1 INTRODUCTION

Smoking constitutes a risk factor for many serious ailments, including cancer, cardiovascular, obstructive pulmonary, and periodontal diseases.1 Electronic cigarettes (ECs) are a drug delivery system that has been originally introduced as a safe substitute to conventional cigarette smoking and as one of the aids to help in smoking cessation.2 It may release nicotine among other chemicals, such as propylene glycol, vegetable glycerin, and flavoring agents in the form of aerosols, while eliminating all chemicals released due to combustion by conventional cigarettes.3 Although its most important feature is the elimination of harmful combustion products, its safety and long-term effects on human body are still not fully elucidated.1-5 Currently, 147 million persons (2.5%) are using Cannabis for different reasons, including recreational and medical reasons, for pain control, or for treating neurological conditions including epilepsy.6 In Egypt, Cannabis is one of the most common illegal drugs abused.7 In a survey on drug abuse in young aged population, hashish was the most used representing almost 90% of drugs abused.6, 7 Gingival and oral tissues are the portal of entry of Cannabis and hence the first affected.6 Nevertheless, the exact mechanism by which Cannabis causes periodontal destruction is not fully understood.6, 8

Gingival tissues are the first to come in contact with the smoke/aerosol6; they are readily accessible and known for their remarkable wound healing properties without scarring.9 Gingival fibroblasts are the most abundant cells in the gingiva.10 Studies demonstrated that gingival connective tissue could be a reservoir for gingival fibroblast/gingival mesenchymal stem cells (GF/G-MSCs) with remarkable regenerative attributes11-14 and resistance to infection and inflammation.15-17 Previous studies demonstrated that smoke and aerosol extracts result in reduced proliferation and impaired function of gingival fibroblast,18 and mesenchymal stem cells,19 with nicotine and other by-products being primarily responsible for this hindered function.18, 20

The aim of the present study was to evaluate and compare the effects of EC aerosol, Cannabis, and conventional cigarettes smoke on the proliferation, viability, and pluripotency gene expression profile (ATM, P21, Oct4, and Nanog) of human GF/G-MSCs of never smokers.

2 MATERIAL AND METHODS 2.1 Study design and settings

The current study was approved by the ethical committee of the Faculty of Dentistry, Cairo University (IRB:18|2|28). After signing the formal written informed consent, gingival biopsies were obtained from 32 healthy non-smoker patients (n = 8/group) (male/female: 3/29) during routine surgical periodontal treatment at the Department of Oral Medicine and Periodontology, Faculty of Dentistry, Cairo University.

2.2 Electronic cigarette aerosol, Cannabis, and cigarette smoke extracts preparation

Generation of EC aerosol, Cannabis, and cigarette smoke was done as described before.2 Cigarettes used were widely available in the market (Cleopatra, 20 pieces with filter in soft pack with cellophane tar 15 mg/cigarette, nicotine 1.0 mg/cigarette, Eastern Company). Cannabis used was in the form of custom-made joints containing 0.25 g of Cannabis (hashish) rolled in a tobacco cigarette.21 A third generation EC device was used to generate the aerosol using e-liquid purchased from the local market (milk and honey flavored; The Illegal Factory) that consisted of 50% vegetable glycerin, 50% propylene glycol, and 3 mg nicotine adjusted on 2.6 V and 24 W. The EC containing the liquid was attached to a 60 ml syringe carrying 10 ml of RPMI 1640 medium + 10% fetal bovine serum + Todd-Hewitt Broth. Activation of EC occurred by applying negative pressure through the syringe, and 50 ml of aerosol was bubbled through the media, followed by syringe agitation on a shaker for 12 s. The aerosol was expelled, and the procedure was repeated for 30 puffs to give 10 ml of media exposed to 1.5 L of aerosol representing the puffs' volume consumed during the vaping session,22 over a duration of 15 min. Similar to previous study,2 1.5 L of aerosol extract was used to reproduce the cigarette smoke extract preparation using one cigarette. All batches of aerosol extract were made following this procedure (Supplement Figure 1).

