Periodontitis (PD) afflicts approximately 64.7 million adults in the U.S. and is one of the most common causes of tooth loss in adults¹⁾. Initiated by polymicrobial biofilms, particularly Porphyromonas gingivalis (P. gingivalis), periodontitis is the most severe form of periodontal disease caused by a disruption in homeostasis and is modulated by genetics, oral hygiene practices, and host responses. Bone loss and destructive inflammation of the surrounding ligaments and soft tissues are hallmarks of the disease. Ultimately, periodontitis can result in partial or total edentulism, which can be debilitating because it decreases the quality of life due to alveolar ridge absorption, ineffective mastication, compromised aesthetics, and speech difficulty²⁾.

Often, endosteal implant placement is required to restore dental function. Like periodontitis, peri-implantitis is an infectious disease resulting in an inflammatory response that affects the gum and bone structure around the implant. Such inflammation causes elevated levels of destructive cytokines such as interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), interleukin-1 alpha (IL-1α), interleukin-6 (IL-6), and interleukin-17 (IL-17), as well as matrix metalloproteinases (MMPs)³⁾. Chronic inflammation in periodontal disease and peri-implantitis disrupts the homeostatic control between pro-inflammatory and anti-inflammatory responses resulting in an imbalance. The shift to a pro-inflammatory state is marked by increased production of pro-inflammatory cytokines and chemokines, such as IL-1β, IL-6, and interleukin-8 (IL-8), which in turn propagates the inflammation, leading to bone resorption and destruction of periodontal tissue⁴⁾. MMPs are also major players during inflammation and are involved in remodeling the extracellular matrix (ECM) by activating growth factors in their proximity, cell surface receptors, and adhesion molecules⁵⁾.

In gingival biopsies, expression of MMP2 (gelatinase) was significantly elevated in failed implants vs healthy implants, owing to MMP2’s capacity to break down the ECM increasing bone loss⁶⁾, which is normally regulated by TIMP (tissue inhibitor of metalloproteinases) to maintain tissue homeostasis⁷⁾. Since many cytokines (i.e. IL-10) behave as anti-inflammatories that can regulate the pro-inflammatory cytokine response⁸⁾. Likewise, during inflammation, MMP activity contributes to tissue remodeling and inflammatory signaling and is regulated by tissue inhibitors of metalloproteinases (TIMPs)⁹⁾. In addition, implants in an inflamed environment, specifically those with peri-implantitis, have been shown to release more ions and metal particles into the surrounding tissues compared to non-diseased implants¹⁰⁾. This can further aggravate the periodontium and lead to more inflammation, which if left untreated, can result in connective tissue destruction, osteoclastic bone resorption, and eventual loss of the implant¹¹⁾.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been used to treat periodontitis, but these drugs can have severe systemic adverse effects, such as kidney disease, gastrointestinal bleeds, increased blood pressure, and reduced wound healing¹²⁾. The regulation of inflammation is key for new therapies targeting peri-implantitis and could serve as a target for therapeutic intervention that could limit the tissue damage caused by inflammation. Therefore, there is a current need for the development of novel anti-inflammatory agents to improve the quality of life and reduce treatment costs for those afflicted with chronic inflammatory conditions, including peri-implantitis.

In recent years, the endocannabinoid system has been extensively studied for its involvement in inflammation. Cannabinoids refer to a group of molecules acting on cannabinoid receptors (CBR) and are classified into three categories: endocannabinoids, phytocannabinoids, and synthetics¹³⁾. CBRs are heterotrimeric Gi/o-protein-coupled receptors: cannabinoid receptor 1 (CB1R; typically expressed in the central nervous system) or cannabinoid receptor 2 (CB2R; mainly expressed on immune and bone cells)¹⁴⁾. Cannabinoids are involved in the inhibition of cytokine production and disruption of immune response affecting cytokine production of subsets of T-regulatory (T-reg) and helper cells (Th1, Th2)¹⁵⁾. The anti-inflammatory effects of cannabinoids include cytokine suppression, induction of regulatory T cells, inhibition of cell proliferation, and apoptosis of activated immune cells^16,17). Recently cannabidiol (CBD), a component of Cannabis sativa, also known as marijuana, has been shown to ameliorate inflammatory disease in clinical and preclinical in vitro and animal studies, which could be applied in both dental and medical practice¹⁸⁾.

