The Role of Different Types of Cannabinoids in Periodontal Disease: An Integrative Review

This integrative review addresses the potential of the Endocannabinoid System (ES) and cannabinoids in the pathogenesis and treatment of periodontal disease (PD). Cannabinoid receptors are expressed in healthy and inflamed periodontal tissues, indicating a potential regulatory role for SEC in oral homeostasis. Healthy periodontal cells express more CB1 receptors, while inflamed sites show increased CB2 receptors. This suggests a dynamic involvement of the SEC in the inflammatory response associated with PD. Cannabinoids such as cannabidiol (CBD) and cannabinoid receptor agonists such as HU-308, anandamide (AEA), and methanamide (Meta-AEA) have demonstrated promising therapeutic potential in studies. CBD has been associated with the control of bone resorption, antibacterial activity, and increased production of gingival fibroblasts, indicating effects in mitigating the progression of PD. HU-308 demonstrated preventive effects against alveolar bone loss, and anti-inflammatory, osteoprotective, and pro-homeostatic properties in animal models of periodontitis. AEA and Meta-AEA have anti-inflammatory effects by reducing pro-inflammatory mediators such as IL-1, IL-6, and TNF-α. The activation of cannabinoid receptors attenuates inflammatory processes, inhibits alveolar bone loss, exerts antibacterial effects, and promotes tissue repair. However, clinical trials are especially needed to validate these results and explore the therapeutic potential of cannabinoids in the treatment of PD in humans.


Introduction
Periodontal disease (PD) is a chronic inflammatory condition that compromises the teeth's supporting tissues.This occurs primarily through the immunoinflammatory responses that are triggered by interactions between pathogenic bacteria and host-derived mediators, and contribute to the exacerbation of inflammation and subsequent tissue destruction [1,2].Some biological systems, i.e., the Endocannabinoid System (ECS), are potential candidates for controlling PD through the modulation of conditions such as pain, inflammation, antimicrobial properties, and tissue repair.This system is a complex signaling network that plays a crucial role in regulating various physiological processes, including immune response, pain modulation, and bone metabolism [3,4].It is composed of endogenous cannabinoids (endocannabinoids), cannabinoid receptors, and enzymes responsible for the synthesis and degradation of endocannabinoids [5].They have roles in the cellular activities of osteoblasts, osteocytes, osteoclasts, and gingival fibroblasts which are important for oral homeostasis, as they assist in tissue remodeling [6].In addition, this system, in isolation or when associated with different types of cannabinoids, has also been purported to hold therapeutic potential for periodontal diseases [6,7].

Eligibility Criteria
Due to the diversity among the studies, an adaptation of the PICOS strategy was employed to consider the eligibility of studies using P, I, and O: P: Population (bacteria, cells, and other structures inherent in diseased and/or healthy periodontal tissue-from humans, mice, or rabbits) I: Intervention (natural, endogenous, and/or synthetic cannabinoids) O: Outcome (anti-inflammatory, antibacterial, or repair-related effects and prevention of soft and hard tissue damage).

Inclusion Criteria
Only in vitro and in vivo studies that evaluated the role of the ECS in tissues, cells, or microorganisms inherent to the periodontium were considered.Restrictions on language, publication time, and geographical scope were not employed.

Exclusion Criteria
Descriptive studies, such as literature reviews, case reports, clinical trials, projects/ protocols, opinion articles, letters, posters, and conference abstracts were excluded.

Data Extraction and Analysis
For the selection of studies, the Rayyan Qatar Computing Research Institute (QCRI) application was used in two phases.In phase one, duplicate articles were identified, and titles and abstracts were independently screened to exclude studies irrelevant to this review.In phase two, the texts were fully read, applying the previously established inclusion and exclusion criteria.The data collected were as follows: (1) Author and year of publication; (2) Type of study; (3) Intervention; (4) Objective; (5) Cannabinoid dose/administration; (6) Results (main outcomes of the articles related to anti-inflammatory, antibacterial, and tissue repair response); (7) Conclusion.

Selection of Studies
After an electronic search in five databases (PubMed, Web of Science, Scopus, Embase, and Lilacs), 270 articles were retrieved, and after the removal of duplicates, 196 remained for us to read the title and abstract.Of these, 21 were included based on the eligibility criteria (Figure 1).