Medias obtained from EC aerosol were sterilized and used immediately (EC group). For the Cannabis (Can) joints and conventional cigarettes (Cig) smoke extracts, the same method of smoke extraction was used to consume one cigarette/joint.23 Briefly, using negative pressure, 50 ml smoke was added to a syringe with 10 ml medium, followed by 12 s of syringe agitation, then the smoke was expelled resulting in Can or Cig smoke extract (Supplement Figure 2).

One milliliter of the freshly prepared aerosol/smoke extracts was added every third day to the cells for 2 weeks.19

2.3 Determination of nicotine, cannabidiol (CBD), and polyaromatic hydrocarbons (PAHs) concentration in the aerosol/smoke extract 2.3.1 PAHs determination using gas chromatography-mass spectrometry (GC-MS) Materials

Fourteen PAHs stock solution (1000 µg/ml) containing naphthalene, fluorene, fluoranthene, benz(a)anthracene, chrysene, pyrene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, acenaphthene, phenanthrene, anthracene, acenaphthylene, and pyrene-d10 (surrogate standard), and reference standards were obtained from Sigma-Aldrich with purity >95%, whereas benzo(g,h,i)perylene and dibenz(a,h)anthracene were purchased as readymade preparations of 100 µg/ml in methylene chloride and indeno[1,2,3-cd]pyrene 200 µg/ml in methanol.

Sample extraction and Chromatographic condition

Following extraction using acetonitrile, three samples/groups were analyzed using GC-MS. The analytical parameters were performed according to a previous study24 and reported in Table 1. The detection limit was 2 µg/L.

TABLE 1. Analytical system and parameters used for the identification of 16 PAHs in EC aerosol/Cannabis and cigarette smoke extracts preparations using GC-MS. (Analysis was performed at Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Food (QCAP), Agricultural Research Center, Ministry of Agriculture and Land Reclamation, Giza, Egypt) PAHs in EC aerosol/Cannabis and cigarette smokes extract GC-MS (Agilent 6890 series gas chromatography/5975 series mass selective detector) (US) Column Agilent GC Column (J&W HP−5 ms Ultra Inert (30 m length, 0.25 mm internal diameter, 0.25 µm film thickness) (US) Injection mode Split-Splitless Injection temperature 260 °C Injection volume 1 µl Carrier gas Helium 99.999%, constant flow Flow rate 1.3 ml/min Oven 90°C (hold 2 min) to180°C at an increasing rate of 15°C/min (hold for 15 min), then to 250°C at an increasing rate 10°C/min (hold 2 min), and then to 290°C as a final temperature at an increasing rate of 10°C/min (hold 10 min) Source temperature 250°C Interface temperature 280°C Run time 36 min Acquisition mode SIM mode Scan range 45–450 m/z 2.3.2 Nicotine and CBD determination using high performance liquid chromatography (HPLC) method Materials

(−) – Nicotine, ˃99%, was obtained pure from Sigma Aldrich. Certified 1.0 mg/ml CBD stock solution from Cerilliant Corporation (Sigma Aldrich) was used. Reagents and solvents used were of analytical reagent and HPLC grade, respectively.

Sample extraction and chromatographic condition

High performance liquid chromatography analysis was performed using an Agilent 1260 series Infinity ǀǀ LC system (Agilent Corporation). A Zorbax Eclipse Plus (Sigma Aldrich) C18 column (4.6 × 250 mm i.d., 5 μm) was used. Chromatographic parameters of the HPLC method for nicotine25 and CBD26 are listed in Table 2.

TABLE 2. Chromatographic parameters of the HPLC method for nicotine and CBD (Analysis was performed at the Chromatography lab, at the National Research Center, Giza, Egypt) Parameters Conditions for nicotine Conditions for CBD Solvent used for sample extraction Dichloromethane Methanol 90% Mobile phase acetonitrile (ACN): sodium hydrogen carbonate (25:75, v/v) (pH = 10.0.03 M) Methanol 90% Flow rate condition Isocratic method Isocratic method Flow rate 1 ml/min 1 ml/min Column temperature 40°C 25°C Injection volume 10 μl 5 μl Ultraviolet detector 250 nm 220 nm Run time 13 min 8 min 2.4 Isolation and identification of GF/G-MSCs

