In this study, we demonstrated that SRP with adjunct supplementation with fish oil rich in omega-3 PUFAs for 6 months had a potential to induce a measurable clinical and microbiological outcomes in comparison to SRP alone. In our study, patients were supplemented with higher doses of omega-3 PUFAs (2.6 g of EPA and 1.8 g of DHA) compared to earlier studies [22-27]. The daily dose was calculated based on the studies concerning other chronic inflammatory systemic diseases. Patients with rheumatoid arthritis were supplemented with up to 7.1 g of omega-3 PUFA per day, while patients with Crohn’s disease with 2.7 g [34,35]. Moreover, taking into account Western diet deficiency in omega-3 fatty acids, with excessive amounts of omega-6 fatty acids [36], administration of high doses of omega-3 PUFAs during treatment of periodontitis, a chronic, multifactorial inflammatory disease, seems reasonable.
We revealed beneficial changes in all clinical parameters in both groups at 3 and 6 months compared to that at baseline. The differences between the groups in favor of the test group were statistically significant mainly at 3 months and in the initially deep pockets (PD≥ 6 mm). Our results after 3 months are in accordance with the recent systematic review and meta-analysis [37] assessing the use of omega-3 PUFAs adjunctively to non-surgical treatment of periodontitis. Authors revealed significant overall PD reduction of 0.42 mm and CAL gain of 0.58 mm, and concluded that the use of omega-3 fatty acids during non-surgical treatment of periodontitis can provide additional benefits in CAL gain and PD reduction compared with subgingival instrumentation alone.
At 6 months, only BOP scores were statistically different when all pockets in the test and control groups were considered. This indicates that supplementation with high-dose omega-3 PUFA as adjunct to non-surgical treatment of periodontitis, has detectable but limited clinical effect in the treatment of generalized periodontitis stage III and IV. A deteriorating compliance to the treatment regimen during 6-month period may be a probable explanation that clinical parameters were not significantly better at 6 months in the test group in comparison to the control group. It is well-known that adherence to medical treatment is one of the issues in the treatment of chronic diseases. It was demonstrated that nearly 50% of patients failed to adhere to their medical directives, and that 25% of patients were nonadherent to prevention and disease management activities including medication taking, appointment keeping, screening, exercise, and dietary changes [38,39]. In our study, the factors which could affect adherence include duration of the treatment, the need to take fish oil two times a day at home without any supervision and, paradoxically, decreased frequency of gingival bleeding. Bleeding during toothbrushing is one of the most common symptoms which worries patients with periodontitis, thus, lack of this sign could also decrease patients’ motivation for regular oil intake with time.
According to 2017 World Workshop of European Federation of Periodontology (EFP), BOP is an important clinical parameter related to assessment of periodontitis treatment outcomes and post-treatment residual disease risk [33]. Moreover, Lang et al. concluded that BOP is a useful prognostic indicator in monitoring periodontal tissue after active therapy and sites with no BOP were not prone to clinical attachment loss [40]. More recent studies also demonstrated that the increase in mean BOP was related to disease severity and periodontal instability irrespective of smoking status during supportive therapy [41] , and that nonbleeding sites can be considered as clinically healthy and periodontally stable [42]. However, sites with residual probing depth ≥6 mm after active periodontal therapy were shown to have higher potential for CAL loss [43]. Therefore, lower BOP rates observed at the end of our study and higher rates of closed pockets at 3 and 6 months in initially deep pockets in the test group were a desirable treatment outcome that could provide long-term disease resolution and less need for retreatment.
Our results obtained at 3 months are in accordance with the study of Deore et al., demonstrating that supplementation with omega-3 PUFA during non-surgical periodontal treatment significantly reduced GI, BOP, PD and CAL [23]. However, our cohorts demonstrated a more severe form of periodontitis, as the initial mean PD in the test and control groups was 5.01±0.49 and 5.02±0.71, while in the study of Deore et al., 4.26±1.10 and 4.05±1.03, respectively. At 6 months, others demonstrated similar clinical results as ours. Keskiner et al. showed no statistically significant differences in clinical parameters like PD, CAL, BOP and GI between the test and control groups [22]. However, patients received anecdotally low-dose of omega-3 PUFAs (19.19 mg of DHA and 6.25 mg of EPA twice daily). Similarly, Martinez et al. revealed no significant differences in the clinical parameters between the test and placebo group at 4 and 12 months independently on the initial PD, but again, patients received rather low doses of omega-3 PUFAs (180 mg of EPA and 120 mg of DHA three times a day) [24].