The cannabis plant contains more than 120 phytocannabinoids (pCBs), and in this study, we explored the anti-inflammatory properties of the phytocannabinoids CBD, cannabidivarin (CBVN), and cannabigerol (CBG)¹⁹⁾. Our labs have previously demonstrated that cannabinoid type 2 receptor (CB2R) ligands HU-308 and SMM-189 promoted inhibition of - IL-6 and MCP-1 production by human periodontal ligament fibroblasts (hPDLFs) stimulated with lipopolysaccharide (LPS), TNF-α, or IL-1β^20,21).

In this study, we explored the immunomodulatory effects of CBD, CBVN, CBG on primary human gingival fibroblasts (HGFs). HGFs are the most abundant resident cells in the periodontium, express many different receptors, and produce pro-inflammatory cytokines, thus playing an important role in host immunomodulation²²⁾. We hypothesized that the immunomodulatory effects of non-psychoactive cannabinoids have a potential for treatment in inflammatory diseases, such as peri-implantitis. To study peri-implantitis in vitro, we developed a peri-implantitis model by stimulating the HGFs with IL-1β and titanium particles²³⁾. Thus, this project will examine the effects of phytocannabinoids and the inflammatory responses in IL-1β stimulated HGFs, with the addition of titanium particles to mimic the microenvironment of peri-implantitis.

1. Cell culture of primary human gingival fibroblasts

Human gingival fibroblasts (HGFs) were purchased from the American Type Culture Collection (HGF-1 ATCC CRL2014) (ATCC, Manassas, VA). Cells were cultured in the manufacturer’s recommended fibroblast basal medium and supplemented with Fibroblast Growth Kit-Low Serum (ATCC) and 1% penicillin, and Streptomycin (Mediatech, Inc. Manassas, VA) (P/S). The cells were originally obtained from a gingival biopsy from a 28-year-old healthy Caucasian male who was negative for HIV-I, Hepatitis B Virus, and Hepatitis C Virus. Furthermore, these cells were validated as fibroblasts via TE-7 (fibroblast marker) by ATCC. The experiments were performed at 70∼80% confluency within passages 3∼5.

2. Titanium particles, phytocannabinoids, and cell viability assay

Cells were plated in 96-well polystyrene flat-bottom plates (Sigma Aldrich; St. Louis MO) for the cell viability assay at cell densities of 10,000 cells/well. Cells were maintained in a full growth medium which contained 1% P/S and Fibroblast Growth Kit-Low Serum for 24 hours followed by a change to fibroblast basal medium (FBM) containing 1% fetal bovine serum (FBS), and 1% P/S for another 24 hr at 37C, 5% CO₂ to synchronize cell activity. Titanium particles (titanium [IV] oxide [TiO2]), rutile powder, <5 µm) were obtained from Sigma Aldrich). Varying concentrations of titanium dioxide (100-40,000 particles/well) were tested for cytotoxicity and a final concentration was made to have a suspension of 0.2 ml/well of titanium dioxide particle suspension (Ti) at a concentration of 100,000 particles/ml of tissue culture medium²³⁾. The cannabidiol, cannabidivarin, and cannabigerol (Cayman Chemicals, Ann Arbor, MI) had been previously screened for effects on viability/proliferation and a final concentration of 1 µg/ml was used²⁴⁾. Control groups were prepared with dimethylsulfoxide (DMSO) at the highest concentrations (1% DMSO) to which the cells were exposed. Assessment of cellular viability was determined using the CCK-8 assay (Dojindo Molecular Technologies, Rockville, MD) by measuring the change in absorbance at 450 nm, using a BioTek Synergy 2 Multidetection Microplate Reader (BioTek Instruments, Inc. Winooski, VT).