Gingival fibroblast/gingival mesenchymal stem cells were isolated as described before.27 Tissues were de-epithelialized and cut into sections (2 × 2 mm), rinsed with RPMI 1640 medium (Thermo Fischer Scientific), and antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin; Biochrom) were added. The samples were placed into dry 50 ml culture flasks (Gibco, Thermo Fisher Scientific) for 30 min allowing adherence. The basic medium consisted of RPMI 1640 medium supplemented with 15% fetal calf serum (HyClone), 400 mmol/ml L-glutamine (Biochrom), 100 µg/ml penicillin, and 100 mg/ml streptomycin (basic medium). The flasks’ incubation was in 5% carbon dioxide at 37°C. Phase contrast inverted microscopy was used to check the flasks, and media were changed every third day.

The cells were washed with 5 ml of phosphate buffered saline (PBS; Biochrom) after reaching 80%–85% confluence. Two milliliters of 0.10% trypsin/ethylenediaminetetraacetic acid (Biochrom) were added, and the culture flasks were incubated for 10 min allowing cells detachment, followed by the addition of 5 ml of the basic medium. The cells-containing medium was transferred to sterile 50 ml polypropylene conical tubes (Becton Dickinson) and centrifuged at 643 g for 10 min. The supernatant was discarded, and the cells were resuspended in 2 ml basic medium. Cells counting and viability testing were done using MTT assay as described before.28

2.5 Flow cytometric analysis

After reaching confluence, third passage GF/G-MSCs were characterized by flow cytometry. Primary antibodies against human CD29, CD34 (eBioscience), CD45, CD73, CD90, and CD105 (Miltenyi Biotec GmbH) were used. Cells were stained with fluorescein-isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated primary antibodies at room temperature. FITC- or PE-conjugated human IgGs were used as isotype controls at a concentration similar to the specific primary antibodies.27 The analyses were done using Cytomics FC 500 Flow Cytometer (Beckman Coulter) and analyzed using CXP Software version 2.2.

2.6 Multilineage differentiation assays

Third passage GF/G-MSCs were cultured in osteogenic medium for 14 days, in adipogenic medium for 21 days, and in chondrogenic medium for 35 days or in basic medium for the same durations as respective controls. Osteogenic differentiation was assessed, using Alizarin Red staining. Areas of osteogenic mineralization were observed as dark orange-red patches, using the inverted light microscopy.19 Intracellular presence of lipid drops indicating adipogenic differentiation was evaluated, using Oil Red O stain. Chondrogenic pellets formed were fixed with 4% formaldehyde solution, rinsed with PBS, and stained with 1% Alcian Blue solution prepared in 0.1 N HCL for 30 min.29

2.7 Experimental groups The study groups comprised GF/G-MSCs obtained from healthy non-smokers. Cells were grouped according to the aerosol/smoke extract to which they were exposed into four groups: EC group: GF/G-MSCs were exposed to EC aerosol extract, Cig group: GF/G-MSCs were exposed to cigarette smoke extract, Can group: Cannabis smoke extract was applied to the GF/G-MSCs, and Control group: only basic medium was added to the GF/G-MSCs. 2.8 Colony-forming unit assay

Cells from all groups were cultured. Clusters of 50 or more cells were recorded as colonies (CFU-F). On the twelfth day, cultures were fixed using 4% formalin, and colonies' numbers were counted and statistically evaluated.29

2.9 MTT assay

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to evaluate the metabolic activity of GF/G-MSCs. Seeding was in duplicates onto flat bottom 96-well plates for all groups. Cells in 90 µl media were incubated with 10 µl of 5 mg/ml MTT substrate solution, thiazolyl blue tetrazolium bromide salt (Sigma- Aldrich) for 4 h. Stopping the reaction occurred by adding 100 µl of solubilizing solution (12 mmol/L HCl, 346 mmol/L SDS, and 5% isobutanol) and incubation for 15 min at room temperature. Absorbance was measured at OD450 nm using a microplate reader 19.