Characteristics of the Studies
The included studies were published between 2005 and 2022.The majority, eleven (52%), had been published in the last 5 years; three (15%) in the last 10 years, and seven (33%) more than 10 years ago.Regarding the study design, fifteen (71%) articles presented in vitro methodologies; four (19%) in vivo, and two (10%) had both methodologies (Table 1).
After an electronic search in five databases (PubMed, Web of Science, Scopus, Embase, and Lilacs), 270 articles were retrieved, and after the removal of duplicates, 196 remained for us to read the title and abstract.Of these, 21 were included based on the eligibility criteria (Figure 1).

Characteristics of the Studies
The included studies were published between 2005 and 2022.The majority, eleven (52%), had been published in the last 5 years; three (15%) in the last 10 years, and seven (33%) more than 10 years ago.Regarding the study design, fifteen (71%) articles presented in vitro methodologies; four (19%) in vivo, and two (10%) had both methodologies (Table 1).

Prevention of Bone Resorption by Cannabinoids
Among the six studies that evaluated the prevention of bone resorption, four used synthetic cannabinoids, including HU-308 [3,13,30] and Meta-AEA [6].HU-308, employed in in vivo studies, significantly reduced alveolar bone loss in animals subjected in parallel to bacterial LPS (4.52 ± 0.19 mm) stimulation, compared with animals receiving LPS alone (5.22 ± 0.14 mm) [13], and similar results were found for Meta-AEA [6].The only study that investigated the role of a phytocannabinoid concluded that rats subjected to experimental periodontitis and systemic CBD injection showed less bone loss in the furcation region (0.5 mm 2 ), compared with those treated with saline (0.9 mm 2 ) [22].

Tissue Repair (Cell Proliferation/Viability) by Cannabinoids
Those studies that analyzed tissue repair capacity (as assessed by cell proliferation or molecule expression) mainly investigated the effects of the endocannabinoid AEA [15,19,27].In human periodontal ligament cells not exposed to bacterial LPS, anandamide had no significant effect on the proliferation/viability of these cells, while in those submitted to LPS, AEA (10-20 µM) induced a significant increase in proliferation/viability [23].In addition, the phytocannabinoid, CBD, at low concentrations (0.01-0.05 µM), increased (by 40%) the production of transforming growth factor β (TGF-β), an important cytokine that controls cell proliferation and differentiation [28].This relationship between receptors and periodontal tissue is illustrated in Figure 4. ) in healthy and diseased periodontal tissues.In healthy periodontal tissues, CB1 receptor expression predominates, while inflamed tissues exhibit a higher tendency for CB2 receptor expression.This shift in receptor expression suggests a potential regulatory role of the Endocannabinoid System in the pathogenesis and progression of periodontal disease.

Antibacterial Effects of Cannabinoids
With regard to the antibacterial activities of cannabinoids, only the phytocannabinoids have been studied, especially CBD, CBC, CBN, and THC [21,25].In addition, one ) in healthy and diseased periodontal tissues.In healthy periodontal tissues, CB1 receptor expression predominates, while inflamed tissues exhibit a higher tendency for CB2 receptor expression.This shift in receptor expression suggests a potential regulatory role of the Endocannabinoid System in the pathogenesis and progression of periodontal disease.

Antibacterial Effects of Cannabinoids
With regard to the antibacterial activities of cannabinoids, only the phytocannabinoids have been studied, especially CBD, CBC, CBN, and THC [21,25].In addition, one study investigated the effect of CBD and CBG when infused in mouthwashes [25].Exposure of the oral biofilm to the phytocannabinoid, CBD (12.5%), resulted in lower bacterial colony counts in samples taken from patients with gingivitis (mean colony count (MCC) = 5), calculusassociated gingivitis (MCC = 4.9), and from patients with severe PD (MCC = 1.5); these values differed from those of samples from patients with severe periodontitis submitted to Oral B (MCC = 29.8).Meanwhile, CBD (12.5%) administration, especially in patients with periodontitis (CMC = 3.1), also lowered bacterial growth averages, compared with those presented by groups treated with Oral B (CMC = 27.3) or Colgate (CMC = 27.7)[25].The direct effects of CBD, CBN, and THC on P. gingivalis were also investigated.CBD, at concentrations of 5.0 and 10 µg/mL, significantly inhibited bacterial logarithmic growth up to 38 h after exposure, and similar results were observed for CBN and THC [21].On the other hand, CBD and CBG, when infused in mouthwashes, showed a similar inhibition of bacterial growth to that of 0.2% chlorhexidine, and greater inhibition compared with the inhibition resulting from alcohol-based rinses with essential oils such as thymol, eucalyptol, and menthol (Product A) or fluoride-and potassium nitrate-based products (Product B) [26].