3. Cytokine and MMP/TIMP determination

The HGFs were plated on 96-well polystyrene flat-bottom plates with a seeding density of 20,000 cells/well. Cells were maintained in full growth medium for 24 hours followed by a change to fibroblast basal medium (FBM) containing 1% FBS, and 1% P/S for another 24 hr at 37C, 5% CO₂ to synchronize cell activity. On the third day, the HGFs treatments included IL-1β (1 ng/ml) with or without Ti, and Ti alone. If treatments included pCBs, IL-1β (1ng/ml) with or without Ti were added an hour before the pCBs treatment and the conditioned medium was aspirated after 24 hours. The conditioned medium was either frozen for 2 weeks at −80°C or directly transferred to Mesoscale Discovery Human V-Plex Cytokine Kit (Interferon (IFN)-γ, IL-10, IL-13, IL-4, IL-6, & TNF-α.), or Cisbio MMP-2 & TIMP1. Determination of cytokines was done in an electrochemiluminescence format to get optimal sensitivity and quantification. Mesoscale Discovery Human Pro-inflammatory panel kits were analyzed for their respective marker levels according to the manufacturer’s instructions using Mesoscale Imager SECTOR 2400 or BioTek Synergy 2 Multidetection Microplate Reader for the Cisbio kits.

4. Statistical analysis

All results represent an average of 4∼6 replicates per determinant. Data were analyzed using Graphpad Prism 6.0 using the One-way analysis of variance (ANOVA) test. Differences were considered significant at P≤0.05.

1. Evaluation of HGFs viability to Ti, IL-1β, and phytocannabinoids

Inflammatory responses of HGFs were initiated by stimulating with either titanium dioxide particles (Ti), IL-1β, or a combination of the two stimuli (IL-1β+Ti). The appropriate dose for titanium dioxide first was determined via a viability (cytotoxicity) assay using increasing concentrations of Ti suspensions (100∼40,000 particles/well) with the CCK-8 assay for 24-hour exposure (Fig. 1). No cytotoxicity was observed at any Ti particle concentration, which can be appreciated from cellular imaging (Supplementary Fig. 1). Recent studies in our labs²⁴⁾ and other literature have reported²⁵⁾ 1 ng/ml of IL-1β to be non-toxic to the HGFs, therefore, the same concentration was also used in this study. After a previous evaluation²⁴⁾ of the cellular viability along with the physiological relevance of pCBs, 1 μg/ml was selected as the concentration for all three of the pCBs for the remainder of this study.

Figure 1. The effects of titanium particles on HGF viability. Cellular viability for Ti particles in primary human gingival fibroblasts (HGF) measured using the CCK-8 assay (24-hr exposure). The Ti particles varied from 100 – to 40,000 particles/well. There was no significant cellular viability change found among the concentrations tested. The data are presented as the mean of 4∼6 biological replicates. Data was analyzed using Graphpad Prism 6.0 using One-way ANOVA test. Differences were considered significant at P≤0.05 & error is the standard error of the mean (SEM). ns: non-significant.

2. Effects of pCBs on proinflammatory proteins IFN-γ, IL-6, TNF-α, & MMP-2 in HGFs

The pro-inflammatory, tissue-destructive responses of the HGFs to IL-1β were tested by analyzing protein concentrations of IFN-γ, IL-6, and TNF-α, as well as MMP2. CBG and CBVN caused significant reductions in IFN-γ and TNF-α protein expression in HGFs stimulated with IL-1β or Ti+IL-1β (Fig. 2A). CBD was ineffective in significantly reducing the levels of IFN-γ or TNF-α produced by HGFs stimulated with IL-1β or Ti+IL-1β (Fig. 2A, 2B). In contrast, in HGFs stimulated with IL-1β or Ti+IL-1β, both CBD and CBG significantly increased IL-6 expression, while CBVN significantly reduced the level of this cytokine (Fig. 2C).