2.10 Mitochondrial membrane potential

In all groups, mitochondrial membrane potential (ΔΨm) was assessed using Mitochondrial Permeability JC-1 dye (JC-1 Mitochondrial Membrane Potential Assay Kit, Cayman Chemical Company). After reaching 75%–80% confluence, cells were cultured in 96 well culture plate at a density of 5 × 104–5 × 105 cells/well in 100 µl of culture medium. Ten microliter of JC-1 staining solution was added to each well and gently mixed. Cells were incubated in carbon dioxide incubator at 37°C for 20 min. The culture plate was centrifuged for 5 min at 400 g at room temperature, and the resultant supernatant was carefully aspirated. Two hundred microliters of assay buffer was added to each well followed by plate centrifuge for 5 min at 400 g at room temperature; the resultant supernatant was also carefully aspirated. The last step was repeated twice, followed by adding 100 µl of assay buffer to each well. Fluorescent plate reader was used to directly analyze the cells. Healthy cells with mainly JC-1 J-aggregates (red) were detected with filter sets designed to detect rhodamine (excitation/emission =540/570 nm), while apoptotic or unhealthy cells with mainly JC-1 monomer (green) were detected using filter sets designed to detect FITC (excitation/emission =485/535 nm). Changes in the ΔΨm were determined as aggregates:monomer ratio, using a fluorescent plate reader.30

2.11 Molecular analysis

Total mRNA were extracted from GF/G-MSCs, using GeneJET RNA Purification Kit (Thermo Scientific), according to the manufacturer's instructions 14 days following the exposure in the respective groups. The extracted mRNA was reversely transcribed with the RevertAid first strand cDNA synthesis kit (Thermo Scientific), using random primers according to the manufacturer's instructions. Using Step One Real-Time PCR system (Applied Biosystems), cDNA was amplified. The cycling conditions were 95°C for 3 min then 40 amplification cycles (95°C denaturation for 3 s, annealing for 30 s at the primers' melting temperature, and extension at 72°C for 30 s), followed by a final cycle at 72°C for 5 min. Relative expression of target genes was conducted according to the comparative ∆∆Ct method, using human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference gene for normalization. Gene expression levels were evaluated for ATM, P21, Oct4, and Nanog (Table 3).

TABLE 3. Primers used for RT-PCR and their IDs Gene Sequence Accession ID ATM

Forward

Reverse

5′-TCCTGCAGTATGCTGTTTGACTTTGGC−3′

5′-CTGTGAAGAATTGGAGGCACTTCTGTGC−3′

NM_000051.4 P21

Forward

Reverse

5′-TGTCCGCGAGGATGCGTGTTC−3′

5 ′-GCAGCCCGCCATTAGCGCAT−3′

NR_164656.1 Oct4

Forward

Reverse

5′-TTC TCA GGG GGA CCA GTG TC−3′ LC006948.1 5′-CCC ATT CCT AGA AGG GCA GG−3′ Nanog

Forward

Reverse

5′-CCA GTG ACT TGG AGG CTG C−3′ NG_054758.1 5′-AAG GAT TCA GCC AGT GTCC−3′ 2.12 Statistical evaluation

Data were coded and entered using IBM SPSS Statistics for Windows, Version 22.0. IBM Corp. Released 2015. Data were checked for normality using Kolmogorov-Smirnov and Shapiro-Wilk tests. Normally, distributed data were expressed using means and standard deviation. Comparisons between the four studied groups were done using analysis of variance with multiple comparisons post hoc test. Non-parametric data were expressed using medians and range, and comparisons were done using Kruskal-Wallis test. Statistical significance was considered when p < .05.

3 RESULTS 3.1 Gingival tissues donors

Gingival tissues were obtained from 32 patients. Male to female ratio was 3:29 with mean age of 21 ± 2.6 years. Each group consisted of eight patients.

3.2 GF/G-MSCs characterization

Third passage GF/G-MSCs showed plastic adherent fusiform morphology (Figure 1A). Cells showed successful osteogenic, adipogenic, and chondrogenic differentiation. Osteogenic differentiation was evident by the calcium deposits stained with Alizarin Red stain. Adipogenic differentiation was demonstrated by the presence of intracellular lipid droplets stained with Oil Red O stain. Chondrogenic differentiation was shown by the formation of glycosaminoglycans stained with Alcian Blue stain (Figure 1B). GF/G-MSCs were positive for CD29, CD73, CD90, and CD105 and negative for CD34 and CD45 (Figure 1C).