Expression of Receptors (CB1 and CB2)
The expressions of CB1 and CB2 receptors were analyzed to assess the role of the Endocannabinoid System in the pathogenesis of periodontal disease in five studies [21,25].Compared with CB1, there was a greater tendency towards CB2 expression in healthy [17], inflammatory [16,27,32,33], and healing conditions [31].Additionally, human gingival fibroblasts exposed to P. gingivalis LPS showed a marked expression of CB1 and CB2 messenger RNAs [32].However, in healthy cells and tissues, CB1 levels were higher compared with CB2 [31,32] although the expression of this receptor was lower or absent in some studies [17,27].
The materials and data for this study are openly available on the Open Science Framework (OSF): https://doi.org/10.17605/OSF.IO/GFDZ2.
The studies summaries are included in Table 2. To evaluate the effect of treatment with the synthetic cannabinoid, Meta-AEA, on the progression of periodontitis in rats, with regard to anti-inflammatory activity and prevention of alveolar bone loss.
In vitro: gingiva from healthy rats were dissected and distributed into the 4 study groups In vivo: rats were divided and daily topical application of serum or Meta-AEA was performed at the LPS injection sites, during the 6 weeks of each experiment.
The use of Meta-AEA, in vivo and in vitro, reduced the levels of proinflammatory cytokines, especially TNF-α.
In vivo use significantly decreased alveolar bone loss and the amount of iNOS.CLPH were cultured in DMEM (in the presence, or absence, of Meta-AEA) and some groups were also subjected to P. gingivalis LPS exposure.

Anti-/Pro-inflammatory effect:
There was no significant effect of Meta-AEA (up to 10 µM) on gene expression levels and IL-6, IL-8, and PQM-1 protein production in healthy cells.In contrast, in CLPH exposed to bacterial LPS, gene expression and the protein production of IL-6, IL-8, and PQM-1 decreased significantly (p < 0.05) with 10 µM Meta-AEA.

Cell proliferation/viability:
Meta-AEA (up to 10 µM) exerted no significant effect, but significantly inhibited at a concentration of 30 µM, compared with the control.
Inflammatory response in CLPH may be influenced by ECS activation, since at certain concentrations, Meta-AEA may contribute to decrease interleukins.

Antibacterial effect:
The highest CMC occurred in the Oral B (29.8) and Colgate (30.6) treatments vs. CBG (3.5) and CBC (3.8), in the periodontitis patient samples.Overall, the CMC in the cannabinoid treatments was significantly lower than that recorded in any of the toothpastes tested.
Cannabinoids have the potential to be used as an effective antibacterial agent against bacteria associated with dental biofilm.To evaluate the bactericidal activity of cannabinoid-infused mouthwashes against dental biofilm bacteria from patients with gingivitis and periodontitis.
An aliquot of biofilm collected from patients with PD was spread on plates containing wells or disk diffusion with rinses.After incubation, the diameter of the zone of inhibition was measured manually.

Antibacterial effect:
The mean zones of inhibition of samples from all patients for CBD-MW (18.1 mm) and CBG-MW (17.7 mm) were similar to that of the 0.2% chlorhexidine group (16.8 mm).
CBD-MW and CBG-MW showed similar or better bactericidal efficiency than the positive control (0.2% chlorhexidine).
KOZONO, 2010 [27] In vitro Test: AEA and 2-AG To elucidate the role of ECS in periodontal healing.
FCG was collected from patients undergoing periodontal surgery.In animals, EP was induced to assess CB1 and CB2 expression.
HGFs were cultured and exposed, or not, to CB1 and CB2 antagonists and agonists (AEA).

Cell proliferation/viability:
In the GFR of patients undergoing surgery, AEA levels were significantly increased after 3 days (p < 0.05), compared with pre-surgery, but 2-AG levels were virtually unchanged.AEA via CB1 and CB2 significantly increased (p < 0.05) the proliferation of HGFs, compared with the control.

Expression of receptors:
Upon EP, CB1 and CB2 were expressed in scar tissue cells, but in healthy tissue expression, expressions were low or absent.
ECS may have an important modulatory role in periodontal healing, through proliferation of gingival fibroblasts via upregulation of CB receptor expression and increased AEA levels at wound sites.Localization of CB1 and CB2 in gingival tissue collected from rats was performed by immunohistochemistry.
HGFs were also cultured in plates containing 6 wells, in which culture media with or without THC were deposited.

