Effects of cannabinoid receptor activation on Porphyromonas gingivalis lipopolysaccharide stimulation in human periodontal ligament stem cells in vitro
Department of Plastic Surgery, Hand Surgery-Burn Center, University Hospital RWTH Aachen, Aachen, Germany.
Department of Orthodontics, University Hospital RWTH Aachen, Aachen, Germany.
Correspondence: Tim Leypold. Department of Plastic Surgery, Hand Surgery–Burn Center, University Hospital RWTH Aachen, Pauwelsstraße 30, 52074 Aachen, Germany. tleypold@ukaachen.de, Tel: +49-241-80-89701, Fax: +49-241-80-82448Abstract
Purpose
Periodontitis is an inflammatory disease that results in the loss of periodontal tissue. The endocannabinoid system has anti-inflammatory properties and displays considerable potential for tissue regeneration. In this study, we aimed to explore whether the activation of this system can alleviate or reverse the inflammatory phenotype of human periodontal ligament stem cells (hPDLSCs) induced by exposure to the inflammagen lipopolysaccharide (LPS).
Methods
We investigated the effects of activating specific cannabinoid receptors (CB1 and CB2) on the inflammatory phenotype of LPS-stimulated hPDLSCs. The exogenous ligands WIN55,212-2 and JWH-133 were employed to target the cannabinoid receptors. We conducted a thorough assessment of cell proliferation, metabolic activity, and adipogenic, osteogenic, and chondrogenic differentiation potential. Additionally, we measured cytokine release using enzyme-linked immunosorbent assays.
Results
Exposure to Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) caused an increase in cell proliferation while decreasing metabolic activity. While this exposure did not influence adipogenic or chondrogenic differentiation, it did result in reduced osteogenesis. Additionally, LPS induced the release of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein 1. Immunolabeling revealed the presence of CB1 and CB2 on the cellular membrane, with these receptors playing distinct roles in hPDLSCs. The CB1 agonist WIN55,212-2 was found to increase metabolic activity and promote adipogenic differentiation, whereas the CB2 agonist JWH-133 promoted cell proliferation and osteogenic differentiation. When hPDLSCs were co-exposed to Pg-LPS and CB ligands, JWH-133 slightly ameliorated the inhibition of osteogenic differentiation and suppressed the release of inflammatory cytokines.
Conclusions
This study clarifies the effects of specific CB receptor activation on hPDLCs and the inflammatory phenotype. Stimulation of the endocannabinoid system through the manipulation of endogenous or the application of exogenous cannabinoids in vivo may represent a potent therapeutic option for combating periodontal inflammatory disorders.
Graphical Abstract
INTRODUCTION
Periodontitis is a chronic inflammatory disease that results from bacterial infection and various other factors, leading to the resorption of alveolar bone along with tooth mobility and migration [1]. Porphyromonas gingivalis has been implicated as the causal pathogen in most cases of periodontal disease [2]. Lipopolysaccharide (LPS), which is present in the outer membrane of the bacterial cell wall, acts as a potent endotoxin. LPS induces cells to secrete inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and IL-8, which are associated with periodontal tissue degradation [3].
Periodontal tissue comprises the periodontal ligament (PDL), cementum, alveolar bone, and gingiva. The PDL consists of fibrous connective tissue that anchors the tooth root to the alveolar bone and contains periodontal ligament stem cells (PDLSCs). PDLSCs, a type of tissue-specific mesenchymal stem cells (MSCs), play key roles in the development and homeostasis of periodontal tissue [4]. Consequently, they have been proposed as cellular tools for tissue regeneration therapies.