In our study, we also analyzed mean scores of clinical parameters separately for moderate (4-5 mm) and deep (≥ 6 mm) pockets. Inter-group comparison for deep pockets after 3 months showed statistically significant differences between groups for PD, REC and their Δ, and percentage of closed pockets, in favor of the test group. Moreover, assessment after 6 months revealed statistically significant differences between the groups for REC and CAL, and in the number of closed pockets also in favor of the test group. However, inter-group comparison for their Δ baseline–6 months did not reveal statistical differences. When comparing our clinical results with the recent systematic review and meta-analysis on subgingival instrumentation in the treatment of periodontitis [44] , mean PD reduction at 3 and 6 months was better in the FO group, 1.3 mm and 1.5 mm, respectively, in comparison to 1.0 mm and 1.4 mm shown in the meta-analysis at 3-4 or 6-8 months, respectively. Furthermore, considering the extent of disease resolution, as measured in terms of percentage of closed pockets, in the FO group 60 % of pockets were closed at 3 months and 62 % at 6 months in comparison to 57 % at 3-4 months and 74 % at 6-8 months shown in the meta-analysis [44].
Recently, EFP provided evidence-based clinical guidelines for treatment of periodontitis concluding that adjunctive use of systemic antibiotics may be considered for particular patients with generalized periodontitis stage III [2]. When comparing our clinical results with the recent systematic review and meta-analysis of the use of systemic antimicrobials in periodontitis therapy [45], we demonstrated that FO resulted in 0.14 mm of additional improvement of PD at 6 months in comparison to systemic antibiotics that resulted in 0.45 mm of additional improvement at 6 months. Furthermore, the adjunctive use of systemic antimicrobials at 6 months resulted in greater additional improvement of CAL than in the FO group in our study (0.39 mm vs. 0.34 mm), pocket closure (14.5% vs. 5.6%), less percentage of sites with BOP (6.6% vs. 2.4%). Summarizing, omega-3 PUFAs together with SRP provide better clinical results than SRP alone. However, adjunctive use of systemic antimicrobials during non-surgical treatment of periodontitis is much more efficient than supplementation with omega-3 PUFAs. However, because of potential adverse effects of systemic antibiotics [45] and worldwide increasing problem with antimicrobial drug resistance, the use of antibiotic should be limited to specific situations.
In this study, we also addressed an antimicrobial effect of omega-3 PUFAs. Quantitative analysis of crucial periodontal pathogenic bacteria P. gingivalis, T. forsythia, T. denticola and A. actinomycetemcomitans demonstrated significantly lower numbers of these bacteria in the FO group in comparison to the control group. While omega-3 PUFA anti-inflammatory activity is well-described, little is known about their effect on the growth and survival of oral bacteria. Recently, it was demonstrated that omega-3 PUFAs and their ester derivatives exhibited strong in vitro antimicrobial activity against various oral pathogens, such as Candida albicans and periodontopathogens, A. actinomycetemcomitans, F. nucelatum, and P. gingivalis [46,47]. Moreover, Ribeiro-Vidal et al. provided evidence that application of EPA or DHA extracts on in vitro multispecies biofilm model of six bacterial species (S. oralis, A. naeslundii, V. parvula, F. nucleatum, P. gingivalis and A. actinomycetemcomitans) for 60 s resulted in an antimicrobial effect [48]. Contrary to those in vitro studies, in a 3-month pilot randomized controlled trial, 2 g of daily DHA with 81 mg of aspirin did not significantly modify total bacterial plaque growth in the patients that were not subjected to mechanical plaque removal [49]. However, an inhibition of P. gingivalis growth was detected, whereas in the placebo group, an increase in the mean counts of P. gingivalis was observed. Furthermore, among all investigated bacterial complexes, red complex tended to drop to a higher degree than the others [49]. Potential antimicrobial action of omega-3 PUFAs against periodontal pathogens seems to be crucial not only in the context of treatment of periodontitis. P. gingivalis, T. denticola and A. actinomycetemcomitans were shown to be linked to various systemic diseases, including cardiovascular diseases, Alzheimer’s disease, rheumatoid arthritis and systemic lupus erythematosus [50,51]. Thus, decrease of key periodontal bacteria abundance support periodontal treatment and protect from other chronic diseases.