Cell proliferation/viability:
Regarding the HGFs exposed to THC, a significant increase (p < 0.05) in the migration of these cells towards this cannabionoid was observed, compared with the control group, after 3 h.

Receptor expression:
CB1 was decreased in periodontal tissues, although its expression was higher in perivascular and immune cells (macrophages, lymphocytes, and dendritic cells).CB2 was identified at high levels in tissues, especially in the EJ, LP, TC, OA.
ECS may be a therapeutic target to treat periodontal diseases and induce periodontal regeneration.
HGFs were cultured in appropriate culture medium and exposed or not to bacterial LPS.
Expression of receptors: CB1 and CB2 were detected in HGFs, especially upon exposure to inflammatory agents (LPS and POLY I:C).In addition, exposure of these cells to CBD resulted in a significant increase (p < 0.001) in intracellular calcium concentration, compared with unstimulated cells.
FGHs express higher amounts of CB1 and CB2 upon inflammatory stimuli and CBD may influence a higher calcium influx into these cells, which demonstrates its participation in cell biological activity.

Discussion
The pathogenesis and therapeutics of PD are still being studied, with the aim of making its prevention and management more efficient.Currently, there are a variety of cannabinoid-based oral products on the market that promise analgesic, anti-inflammatory, and antibacterial efficacy; such products include chewing gums, dentifrices, oils, capsules, sprays, and mouthwashes [34].However, no evidence or clinical studies are available that directly involve the use of these products for the prevention or treatment of this condition.Thus, this integrative review of preclinical studies investigated the role of the ECS and different types of cannabinoids in the pathogenesis and therapy of periodontal disease.
Since 2006, in vitro and in vivo studies have been investigating the performance of natural, synthetic, and endogenous cannabinoids, with regard to their anti-inflammatory, antibacterial properties, participation in tissue repair, and prevention of alveolar bone loss, in relation to periodontal disease [13,15,16,21,32].Through this review, the role of cannabinoids in the prevention of alveolar bone resorption and the modulation of inflammatory responses, tissue repair, and antimicrobial effects was synthesized.The authors identified the potential of endogenous, synthetic, and natural cannabinoids in reducing inflammatory processes, exhibiting antibacterial activity, reducing alveolar bone loss, and promoting tissue repair in both in vitro and in vivo studies.However, none of the included studies directly investigated the analgesic potential of different types of cannabinoids on periodontal tissues.This may be justified by the profile of the studies (mostly in vitro), but is also due to the characteristics of periodontal disease, as the patient usually does not manifest pain, except in advanced and/or acute stages [35].
Proportionally, most studies have investigated the anti-inflammatory capacity of synthetic and endogenous cannabinoids and, to a lesser extent, phytocannabinoids.The findings reveal that the exposure of rat or human periodontal tissue/cells to these substances significantly reduces the expressions of cytokines relevant to periodontal destruction, such as TNF-α and IL-1β [3,12,20,24].The importance of these findings is supported by evidence, for example, that TNF-α participates in alveolar bone resorption through different pathways, such as osteoclast differentiation and maturation [36].IL-1β increases the expression of collagenolytic enzymes and matrix metalloproteinases (MMPs), contributing to the degradation of the extracellular matrix and, in turn, leading to bone resorption and tissue destruction [37].As such, these anti-inflammatory findings align with the observed decrease in alveolar bone loss observed in animals that underwent cannabinoid treatment for experimental periodontitis [6,13,22].
With regard to the healing, repair, and regeneration of periodontal tissues, the effects of endogenous and synthetic cannabinoids on the proliferation and cell viability of human gingival fibroblasts and periodontal ligament cells [15,23,27] were mainly investigated.This may represent an important indicator, as gingival fibroblasts participate in tissue repair and remodeling, after procedures such as scaling and root planning and periodontal surgery.Furthermore, in general, periodontal ligament cells constitute the supporting tissue of the teeth in the alveolus, participate in the sensory function due to their abundant innervation, contribute to the dissemination of occlusal forces to the supporting bone, and favor cell formation and nutrition of bone, cementum, and gingiva [27].
The role of CBD in the therapy of several diseases (rheumatoid arthritis, multiple sclerosis, Alzheimer's disease, anxiety disorders) is consolidated in the medical literature due to its analgesic, antitumor, anti-inflammatory, and central nervous system effects, among others [38].However, only 5 of the 21 studies included in this review studied this phytocannabinoid with regard to its effects on the control of bone resorption and repair [16,22], antibacterial activity [21,25,26], and cell proliferation [28].Traditionally, the main objective when studying the management of PD is to reduce the microbial load, especially periodontopathogens.Phytocannabinoids (CBD and THC) have already shown efficacy against gram-positive microorganisms such as Staphylococcus aureus [39] and, similarly, CBD reduced the growth of some gram-positive and negative microorganisms (P.gingivalis and Filifactor alocis) [19].In addition, phytocannabinoids (isolated or infused in mouthwashes) also demonstrated antimicrobial activity that was superior or equal to that of traditional oral products such as 0.2% chlorhexidine and dentifrices, suggesting that these substances hold antibacterial potential [25,26].
With regard to the expressions of CB1 and CB2, there was no consensus among the studies, and differences were observed in the expressions of these receptors according to the characteristics of periodontal tissues [17,31].This may be related to the types of stimuli examined, such as experimental periodontitis [27] and mechanical stress [31] or due to differences in cells and tissues, as some studies evaluated gingival tissue from rats [17,27] and humans [31], as well as gingival fibroblasts [16,32] and periodontal ligament cells from humans [31].
Despite the potential role of cannabinoids in preventing alveolar bone resorption, modulating inflammatory responses, promoting tissue repair, and exerting antimicrobial effects, some disadvantages should be considered.The long-term effects of cannabinoids on periodontal tissues remain unknown, and predicting their effectiveness and appropriate dosage is challenging due to the close relationship between the actions of these substances and the individual's ECS and immune system.Moreover, the lack of standardization in dosing inherent to cannabinoid-based products may lead to inconsistent therapeutic outcomes [4].
The clinical relevance of these findings suggests potential improvements in periodontal clinical parameters, which may prevent, reduce, or limit the destruction of periodontal tissues inherent in the immune-inflammatory response of periodontal disease.However, although in vitro and in vivo studies provide contributions to the development of clinical trials, the limitations of these trials include the irreproducibility of the interaction between the products and substances tested with the human host, since the tests are restricted to inoculation in groups of cells isolated from humans or other animals and to cultivation in tubes or glass plates, for example.Finally, other limitations exist, such as the lack of standardization of studies, techniques, and analyses, which could provide greater data replicability, as well as the fact that laboratory conditions do not always occur simultaneously with real conditions.Further randomized and controlled clinical studies are needed to confirm the safety, tolerability, toxicity, efficacy, and optimal dosages of these compounds.