Figure 2. The effects of CBD, CBVN, and CBG with Ti particles with/without IL-1β stimulation on HGF production of cytokines IFN-γ, TNF-α, & IL-6. (A) IFN-γ, (B) TNF-α, (C) IL-6. For effects on IL-1β -stimulated cytokine production, HGFs were treated with IL-1β 1 hr before the addition of Ti particles and/or CBD, CBVN, and CBG, then assayed for expression of cytokines 24 hr later in Mesoscale Discovery Human Cytokine kits. IL-1β significantly increased the protein concentration of each cytokine compared to control levels. The data are presented as the mean of 4∼6 biological replicates and the error is the SEM. The U-shaped line (Π) describes the significance of IL-1β from control, Capped lines (ꟷ) describe the significance from IL-1β alone to phytocannabinoids+IL-1β, double lines (═) measure the significance from Ti+IL-1β to phytocannabinoids with Ti+IL-1β. (*) P≤0.05, (**) P≤0.01, ns: not significant. Data were analyzed using GraphPad Prism 6.0 using the One-way ANOVA test. Differences were considered significant at P≤0.05.

Fig. 3 shows the effects of the pCBs, titanium particles, and IL-1β, alone and in combinations, on MMP-2 production. Titanium particles alone had no effect on the basal level of MMP-2 produced by HGFs; however, it was significantly reduced by CBD or CBG, but not CBVN (±titanium particles). IL-1β (±titanium particles) significantly increased MMP-2 expression, and treatment with all three pCBs significantly reduced it in all instances.

Figure 3. The effects of CBD, CBVN, and CBG with Ti particles with/without IL-1β stimulation on HGF production of MMP-2. For effects on IL-1β-stimulated MMP-2 production, HGFs were treated with IL-1β 1 hr before the addition of Ti particles and/or CBD, CBVN, and CBG then assayed for expression of MMP-2 24 hr later in the Cisbio human MMP-2 kit. The data are presented as the mean of 4-6 biological replicates and the error is the SEM. The U-shaped line (Π) significance of IL-1β from control. The capped lines (ꟷ) describe the significance of IL-1β alone to phytocannabinoids+IL-1β or Ti+IL-1β to phytocannabinoids with Ti+IL-1β. (*) P≤0.05, (**) P≤0.01, (****) P≤0.0001. ns: not significant. Data were analyzed using Graphpad Prism 6.0 using the One-way ANOVA test. Differences were considered significant at P≤0.05.

3. Evaluating the effects of pCBs on proposed anti-inflammatory proteins IL-4, IL-10, IL-13, and TIMP-1 in HGFs

The anti-inflammatory and tissue-protective of the HGFs were tested by analyzing protein concentrations of IL-4, IL-10, IL-13, and TIMP-1 under IL-1β stimulation. All of the cytokine treatments were within the detection limit other than IL-10, in which only a few were above the detection thresthold. All cannabinoids significantly reduced IL-4 protein expression after IL-1β stimulation, but CBVN was the only pCB that caused a significant reduction in IL-1β+Ti stimulation (Fig. 4A). In the IL-10 assay, the majority of the experimental group protein levels were beneath the detection limit (shown in figure), and treatment with CBD in conjunction with IL-1β and IL-1β+Ti caused significantly more expression of IL-10 than in the control group (Fig. 4B). All cannabinoids reduced IL-13 protein expression in IL-1β treated fibroblasts (Fig. 4C). CBVN showed the most significant reduction, followed by CBG, and then CBD. The same pattern was noted for IL-1β+Ti treated HGFs, but these results were not significant. Although there was a subtle increase in TIMP-1 expression caused by pCBs, none of the treatments resulted in a significant effect on TIMP-1 (Fig. 5).