Phase contrast inverted light microscopy images (magnification 200x) showing fusiform morphology of GF/G-MSCs. EC, Cig, and Can smoke extracts alter cells morphology. High magnification DIC images are shown in the lower panel (1A). Phase contrast inverted light microscopy images (magnification 100x) showing multilineage differentiation potential of GF/G-MSCs (1B) GF/G-MSCs control (a) and their Alizarin Red staining of the osteogenic stimulated GF/G-MSCs (b). GF/G-MSCs control (c) and their Oil Red O staining of the adipogenic stimulated GF/G-MSCs (d). GF/G-MSCs control (e) and their Alcian Blue staining of the chondrogenic stimulated GMSCs (f). Flow cytometric analysis (1C)

3.3 Nicotine, CBD, and PAHs concentration in the aerosol/smokes extract

The standard curve of nicotine showed linearity in the range of 12.5–50 µg/ml with r2 = .999, while that of CBD was in the range of 2.5–10 µg/ml, with r2 = .999 as obtained from the linear regression analysis. The analysis revealed a statistically significant difference between the median concentration of nicotine in the EC aerosol extract (1.7 (1.35–2.25) µg/ml) (median (minimum-maximum)), cigarette smoke extract (8.2 (6.53–9) µg/ml), and Cannabis smoke extract (4.17 (3.65–4.31) µg/ml; p = .02). Median CBD concentration in the Cannabis smoke extract was 0.59 (0.45–1.4) µg/ml.

The standard curve of PAHs showed linearity in the range of 2–500 µg/L with r2 = .998. The total of 16 PAHs (Ʃ16 PAHs) concentration as Benzo(a)pyrene was calculated according to the total equivalent toxic factor for conversion.31 The median Ʃ16 PAHs concentration as Benzo(a)pyrene in Cannabis smoke extract was 0.883 (0.501–1.414) µg/L. All analytes were below the detection limit in the EC aerosol and the cigarettes smoke extracts.

3.4 Cellular proliferation, mitochondrial membrane potential, and viability following smoke and aerosol exposure

Compared to the control group (14.3 ± 3), CFU-Fs were significantly reduced in the Cig (5.5 ± 1.5, p < .00001) and Can groups (2.3 ± 1.2, p < .00001), with no statistically significant difference to the EC group (10.8 ± 2.5, p = .05). CFUs in Cig and Can groups were significantly different to the EC group (p = .001 and p < .00001, respectively; Figure 2A).

Colony-forming unit assay (A). Comparison of mitochondrial membrane potential in all studied groups (B). Comparison between cellular viability of all studied groups by MTT assay at 24 and 48 h (C). Comparison between cellular viability of all studied groups by MTT assay at 48 h (D). Data were expressed as Mean ± SD; p value < .05 was significant (n = 8). * denotes significant difference versus control group, # denotes significant difference versus EC group, and $ denotes significant difference versus Can group

Mitochondrial membrane potential assay showed significant decrease of JC-1 aggregates fluorescence ratio, indicating reduced mitochondrial potential in Can (0.47 ± 0.05, p = .001) and Cig (0.36 ± 0.04, p = .001) compared to control group (1.74 ± 0.26), with no difference to the EC group (1.6 ± 0.4, p = .09). Moreover, Can and Cig groups exhibited significant decrease compared to EC group (p = .008 and p = .006, respectively; Figure 2B).

MTT assay showed significant reduction in cell viability (OD450 nm) at 24 and 48 h in Can (0.47 ± 0.21, 0.77 ± 0.28) and Cig (0.59 ± 0.13, 0.85 ± 0.46) compared to control group (1.33 ± 0.12, 2.06 ± 0.5, p < .05), with no difference to the EC group (1.27 ± 0.47, 2.17 ± 0.69, p = .9). Can and Cig groups showed a significantly lower cellular viability compared to EC (p < .001; Figure 2C).