The endocannabinoid system (ECS) functions through cell-modulating lipid mediators known as endocannabinoids and is considered to regulate homeostatic processes across various tissues [5]. Following periodontal surgery, the ECS is activated, as evidenced by elevated endocannabinoid levels in gingival crevicular fluid and increased expression of cannabinoid receptors on dental fibroblasts and macrophage-like cells [6]. Within the oral cavity, endocannabinoids appear to function as immune and pain regulators and may also influence tissue regeneration. Histological studies have identified the cannabinoid receptors CB1 and CB2 in human periodontal biopsies, with CB2 signals being upregulated in diseased states and CB1 expression confirmed on gingival fibroblasts, oral epithelial cells, and PDLSCs [7]. However, the precise physiological functions of CB1 and CB2 remain unknown. Building on earlier findings in other tissues, we hypothesize that activating these receptors may offer a promising therapeutic approach to influence the progression of periodontitis, potentially by reducing pathological inflammation or impacting cell differentiation. Thus, the objective of this study was to first investigate the cellular characteristics of PDLSCs following exposure to LPS. Subsequently, we confirmed the expression of both cannabinoid receptors, CB1 and CB2, on PDLSCs and sought to elucidate their physiological functions. These preliminary tests laid the groundwork for examining the anti-inflammatory effects of ECS activation in PDLSCs within an artificial inflammatory environment in vitro.
We employed various concentrations of P. gingivalis LPS (Pg-LPS) to characterize the inflammatory phenotype of primary human PDLSCs (hPDLSCs). Specifically, we assessed its impact on cell vitality, including proliferation and metabolic activity, as well as its influence on trilinear differentiation—osteogenic, chondrogenic, and adipogenic—and secretory activity. Subsequently, we investigated receptor-specific responses to ECS stimulation in hPDLSCs, utilizing the same outcome measures. Finally, we explored the potential modulation of LPS-induced effects through co-stimulation of CB1 and/or CB2 receptors.
MATERIALS AND METHODS
Ethical approval and informed consent
All procedures performed were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent was obtained from all study participants and/or their legal guardian(s). The research protocol received approval from the regional ethics committee (Ethics Committee of RWTH Aachen University; EK374/19).
Materials
Resazurin sodium salt was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Fetal bovine serum (FBS), ITS 1 premix, and high/low glucose medium (4.5 g/L; 1 g/L) were acquired from Life Technologies (Darmstadt, Germany). LPS from P. gingivalis, safranin O, rosiglitazone, ascorbate 2-phosphate, β-glycerophosphate, paraformaldehyde (PFA), trypsin–EDTA, 2-amino-2-methyl-1-propanol (AMP), o-cresolphthalein complexone, 8-hydroxyquinoline, bovine serum albumin, and Tween 20 were sourced from Sigma-Aldrich (Taufkirchen, Germany). L-proline, acetic acid, hydrochloric acid (HCl), and crystal violet were procured from Roth (Karlsruhe, Germany). Insulin was supplied by Roche (Mannheim, Germany). Transforming growth factor-β3 (TGF-β3) was obtained from PeproTech (Hamburg, Germany). Isopropyl alcohol and Oil Red O were purchased from Merck (Darmstadt, Germany). Phosphate-buffered saline (PBS) was obtained from Biochrom (Berlin, Germany).
Cell isolation and cultivation
hPDLSCs were isolated and cultured according to methods described elsewhere [8]. In brief, hPDLSCs were harvested from the premolars of 7 donors, aged 20 to 25 years, who exhibited no clinical signs of periodontitis. The cells were scraped from the middle third of the tooth root to minimize contamination with gingival or apical tissue. These teeth had been extracted for orthodontic reasons. The isolation procedures took place in the Department of Orthodontics at the University Hospital RWTH Aachen, Germany. The hPDLSCs were cultured under standard conditions (37°C, 5% CO2) using a proliferation medium composed of high-glucose Dulbecco modified Eagle medium, 10% FBS, and 50 mg/L L-ascorbic acid. All experiments were performed using cells from passages 5–8, which were seeded at a density of 3×104 cells per cm2 in 1 mL of medium.
Immunocytochemical staining was employed to assess the surface expression levels of the cannabinoid receptors CB1 and CB2. Cells were seeded at a density of 10,000 per cm2 and cultured for 3 days until they reached 60% to 80% confluency. Next, the cells were rinsed with PBS, fixed with 4% PFA for 10 minutes, and then washed again with PBS. The cells were subsequently blocked with 10% FBS in PBS for 1 hour at room temperature (RT), then immunolabeled with primary conjugated antibodies. These antibodies, which were diluted to 10 µg/mL in the blocking buffer, included anti-CB1-Alexa Fluor 488 (Santa Cruz Biotechnology) and anti-CB2-Alexa Fluor 555 (Biozol, Eching, Germany). Cells were incubated with the antibodies for 3 hours at RT. Finally, the wells were overlaid with a mounting medium that contained DAPI (ibidi GmbH, Martinsried, Germany).
