which was well within the range of safety dose.

Duration of study was 3 months as long-term therapy with ω-3 PUFAs can alter platelet function and pose risk of undue bleeding, which was reported in a study by Thorngren as early as 11 weeks.[24] Furthermore, the incorporation half-lives of EPA into the human body are about 5 days in serum. Incorporation of DHA was dose-dependent as values were higher for every g/day ingested.[25]

Saliva was preferred to evaluate levels of IL-1β as the dietary supplements were given orally and will act systemically. Furthermore, saliva is present for sample collection in sufficient amount and collection procedure is relatively less technique sensitive. Furthermore, it is found that constituents of GCF ultimately merge with saliva.[26] In addition, as whole saliva represents a pooled sample from all periodontal sites, the study of biomarkers in saliva may deliver overall valuation rather than site-specific GCF analysis.[27] In this study, ω-PUFAs acts through systemic circulation and do not possess any local or topical action; therefore, saliva was preferred over GCF.

Of all the ILs studied to date in crevicular fluid and/or the saliva of patients with gingivitis, IL-1β has been the most analyzed and that most authors agree that it is higher in gingivitis and experimental gingivitis. Salivary IL-1β levels and several periodontal pathogens were found to be linked with periodontitis to the same extent.[2628] Therefore, IL-1β was used as a diagnostic marker of inflammation in this study to gauge anti-inflammatory and pro-resolution effects of ω-3 PUFA supplementation. We found that levels of IL-1β were high at baseline in both groups and after SRP their levels were reduced. The reduction in test group was statistically significant when compared to control group at 3 months' interval. Our results were in accordance with Ulker et al. and Engebretson et al.[2930] Based on these findings, we can infer that reduction in markers of inflammation like IL-1β may be attributed to effects of ω-3 PUFA supplementation.

The strength of this study is similar age and gender distribution and all participants being nonfish eaters. In addition, low-dose aspirin was not given in this study, as it can have similar anti-inflammatory action as ω-3 PUFAs and can act as a confounding factor. Furthermore, the study design was randomized, examiner-masked, controlled trial. The strong suit of the study also lies in the fact that all intraoral examinations were performed by a calibrated examiner, and SRP treatment was given by another operator for all the patients. SRP for all patients was done in the same setting with the same ultrasonic device by this operator. Compliance of the patient was checked at all the scaling and maintenance recall visits; plus, oral hygiene instructions were reinforced at all visits. Thus, we can propose that ω-3 PUFA supplementation may reduce inflammation, but its anti-inflammatory and pro-resolution effects were not reflected on the clinical outcomes.

CONCLUSION

ω-3 PUFA dietary supplementation as an adjunct to SRP can reduce gingival inflammation by modulating cytokine profile. Therefore, this may be used as host modulatory agent in the adjunctive management of chronic periodontitis. Further multicentric, controlled clinical trials addressing issues such as safety, risk-benefit analysis, and dosage are required to support these findings.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.