3.5 Gene expression

Significant upregulation of ATM gene expression (gene copies/GADPH) was notable in Can (1.52 ± 0.75) and Cig (1.5 ± 0.42) compared to the control group (0.46 ± 0.26; p = .001), with no elevation in the EC group (0.63 ± 0.1, p > .05). Compared to EC group, Can and Cig groups exhibited significant upregulation in ATM expression (p = .001), with no difference between Can and Cig groups (p > .05). EC (1.22 ± 0.46), Can (0.71 ± 0.24), and Cig groups (0.83 ± 0.54) downregulated p21 gene compared to the control group (2.25 ± 1.74, p = .053). Can (0.7 ± 0.14) and Cig (0.82 ± 0.44) significantly upregulated Oct4 gene expression compared to the control group (0.21 ± 0.06, p = .001). Only Cig significantly upregulated Oct4 gene expression compared to EC group (0.31 ± 0.12, p = .01).

Can (1.48 ± 0.79) and Cig (1.54 ± 0.52) significantly upregulated Nanog gene expression compared to the control group (0.62 ± 0.18, p = .007), while EC (0.64 ± 0.46) group failed to show significant difference (Figure 3).

Comparison of p21 gene (A), ATM gene (B), Oct4 gene (C), and Nanog gene (D) expression in all studied groups. Data were expressed as Mean ± SD, p value < .05 was significant. *Denotes significant difference versus control group; # denotes significant difference versus EC group

4 DISCUSSION

Gingival fibroblast/gingival mesenchymal stem cells' are key players in periodontal repair and regeneration.13, 32 Their unique properties make them ideal candidate for tissue engineering, regenerative procedures, and cellular and gene therapies.13, 33 Previous investigation suggested that cigarette smoke increases the permeability of the oral epithelial cells by upregulating the desquamation and affecting the tight junctions between cells, leaving the underlying GF/G-MSCs prone to the negative effects of smoke.34 The present study aimed to evaluate and compare the effects of EC aerosol, Cannabis, and cigarettes smoke on the proliferation, viability, and gene expression profile (ATM, P21, Oct4, and Nanog) of GF/G-MSCs of never smokers.

Similar to earlier studies, GF/G-MSCs were positive for CD29, CD73, CD90, CD105, while being negative for CD34 and CD45.13, 14 Moreover, they showed the capacity of colony formation, along with remarkable multilineage differentiation ability into osteogenic, adipogenic, and chondrogenic directions.14, 29

The present investigation demonstrated a significantly reduced CFU-F ability in GF/G-MSCs exposed to cigarette smoke compared to controls, in accordance with a previous study on CFU-F from mice stem cells35 and mice embryonic stem cells.36 It could be attributed to several mechanisms, including the oxidative stress resulting in reactive oxygen species (ROS) production, which affects the equilibrium between proliferation and apoptosis, genetic damage, as well as the effect of the aldehydes content, resulting in reduced defense mechanisms, cell death, and direct cell membrane damage.37 To our knowledge, this is the first study on the effect of Cannabis (hashish) smoke on GF/G-MSCs. The detrimental effect of the Cannabis smoke on the colony formation ability of GF/G-MSCs could be partly attributed to the nicotine content in the joint and the negative effect of CBD on cell proliferation,38 in addition to 9-tetrahydrocannabinol (THC) as a mitochondrial inhibitor, negatively affecting their viability and proliferation,39, 40 with the least viability notable in the Cannabis group. Additionally, cellular exposure to Cannabis smoke results in reduced levels of IL-8 and ΔΨm, which are involved in wound healing.40 Yet, the current findings are in contrast to previous results by Sazmand et al.,41 where the addition of different concentration of Cannabis to rat bone marrow MSCs resulted in an increased cell viability compared to the unexposed controls at 24 h period. The results could be attributed to the beneficial effect of Cannabis on growth factors, as well as its antioxidant and anti-inflammatory effects. The contradictory results seen in the present study during the 24 h period might be due to the different method of Cannabis application: plant in powder form in the previous study versus smoke in the present study allowing the delivery of harmful combustion products in addition to the THC.42 In the present study, the EC group demonstrated the highest cellular viability and a nonsignificant reduction in the number of CFU-F compared to the control group. These findings are in agreement with Shaito et al.,19 demonstrating that the effects of EC aerosol on MSCs proliferation were significantly lower than those of cigarette smoke. It was suggested that EC aerosol could have a cytotoxic effect on human cells, resulting in necrotic cell death.2 Necrotic cells lose the integrity of their membrane, releasing their intracellular content, stimulating pro-inflammatory cytokines production such as IL-1, IL-6, IL-8, and PGE2,43, 44 which could amplify the reparative actions of stem cells, and upregulating their proliferation and differentiation capacities.16 This could explain the similarity in the cells proliferation rate between the EC and control groups. This could be further attributed to the low levels of nicotine in the EC aerosol extract, thus reducing its negative effects on the cells’ proliferation.