Cell stimulation
LPS from P. gingivalis was utilized as an exogenous toll-like receptor 4 (TLR4) ligand at concentrations ranging from 0.01 to 1 µg/mL. These concentrations were chosen to evaluate potential concentration-dependent effects on the viability and differentiation of hPDLSCs. WIN55,212-2 and JWH-133 served as exogenous ligands for the cannabinoid receptors CB1 and CB2, respectively. WIN55,212-2 (hereafter “WIN”) exhibits a higher binding affinity for CB2 (ki=0.29 nM) compared to CB1 (ki=9.87 nM) [9], while JWH-133 (hereafter “JWH”) is a CB2-selective agonist (ki=3.4 nM; CB1 ki=677 nM) [10]. Both agents were applied at a concentration of 3 µM, as previously described [11]. Each compound was dissolved in ethanol and diluted with double-distilled water to achieve a final ethanol concentration of 0.01% (v/v). Double-distilled water containing the corresponding ethanol concentration served as the vehicle control (Veh). To ensure complete cell adherence, all compounds were freshly added to the culture medium 3 days after cell seeding and with each subsequent medium change.
Proliferation
Cell proliferation was assessed by quantifying cells using crystal violet staining, following the previously described protocol [12]. Cells were rinsed with PBS and fixed with isopropyl alcohol for 10 minutes at RT. After another rinse with PBS containing 0.05% Tween20, the cells were stained with 0.1% crystal violet for 20 minutes and subsequently washed with distilled water. The absorbed stain was solubilized in 33% acetic acid with a 15-minute incubation. Aliquots of 70 µL were transferred in duplicate to a microplate, and the absorbance was measured at 620 nm using a BMG Labtech microplate reader (Ortenberg, Germany).
Differentiation
To assess differentiation, cells were exposed to 3 specific media [14]. The adipogenic medium consisted of high-glucose medium supplemented with 2% FBS, 10 µM insulin, and 0.28 µM rosiglitazone. Adipogenic differentiation was evaluated using Oil Red O staining as previously described [11]. Cells were washed with PBS and fixed for 20 minutes in 4% PFA. Subsequently, the cells were washed again in PBS and stained with 0.2% Oil Red O for 15 minutes. Excess dye was removed by washing, and the bound Oil Red O was eluted with 100% isopropyl alcohol. Absorbance of the eluted dye was measured in triplicate, using 100-µL aliquots, at a wavelength of 540 nm.
The osteogenic medium consisted of low-glucose medium supplemented with 2% FBS, 0.8 mM pyruvate, 0.25 µM dexamethasone, 10 mM β-glycerophosphate, and 200 µM ascorbate-2-phosphate. As an indicator of osteogenesis, cresolphthalein staining was used to determine the calcium content of differentiated hPDLSCs [11]. The cells were washed with PBS and fixed with 4% PFA. They were then incubated in a cresolphthalein buffer solution—prepared by dissolving 50 mg of o-cresolphthalein complexone and 500 mg of 8-hydroxyquinoline in 30 mL of 37% HCl and diluting the mixture to 500 mL with double-distilled water—for 5 minutes. Subsequently, the cells were incubated in AMP buffer (76 mL of AMP in 500 mL of double-distilled water, adjusted to a pH of 10.7 with HCl) for 15 minutes. The absorbance was measured in triplicate at 580 nm using 100-µL aliquots.
The chondrogenic differentiation medium consisted of low-glucose medium supplemented with 0.8 mM pyruvate, 5 ng/mL TGF-β3, 1 µM dexamethasone, 0.17 mM ascorbate-2-phosphate, 350 µM proline, and 50 µg/mL ITS 1 Premix. Chondrogenic monolayers were fixed with PFA and stained with safranin O for 30 minutes. Excess dye was removed by washing with isopropanol, and the absorbance was measured at 540 nm.