Figure 4. The effects of CBD, CBVN, and CBG with Ti particles with/without IL-1β stimulation in on HGF production of cytokines IL-4, IL-10, & IL-13. (A) IL-4, (B) IL-10, (C) IL-13. HGFs were treated with IL-1β 1 hr before the addition of Ti particles or CBD, CBVN, and CBG then assayed for expression of cytokines 24 hr later in Mesoscale Discovery Human Cytokine kits. The data are presented as the mean of 4∼6 biological replicates and the error is the SEM. The U-shaped line (Π) describes the significance of IL-1β from control, Capped lines (ꟷ) describe the significance from IL-1β alone to phytocannabinoids+IL-1β, double lines (═) measure the significance from Ti+IL-1β to phytocannabinoids with Ti+IL-1β. (*) P ≤0.05, (**) P≤0.01, ns: not significant. Data were analyzed using GraphPad Prism 6.0 using the One-way ANOVA test. Differences were considered significant at P≤0.05

Figure 5. The effects of CBD, CBVN, and CBG with Ti particles with/without IL-1β stimulation on HGF production of TIMP-1. HGFs were treated with IL-1β 1 hr before the addition of Ti particles or CBD, CBVN, and CBG then assayed for expression of TIMP-1 24 hr later in the Cisbio human TIMP-1 kit. The plates were analyzed for their respective marker levels according to the manufacturer’s instructions using a BioTek Synergy 2 Multidetection Microplate Reader. The data are presented as the mean of 4-6 biological replicates and the error is the SEM. No significant change in TIMP-1 protein levels was observed in any condition compared to control. ns: not significant. Data were analyzed using GraphPad Prism 6.0 using the One-way ANOVA test. Differences were considered significant at P≤0.05.

Periodontal disease (PD) is one of the most common conditions in humans. Data from the 2009 and 2010 National Health and Nutrition Examination Survey (NHANES), which estimated the prevalence, severity, and extent of PD in the adult U.S. population, showed that more than 47% of the sample, representing 64.7 million adults, had PD, distributed as mild (8.7%), moderate (30.0%), and severe (8.5%)¹⁾. Although the primary etiological factor of PD and peri-implantitis is bacterial plaque (primarily red and orange complex pathogens), both are considered multifactorial diseases^26,27). Factors such as genetics, smoking, diabetes, and oral hygiene habits contribute to PD, which itself is a factor associated with the development of peri-implantitis^26-29). Recently, it has been reported that up to 90% of implants exhibit some inflammation in their surrounding tissues³⁰⁾. PD and peri-implantitis can be described as states of chronic inflammatory dysbiosis, which ultimately lead to soft and hard tissue breakdown and loss of teeth or implants, respectively^2,26). Cannabinoid receptors can contribute to protection against inflammation²⁰⁾ which has led to further research interest in investigating the use of cannabinoids to treat oral inflammation. Current shortcomings in understanding the clinical effects of marijuana or its components are limited because of it being a schedule I controlled substance (Federal status) thus, the comprehension of the biologically complex activities of phytocannabinoids (pCBs) are limited and are not fully known. There are active reports of marijuana being a risk factor for periodontal disease, however, the reported effects by many cannabinoids also are known to mitigate inflammation and pain³¹⁾. Meanwhile, altering inflammation and immune responses to oral microflora in a healthy subject does negatively influencing the oral cavity and that most likely accelerates the onset of periodontal disease, peri-implantitis, or exacerbate other existing conditions³²⁾.