ΔΨm is a key factor that evaluates mitochondrial function, and reduced ΔΨm with increased depolarization is connected to apoptosis.30, 45, 46 In that context, control and EC groups demonstrated similar results in agreement with a previous investigation.30 On the contrary, a recent investigation showed reduced ΔΨm and increased mitochondrial depolarization, when epithelial cells were exposed to cinnamon flavored EC aerosol.47 The difference between the results could be attributed to the cinnamon flavor, being an aldehyde capable of reacting with propylene glycol to form acetals resulting in oxidative stress.

The ATM gene has a crucial role in repairing double strands breaks of DNA, while the p21 gene inhibits DNA synthesis and plays a role in apoptosis and DNA repair.48 The least ATM gene expression was observed in EC group suggesting the absence of DNA damage and reduced amount of ROS production, whereas the Cannabis and cigarette groups showed significantly higher expression, indicating the role of ATM gene in repairing damaged DNA,49 as well as its role in controlling ROS levels,50 thus protecting other biomolecules in the cells from the oxidative damage caused by cigarette and Cannabis smoke.45, 51 The current findings contradict a previous investigation, where ATM gene expression was significantly downregulated in the epithelial cells of the oral mucosa of chronic smokers, which could be attributed to the advanced maturation stage of epithelial cells and their lower metabolic activity compared to the GF/G-MSCs used in this study.52

Normally, p21 gene is downregulated in response to severe DNA damage, to allow the induction of the apoptotic process.53 In accordance with an earlier investigation,54 present results revealed that the lowest p21 gene expressions were in the Cannabis and cigarette groups, which can be attributed to the high levels of oxidative stress released by the smoke. These findings contradicted a previous study,55 where an upregulation of p21 gene expression was observed in mouse lung cells exposed to cigarette smoke. This difference might be explained by the variation in cell types (GF/G-MSCs versus lung cells), the culture condition (in vivo vs. in vitro), and the method of exposure to smoke.

Oct4 and Nanog are pluripotency genes regulating and maintaining the cellular pluripotency and self-renewal. Their expression is limited to pluripotent cells and is reduced upon differentiation.56 Oct4 and Nanog genes should be maintained in a balanced state.57, 58 The EC group showed almost similar expression to the control group, suggesting no effect of ECs on the pluripotency of GF/G-MSCs. In accordance with a recent investigation,59 the highest value of Oct4 and Nanog genes were found in the cigarettes and the Cannabis groups. This upregulation of the pluripotency markers may direct the cells’ dedifferentiation into a more primitive form, which eventually could result in malignant transformation.60

The results presented here should be cautiously interpreted in light of the current study's limitations. First, using positive control groups like gingival tissues collected from smokers/vapers could have made the present results more credible and could be recommended for future studies. Second, examining multiple concentrations of the used extracts would have allowed further investigation of the effects of the different chemical compounds on the used cells. Third, the exploration on protein level, not only their mRNA, could have been conducted to give a better understanding of the cells’ response to these chemicals. Finally, the in vitro trials never full reproduce the complex biodynamic systems of the human body.

5 CONCLUSION

The present investigation shows that Cannabis and cigarette smoke extracts negatively affect the cellular proliferation, viability, and mitochondrial membrane potential of GF/G-MSCs, in contrast to the specifically used concentration of EC aerosol extract in the current investigation, which had less effect on these attributes. The difference observed in gene expression further underlines the damaging effects of the Cannabis and cigarette smoke extracts on the GF/G-MSCs, driving their dedifferentiation. Within the limits of the current investigation, an adverse change in GF/G-MSCs mediated healing process could be anticipated in Cannabis and cigarette users.

CONFLICT OF INTERESTS

The authors declare no conflict of interest.