Cytokine quantification using enzyme-linked immunosorbent assay
After 24 hours of exposure to Pg-LPS and/or cannabinoids, the cell supernatant from each well (containing 5×104 cells in 2 mL of medium, in a 24-well plate) was collected to measure the concentrations of IL-6, IL-8, macrophage migration inhibitory factor (MIF), and monocyte chemoattractant protein-1 (MCP-1) using DuoSet enzyme-linked immunosorbent assay systems (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. Extinction was measured in duplicate for each well using a FLUOstar Optima microplate reader (BMG Labtech, Ortenberg, Germany).
Analysis and statistics
The data from all experiments were aggregated to evaluate the results for each experimental type and treatment. Using the Kolmogorov-Smirnov test, the data were assessed regarding adherence to a normal distribution. The results were presented as box plots, with the median represented by the central line and the 25th and 75th percentiles defining the box boundaries. Statistical analysis for the Pg-LPS experiments employed the Kruskal-Wallis H test, followed by pairwise comparisons with the Mann-Whitney U test, with the Bonferroni correction applied. To evaluate differences between Veh and WIN/JWH, the Mann-Whitney U test was used (SPSS 24; IBM Corp., Armonk, NY, USA). P-values of less than 0.05 were considered to indicate statistical significance. The figures were generated using Corel Draw X5 (Corel Corporation, Ottawa, Canada).
RESULTS
Effects of Pg-LPS on proliferation and metabolic activity of hPDLSCs
Upon exposure to increasing concentrations of LPS, we assessed cellular viability by using crystal violet staining to evaluate the proliferation of hPDLSCs. Additionally, we investigated metabolic activity through resazurin conversion assays conducted at 7 and 14 days. The Kruskal-Wallis H test revealed significant differences between treatments in terms of cell numbers (day 7: P=0.019; day 14: P=0.002) and resazurin conversion (day 7: P<0.001; day 14: P=0.013). Subsequent pairwise comparisons indicated a significant increase in cell number at the low LPS concentration (0.01 µg/mL) on day 7 (Figure 1A), as well as significantly elevated cell numbers at the high LPS concentration (1 µg/mL) after 14 days (Figure 1B). In contrast, LPS exposure reduced the metabolic activity per cell, with significant reductions observed at both 7 and 14 days after application of 1 µg/mL LPS (Figure 1A and B).
Effects of Pg-LPS on hPDLSC differentiation
We also investigated the impact of increasing LPS concentrations on the adipogenic, chondrogenic, and osteogenic differentiation of hPDLSCs (Figure 2A-C). The total cellular lipid content was measured using Oil Red O to assess the degree of adipogenic differentiation after 14 days of stimulation. To measure chondrogenic differentiation, proteoglycan accumulation was determined via safranin O staining after 5 days, as previously described [15]. Osteogenic differentiation was evaluated with cresolphthalein staining to detect extracellular matrix mineralization in hPDLSCs after 14 days. The Kruskal-Wallis H test revealed no significant impact of LPS on adipogenic and chondrogenic differentiation (Figure 2A and B). However, LPS application did reduce the calcium content in osteogenically differentiated cells. The inhibitory effect was strongest at a low LPS concentration (0.01 µg/mL) and declined at higher concentrations (Figure 2C).
Cannabinoid receptor expression and effects on MSC viability
As revealed by immunolabeling of hPDLSCs, both cannabinoid receptors were expressed on the plasma membranes of the cells. The staining for CB2 was more pronounced than that for CB1 (Figure 3A). The viability of hPDLSCs was assessed after exposure to WIN or JWH, each at a concentration of 3 µM, on day 7 and day 14. WIN exposure did not significantly alter the cell count of hPDLSCs, but it did increase their metabolic activity (Figure 3B and D). Pairwise comparison indicated a significant difference between the Veh- and WIN-treated cells. In contrast, the CB2 agonist JWH significantly increased proliferation but reduced the metabolic activity of hPDLSCs with long-term exposure (Figure 3C and E).