Therefore, because pCBs have anti-inflammatory effects, the objective of this study was to determine if targeting the endocannabinoid system (ECS) with pCBs could offer a new therapeutic approach for treating chronic inflammation associated with peri-implantitis. CBG and CBVN exhibited a significant reduction in IFN-γ and TNF-α protein expression in IL-1β-stimulated HGFs. The reduction of the inflammatory response with cannabinoids may potentially influence bone levels in peri-implantitis by promoting osteoblastic activity³³⁾. Proinflammatory cytokines are produced predominantly by activated macrophages and are involved in the up regulation of inflammatory reactions. There is abundant evidence that certain pro-inflammatory cytokines such as IFN-γ, IL-6, and TNF-α are involved in the process of pathological pain⁸⁾. The pro-inflammatory cytokines IL-1β and TNF-α are pivotal because they are involved with tissue destruction, alveolar bone resorption, and production of pro-inflammatory molecules by fibroblasts^34,35). Daily administration of the anandamide (an endocannabinoid) analog methanandamide in a rat PD model reduced TNF-α expression and significantly diminished alveolar bone loss³³⁾. Likewise, salivary IFN-γ is an indicator for periodontal breakdown since it is a potent inducer of inflammatory cytokines and chemokines and can upregulate proteins such as major histocompatibility complex (MHC) antigens, Fc receptors, and leukocyte adhesion molecules³⁶⁾. Moreover, increased levels of IFN-γ in gingival crevicular fluid samples have been found in chronic PD lesions³⁷⁾.

CBD and CBG significantly increased IL-6 expression, while CBVN was the only pCB significantly reducing IL-6 production by fibroblasts stimulated with both IL-1β and Ti+IL-1β. The reduction of IL-6, a potent activator of osteoclasts, by CB2R-selective ligands may help support bone formation³⁸⁾. IL-6 released by immune cells activates osteoclastogenesis and induces bone resorption, and IL-6 levels are higher in patients with PD compared to patients with less severe gingivitis^39-43). Therefore, IL-6 production by fibroblasts can contribute to periodontal inflammation cytokine, exacerbating the host immune response. In this study, only CBVN exhibited suppression of IL-6, thereby showing its promise for combatting destructive peri-implant inflammation.

Treatment with all three pCBs significantly reduced MMP-2 levels in fibroblasts stimulated with IL-1β and Ti+IL-1β. The basal level of MMP-2 was also significantly reduced by CBD and CBG, but not CBVN. MMP-2 expression was increased in the peri-implant crevicular fluid of patients with peri implantitis, and was also increased in human macrophages infected with P. gingivalis⁴⁴⁾. MMP-2 cleaves type IV collagen, a major structural component of the basement membrane providing support to the extracellular matrix and are abundant gingival connective tissue matrix⁴⁵⁾. MMP-2 expression is disturbed in peri-implantitis and in a 2013 study by Ai-Duboni⁶⁾, the expression of MMP-2 was significantly elevated in failed implants versus healthy implants. In addition, MMP-2 was detected in peri-implant sites with ongoing inflammation, bone loss, and cavitations⁶⁾. Therefore, reduction of MMP-2 levels with pCBs can be a potential treatment method to reduce connective tissue loss in peri-implantitis. While MMP activity contributes to inflammatory signaling and tissue remodeling, there is a regulatory mechanism by which its activity is suppressed known as tissue inhibitors of metalloproteinases (TIMPs)⁹⁾. Although pCBs caused subtle increases in TIMP-1 expression, these effects were not statistically significant, suggesting that cannabinoids might not directly impact TIMP-1 levels. Studies have demonstrated the involvement of MMPs and TIMPs in the destruction of periodontal tissues⁴⁶⁾. However, regarding peri-implant tissues, it is important to note that there is little scientific information published on this subject and the results are contradictory.