Animal studies provided more evidence for possible role of omega-3 PUFAs in bacterial clearance in periodontitis. It was demonstrated that topical treatment with RvE1 inhibited tissue and bone damage, and resulted in eradication of P. gingivalis [52]. The authors hypothesized that resolution of inflammation effectively eliminates P. gingivalis from the lesion by removing the nutritional source for the bacteria. P. gingivalis as a Gram-negative, obligate anaerobic, asaccharolytic organism is unable to use sugars for growth and metabolizes essential amino acids and hem coming from periodontal tissue breakdown. In another study with experimental periodontitis, marked changes in subgingival microbiota after RvE1 treatment were revealed [53]. The authors also suggested that resolution of local inflammation had a major role in shaping the composition of the subgingival microbiota. In our study at 6 months, the level of key periopathogenes was reduced significantly only in the test group, but microbiological result was in accordance with clinical improvement in the control group, thus inhibition of bacterial growth cannot be contributed to the resolution of inflammation only. Although bacterial clearance was combined with statistically significant CAL improvement in deep pockets ≥ 6mm at 3 and 6 months and with statistically significant PD reduction at 3 months in the FO group, overall clinical improvement in the control group should be accompanied by better reduction of subgingival periopathogens. Therefore, it seems that in our study, omega-3 PUFAs had an activity against periodontal bacteria independently of their anti-inflammatory effects promoting resolution of inflammation and, in a consequence, inhibition of ‘inflammophilic’ bacteria. More studies are needed to investigate in vivo antimicrobial activity of omega-3 PUFAs.
Optimal plaque control is crucial factor of long-term effects of periodontal treatment. In this study, we demonstrated a statistical difference in FMPI between groups at 3 and 6 months. This difference may result from better motivation and involvement in the treatment process of the patients from intervention group, because they received fish oil as an adjunct to SRP. Alternatively, fish oil intake may interfere with supragingival dental plaque formation resulting in lower FMPI in the test group. Nevertheless, we cannot fully exclude that better clinical outcome in the test group may be partially attributed to lower FMPI in comparison to the control group.
In this study, we used fish-derived oil mixture as a source of EPA and DHA. Natural fish oil was chosen to mimic dietary conditions where a range of essential oils are consumed. To address, how fish oil consumption affected serum lipid composition, gas chromatography/mass spectrometry analysis was performed. We found that FO intake resulted in increased proportions of serum n-3 PUFAs and decreased proportions of n-6 PUFAs. This shift leads to increase in the proportion of EPA and DHA in the membranes of inflammatory cells and thus, by competing reduces the level of arachidonic acid [35]. This, in turn, results in decreased synthesis of proinflammatory mediators derived from arachidonic acid which is a major product of omega-6 PUFA metabolism in the body [54,55].
The present study has several limitations. The first one is the initial difference between the test and control groups in terms of age. This was a result of a relatively high dropout rate, mainly due to SARS-CoV-2 pandemic-related lockdown. When the baseline data of all fifty initially enrolled participants were taken into account, no statistical differences between the groups were noted. Even though at baseline the advancement of periodontitis was similar in both groups, the treatment response might be different as periodontal tissue healing ability may deteriorate with age [56]. Another limitation is the use of fish-derived oil mixture instead of isolated EPA and DHA, thus other components of the fish oil may affect the results of study. Specifically, squalene and alkylglycerols present in the fish oil was demonstrated to activate Th1-type IL-12 and IFN-γ cytokine production and increase antioxidant activity of serum [57,58]. It is also worth mentioning that the amounts of vitamin A and D consumed with fish oil in the test group were 3-20 times below recommended daily doses. Since fish oil was chosen as a source of EPA and DHA in our study, no placebo was employed and patients were aware of treatment allocation. From our experience, even when fish oil is taken in capsules, its taste cannot be masked because of characteristic fish burps.