Effects of cannabinoid receptor activation on hPDLSC differentiation
The effects of cannabinoid receptor activation on the trilinear differentiation of hPDLSCs were investigated using 3 µM WIN or JWH. Exposure to WIN significantly increased lipid staining compared to Veh (P=0.002) (Figure 4A), while CB2 receptor activation by JWH did not significantly impact adipogenesis. At the conclusion of the chondrogenic differentiation experiments, hPDLSCs exhibited accumulated glycosaminoglycans, proteoglycans, and collagens, which were stained with safranin O (Figure 4B). Both WIN and JWH exposure significantly inhibited chondrogenic differentiation. Osteogenic differentiation was quantified by cresolphthalein staining. WIN exposure did not significantly impact osteogenesis, whereas JWH significantly increased the osteogenesis of hPDLSCs after 14 days (Figure 4C). To determine whether the pro-osteogenic effect of JWH could counteract the LPS-induced inhibition of osteogenesis, a control experiment was conducted in which cells were co-exposed to both agents (Figure 5). While 0.01 µg/mL LPS significantly reduced calcification, CB2 activation by JWH did not restore the osteogenic differentiation potential of the cells.
Effects of Pg-LPS and cannabinoid exposure on cytokine release
The results regarding cytokine secretion are summarized in Figure 6. The Kruskal-Wallis H test revealed significant differences between treatments for IL-6 (P<0.001), IL-8 (P<0.001), and MCP-1 (P=0.032) release following 48 hours of LPS application in hPDLSCs. Pairwise comparison indicated a significant increase in IL-6 secretion (Figure 6A), with median levels rising from 44 pg/mL to 67 pg/mL for the LPS+Veh group. This effect of LPS was significantly counteracted by JWH (P=0.03), whereas co-exposure of LPS with WIN did not have a significant impact. The same pattern was observed for IL-8 release, which was significantly elevated to 126 pg/mL by LPS treatment (Figure 6B) but reached a much lower level, 99 pg/mL, when co-stimulated with JWH (P=0.021). In contrast, MIF release was not meaningfully affected (Figure 6C). LPS significantly increased MCP-1 secretion from 149 pg/mL to 184 pg/mL (Figure 6D), and this increase was not significantly altered by co-exposure to either WIN or JWH.
DISCUSSION
Numerous studies have investigated the effects of inflammatory stimuli on MSCs derived from various oral tissues. This research has primarily examined how inflammatory stimuli influence MSC characteristics, including cell proliferation, migration and homing, differentiation, and cytokine secretion [16]. However, the findings are somewhat conflicting. Factors such as the source of the MSCs, the type and intensity of the inflammatory stimulus, and the methodologies used to assess cell growth have likely contributed to the variability in results [1718].
Escherichia coli-derived LPS (Ec-LPS) influences the growth of dental pulp stem cells (DPSCs), with concentration-dependent effects. High concentrations of Ec-LPS (10 μg/mL or higher) generally inhibit the proliferation of DPSCs, while lower concentrations (0.1 μg/mL or higher) appear to promote it [17]. Similarly, the proliferation of bone marrow mesenchymal stem cells (BM-MSCs) is affected by varying concentrations of Pg-LPS. A concentration of 0.1 μg/mL of Pg-LPS fosters cell growth, but a higher level (10 μg/mL) leads to reduced proliferation [18]. Regarding gingival MSCs (GMSCs), prior findings indicate that GMSCs from inflamed tissue exhibit a higher proliferation rate than those derived from healthy tissue [19]. In contrast, the proliferation of dental follicular stem cells is not significantly altered upon exposure to Pg-LPS concentrations ranging from 1 to 50 μg/mL [20].
In the present study, we observed an increase in cell number at low LPS concentrations (ranging from 0.01 to 1 µg/mL) after exposure periods of 7 and 14 days. This proliferation was accompanied by a reduction in metabolic activity per cell. Our results align with previous research, which demonstrated that Pg-LPS stimulation promotes the proliferation of hPDLSCs at concentrations of 0.0001 to 20 μg/mL [321]. Pg-LPS upregulates the expression of cyclins and cyclin-dependent kinases, which are key regulators of cell cycle progression; these include cyclins D1, A, and B1. This upregulation may contribute to the increased proliferation of PDLSCs in response to Pg-LPS stimulation [22].