IL-4, IL-10, and IL-13 possess anti-inflammatory capacity, especially IL-10 which suppresses proinflammatory responses⁸⁾. In our study, IL-1β significantly upregulated IL-4 production, which was significantly reduced by all pCBs. However, CBG and CBVN produced a more robust reduction of IL-1β-stimulated IL-4. In fibroblast cultures exposed to both IL-1β+Ti, however, only CBVN caused a significant reduction in IL-4 levels. Treatment of the fibroblasts with CBD in conjunction with IL-1β and IL-1β+Ti resulted in significantly greater expression of IL-10 than in the control group. If IL-10 upregulation is a desired pro-resolving therapeutic strategy to target peri-implant inflammation, CBD could be a promising molecule for this purpose. All cannabinoids reduced IL-13 protein expression, which was elevated by IL-1β stimulation in HGFs, and under IL-1β+Ti it showed the same trend, but it was not significant. While the roles of pro-inflammatory markers and cytokines are more straightforward in their contribution toward the destruction of periodontal and peri-implant tissues, the roles of IL-4, IL-10, and IL-13 are also worthy of more investigation, since these cytokines mediate immunity and contribute to maintaining homeostasis^47,48). However, it is largely unknown how these cytokines influence fibroblast activity in the periodontium, as it is known that periodontal fibroblasts are immune like²²⁾ and can interact with immune cells to modulate inflammatory responses⁴⁹⁾, however, the gingival fibroblast do not necessarily show the same properties as does it periodontal fibroblast during inflammatory challenge⁵⁰⁾.

Our study demonstrates that cannabinoids may act as effective anti-inflammatory agents in stimulated HGFs; however, the molecular bases underlying the effects of CB2R activation in stimulated HGFs remain largely unknown. CB1R and CB2R are upregulated in inflamed gingival fibroblasts which is precisely the oral scenario presented in cases of peri-implantitis⁵¹⁾. CBD does not activate either CB1R or CB2R directly, but can indirectly upregulate endocannabinoid activity by binding to fatty acid amide hydrolase (FAAH) and reducing the breakdown of the endocannabinoid anandamide⁵²⁾. CBG and CBVN have shown to be agonists at the CB2R in our functional studies with the CB2R-ACTOne cell line¹⁹⁾, which is a promising target for cannabinoid therapy due to its widespread expression in peripheral tissue and the lack of adverse psychotropic effects associated with CB1R agonists⁵³⁾. Targeting CB2R has been investigated by Ossola et al.³³⁾ wherein the administration of HU-308, a selective CB2R agonist, exhibited anti-inflammatory and osteoprotective effects in the rat periodontitis model. The signaling pathways activated and/or inhibited by CB2R agonists and inverse agonists are under extensive investigation.

Targeting the cannabinoid receptors in peri-implantitis, specifically, by pCBs tested in this study may have additional benefits, which are outside the scope of this study but warrant discussion. Cannabinoid receptors are involved in bone metabolism and are widely expressed in osteoblasts and osteoclasts⁵⁴⁾. Geng et al.⁵⁵⁾ demonstrated that titanium wear particles can result in osteolysis and could result in aseptic loosening of the implant. CB2R inactivation by AM630, a CB2R selective inverse agonist (antagonism), effectively inhibited osteoclastogenesis, thus preventing bone loss induced by titanium particle irritation. Although the effects of the activation of the endocannabinoid system are still unknown and remain subject to continued research, many in vivo and in vitro studies have demonstrated that the stimulation of both CB1R and CB2R reduces the level of inflammatory mediators and bone resorption in animal tissues³³⁾. These studies suggest that cannabinoids and the endocannabinoid system (ECS) have a functional role in regulating inflammation in patients with diseased periodontal tissues, potentially including those with peri-implantitis. Given the destructive effects of cytokines, our study supports the use of pCBs to manage the pro-inflammatory activity and holds a promise as an effective treatment for peri-implantitis and the general preservation of periodontal health.

In conclusion, the present study provides support that the pCBs namely CBG, CBD, and CBVN are effective anti-inflammatory agents that could serve to mitigate inflammation and connective tissue destruction in peri-implantitis. Peri-implant mucositis to peri-implantitis is inflammatory in nature and the pCBs alone or combination may provide benefits to improve oral health. Overall, the activity of pro-inflammatory cytokines decreased with pCB treatment, with CBVN being the most effective pCB in our study to decrease inflammatory activity. Our findings suggest that targeting the endocannabinoid system (ECS) shows promise for therapeutic strategies in periodontal therapy.