Conclusions
Preclinical studies evidence the positive role of the Endocannabinoid System (ECS) and different types of cannabinoids-such as endogenous (anandamide (AEA)), synthetic (methanandamide (Meta-AEA), and HU-308), and natural (∆9-tetrahydrocannabinol (THC), cannabidiol (CBD))-in the pathogenesis and therapeutics of conditions affecting oral tissues and cells.Notable contributions of this system and its ligands include the potential for preventing bone resorption, anti-inflammatory effects, tissue repair capabilities, and antimicrobial effects.However, future applications must consider limitations, such as the safety of natural, synthetic, and/or endogenous cannabinoids and their products, optimal dosages, and interactions between products, which need to be verified for the treatment of periodontal disease.

Figure 1 .
Figure 1.Flow diagram of the selected studies.

Figure 1 .
Figure 1.Flow diagram of the selected studies.

Pharmaceutics 2024 , 23 Figure 4 .
Figure 4. Schematic representation of the differential expression of cannabinoid receptors (CB1 and CB2) in healthy and diseased periodontal tissues.In healthy periodontal tissues, CB1 receptor expression predominates, while inflamed tissues exhibit a higher tendency for CB2 receptor expression.This shift in receptor expression suggests a potential regulatory role of the Endocannabinoid System in the pathogenesis and progression of periodontal disease.

Figure 4 .
Figure 4. Schematic representation of the differential expression of cannabinoid receptors (CB1 and CB2) in healthy and diseased periodontal tissues.In healthy periodontal tissues, CB1 receptor expression predominates, while inflamed tissues exhibit a higher tendency for CB2 receptor expression.This shift in receptor expression suggests a potential regulatory role of the Endocannabinoid System in the pathogenesis and progression of periodontal disease.

Table 2 .
Summary of included studies.