Conversely, Diomede et al. [22] and Chen et al. [23] observed a decrease in the proliferation of hPDLSCs when stimulated with 1 to 5 µg/mL Pg-LPS. However, the assays used in these studies to measure cell proliferation were based on tetrazolium conversion, which could account for the conflicting results. Tetrazolium assays, such as MTT and CCK8, assess metabolic activity as an indicator of proliferation. Therefore, these methods are not valid for examining compounds that influence cellular metabolism, as demonstrated in the present study.
LPS plays a key role in the breakdown of periodontal tissue in periodontitis. These molecules suppress osteoblastic differentiation, which is crucial for bone formation, and promote the release of harmful proinflammatory cytokines that can exacerbate tissue damage [3]. The potential to regulate these processes is of considerable medical interest. LPS binds to TLR4, triggering inflammatory stress by activating the extracellular-signal regulated kinase and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways [24]. One result is inhibition of the expression of Runt-related transcription factor 2 (Runx2), a master regulator of osteogenesis [23]. This leads to decreases in alkaline phosphatase activity, COL1A1 production, osteocalcin release, and hPDLSC mineralization [3]. Yu et al. [25] observed similar inhibitory effects on osteogenesis at Pg-LPS concentrations of 0.01 µg/mL. These earlier findings are consistent with our own, as we observed that Pg-LPS exposure diminished osteogenic differentiation. Traditional in vitro studies have consistently demonstrated a reciprocal relationship between the commitment of stem or stromal cells to adipogenic and osteogenic lineage pathways. In essence, certain agents may promote or induce differentiation towards a certain lineage, often at the expense of suppressing differentiation into the other [26].
Therefore, Ec-LPS has a dual effect on the differentiation of MSCs from bone marrow and adipose tissue: it suppresses adipogenic differentiation while encouraging osteogenic differentiation [27]. Interestingly, at a concentration of 1 µg/mL, Ec-LPS also exhibits biphasic activity on hPDLSCs, inhibiting osteogenic differentiation but supporting adipogenic and chondrogenic differentiation [28]. However, we did not observe a similar impact on the adipogenic differentiation capacity of hPDLSCs when exposed to Pg-LPS. Likewise, Pg-LPS did not influence chondrogenic differentiation. Thus, it appears that LPS derived from specific bacteria can elicit diverse effects on PDLSCs, despite all variants binding to TLR4.
Separately, Raicevic et al. [29] observed that the functional properties of human MSCs can vary substantially depending on their tissue of origin. They examined the impact of TLR ligation on the phenotype and function of MSCs derived from bone marrow, adipose tissue, and Wharton jelly. The findings revealed considerable variations in the response of these cells to LPS stimulation, particularly concerning cytokine secretion. We propose that the disparate effects of LPS on the differentiation of hPDLSCs compared to other MSCs might also be attributed to differences in cell source. Li et al. [30] reinforced this notion by demonstrating that osteogenesis in PDLSCs and BM-MSCs was differentially influenced by LPS exposure.
Activation of the CB2 receptor led to an increase in the proliferation of hPDLSCs; however, it also reduced their metabolic activity with prolonged exposure. Conversely, ligation of the CB1 receptor did not influence proliferation, but it did increase metabolic activity. Previous research has similarly highlighted the impact of cannabinoid receptors on MSC proliferation. For instance, exposure of mouse adipose tissue-derived MSCs (atMSCs) to the CB1 antagonist rimonabant (Rim) at concentrations ranging from 10 to 400 nM has been shown to markedly decrease cell proliferation [31]. In contrast, human BM-MSCs are responsive to concentrations greater than 30 μM [32]. Additionally, treatment with Δ9-tetrahydrocannabinol (THC), a non-selective agonist for CB1/2 receptors, has been shown to reduce the proliferation of murine BM-MSCs [33].
We observed a CB1-dependent decrease in the count of human atMSCs with increasing concentrations of WIN, which is antagonized by blocking CB1 [11]. In contrast, CB2 activation leads to an increase in the number of atMSCs, aligning with the present findings on CB2 agonism in hPDLSCs. Nevertheless, the specific intracellular mechanisms by which cannabinoid receptors affect MSC proliferation remain unclear and require further investigation.
Exposure to WIN significantly increased lipid staining, while activation of the CB2 receptor by JWH did not affect adipogenesis. Consequently, adipogenesis in hPDLSCs may be regulated by the CB1 receptor. This phenomenon has been observed in other types of MSCs [1112]. Pagano et al. [34] found that exposure of atMSCs to WIN results in increased glucose uptake, calcium influx, and PPAR-γ expression, which collectively promote adipogenic differentiation. We propose that a similar mechanism may be responsible for the effect of WIN on adipogenesis in hPDLSCs. However, our findings contrast with those of Yan et al., [36] who reported that silencing the CB1 gene in hPDLSCs suppresses osteo/dentinogenic differentiation, whereas overexpression of CB1 produces the opposite effect. These conflicting findings are challenging to reconcile, but they may highlight the fundamental differences between genetically modified models and pharmacological compounds, both of which can have the drawback of unintended off-target effects. Given that CB1 plays a critical role in the viability of various cell types (as mentioned previously), it is plausible that the observed differences in adipogenesis in CB1-silenced or overexpressing cells could be due to secondary effects on cellular proliferation, an aspect not addressed in the report by Yan et al. [35].
Increased osteogenic differentiation of osteoblasts has been reported to be stimulated by both CB1 and CB2 activation [36]. Moreover, based on existing studies, it is highly probable that the activation of CB receptors also promotes osteogenic differentiation in various cell types [3336]. Osteoblast precursor cells exhibit increased alkaline phosphatase activity and matrix mineralization upon CB2 activation [37]. In this study, the administration of JWH, a highly selective CB2 agonist, led to increased osteogenic differentiation of hPDLSCs. These findings align with those of Qian et al. [38], who demonstrated that the application of HU-308, a highly CB2-specific agonist, upregulated osteogenic genes in hPDLSCs. Those genes included runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP), osteopontin (OPN), alkaline phosphatase (ALP), osteocalcin (OC), and collagen type I (COL I). Ultimately, this results in calcium depletion and osteogenic differentiation.
On the contrary, neither WIN nor JWH appears to impact the osteogenic differentiation of atMSCs [11]. Research has suggested a potential inverse relationship between adipogenic and osteogenic differentiation in MSCs, as previously discussed. Therefore, these cells’ inherent propensity for osteogenic differentiation—stronger relative to atMSCs—coupled with a reduced inclination toward adipogenic differentiation could account for the increased osteogenic differentiation observed in hPDLSCs following CB2 activation. Stimulating the osteogenic differentiation of periodontal cells and promoting the regeneration of periodontal tissue is viewed as a promising therapeutic strategy to mitigate the harmful effects associated with periodontitis.
Our understanding of how cannabinoids affect bone remodeling and osteogenesis is well-developed; however, their impact on cartilage tissue has not been as extensively studied or understood [36]. Few studies have investigated the effects of cannabinoid exposure on chondrogenesis. Research measuring collagen II expression and proteoglycan deposition in the extracellular matrix has demonstrated that THC can promote chondrogenic differentiation [39].
Cannabinoids are believed to modulate the activation of the signal transducer and activator of transcription pathways. These then regulate the expression of SOX9, the master regulator of chondrogenesis, along with other components specific to the cartilage matrix [40]. Consequently, chondrogenic differentiation in atMSCs appears unaffected by CB2 receptor ligation. However, exposure to 3 μM WIN results in an observed increase in chondrogenic differentiation, indicating that the CB1 receptor may participate in chondrogenesis [11]. Nevertheless, these findings are at odds with the effects of WIN and JWH on chondrogenic differentiation of hPDLSCs in the present study, as both receptor ligands inhibited chondrogenic differentiation. These disparate results suggest that the ECS may elicit varying effects on MSC differentiation, depending on their tissue of origin.
As discussed above, Ec-LPS inhibits adipogenesis in atMSCs. This inhibition can be reversed by CBD, presumably through its interaction with PPAR-γ, the master regulator of adipogenesis [27]. We observed an osteogenesis-inhibiting effect of Pg-LPS in hPDLSCs, whereas activating CB2 increased osteogenic differentiation; thus, we examined the possibility of an antagonistic effect. However, CB2 activation by JWH did not restore the osteogenic differentiation potential of the cells. While CB2 activation does increase Runx2 activity, osteogenesis is not solely dependent on this master regulator, which may explain why the LPS effect was not mitigated or reversed. It is possible that a higher concentration of the CB2-receptor ligand could have more effectively antagonized the osteogenic inhibition by LPS.
Pg-LPS triggers the release of inflammatory cytokines by hPDLSCs, which amplifies the inflammatory response in the oral cavity. The release of pro-inflammatory cytokines is mediated by the NF-kB pathway [3]. Over time, such persistent inflammation is associated with the degradation of periodontal tissue [3]. Consequently, targeting factors that influence this process may provide a basis for the development of new therapeutic options. The ECS plays a key role in modulating the immune system by regulating cytokine release, thereby helping to suppress inflammatory responses. Thus, managing oral inflammation through the activation of the ECS represents a potentially promising therapeutic approach.
The endocannabinoid anandamide (AEA), which binds to CB1, CB2, and other non-classical cannabinoid receptors, has been demonstrated to inhibit NF-κB activation and reduce the production of pro-inflammatory cytokines in human gingival fibroblasts upon exposure to Pg-LPS [41]. This anti-inflammatory effect of AEA, as well as that of 2-AG, on hPDLSCs has been supported by findings from other researchers [42]. However, the precise roles of CB1 and CB2 receptors in this context remain uncertain, as only nonselective cannabinoid agonists have been tested to date. In the present study, we confirmed that Pg-LPS stimulation elevates the release of pro-inflammatory cytokines. We observed that JWH, but not WIN, reduced the secretion of the inflammatory cytokines IL-6 and IL-8. This suggests that the anti-inflammatory activity may be attributed to selective CB2 activation, likely through the inhibition of the NF-κB pathway, as previously demonstrated for AEA.
The findings are consistent with prior research, which indicates that activation of the CB2 receptor is linked to reduced secretion of pro-inflammatory cytokines in various cell types [43].
Pg-LPS exhibits concentration-dependent effects on the proliferation, metabolic activity, and differentiation potential of hPDLSCs. Concentrations of 0.1 µg/mL or less exclusively inhibited osteogenesis, while higher concentrations, above 0.1 µg/mL, promoted proliferation with long-term stimulation. Notably, even at a very low concentration of 0.01 µg/mL, Pg-LPS increased the release of pro-inflammatory cytokines. Given that persistent inflammation is harmful to periodontal tissues, we investigated the regulatory role of the ECS in hPDLSCs. Activation of CB1 led to increased metabolic activity and adipogenic differentiation, whereas CB2 ligation promoted proliferation and osteogenic differentiation. Moreover, our findings suggest that CB2 activation may not sustain bone tissue regeneration by restoring osteogenic differentiation in LPS-stimulated hPDLSCs. In contrast, CB2 ligation was effective in suppressing the release of inflammatory cytokines, which could help further the treatment of periodontitis.
This study explored the concentration-dependent effects of Pg-LPS on the cellular characteristics of hPDLSCs. At lower concentrations, Pg-LPS was associated with increased cell proliferation, while at higher levels, it inhibited osteogenic differentiation. Importantly, activation of the CB2 cannabinoid receptor appeared to mitigate some of the detrimental impacts of Pg-LPS by promoting osteogenesis and significantly reducing the secretion of pro-inflammatory cytokines. These findings not only emphasize the importance of CB2 receptor modulation in reducing inflammation but also highlight its potential role in facilitating periodontal regeneration. Thus, therapies targeting the CB2 receptor could offer a two-pronged approach to treating periodontitis, addressing both the underlying pathology and the symptoms of the disease.
Moreover, the differential effects observed upon activation of the CB1 and CB2 receptors underscore the potential for targeted therapeutic approaches that harness the specific anti-inflammatory and regenerative properties associated with CB2 activation. This specificity could improve treatment outcomes while minimizing the adverse effects commonly associated with more generalized cannabinoid therapies. The promising implications of these findings for periodontal therapy underscore the necessity for additional research. Various methods of application, including topical forms like toothpaste or gels as well as systemic routes such as tablets, should also be investigated to determine the most effective and practical mode of delivery.