The purpose of this study was to determine the possible deleterious effects of e-cigarette vapor on osteoblast interaction with dental implant material. Osteoblasts were cultured onto Ti6Al4V titanium implant disks and were then exposed or not to whole cigarette smoke (CS), as well as to nicotine-rich (NR) or nicotine-free (NF) e-vapor for 15 or 30 minutes once a day for 1, 2, or 3 days, after which time various analyses were performed. Osteoblast growth on the titanium implant disks was found to be significantly (P < .001) reduced following exposure to CS and to the NR and NF e-vapors. Osteoblast attachment to the dental implant material was also dysregulated by CS and the NR and NF e-vapors through a decreased production of adhesion proteins such as F-actin. The effects of CS and e-cigarette vapor on osteoblast growth and attachment were confirmed by reduced alkaline phosphatase (ALP) activity and tissue mineralization. The adverse effects of CS and the NR and NF e-vapors on osteoblast interaction with dental implant material also involved the caspase-3 pathway, as the caspase-3 protein level increased following exposure of the osteoblasts to CS or e-vapor. It should be noted that the adverse effects of CS on osteoblast growth, attachment, ALP, and mineralized degradation were greater than those of the NR and NF e-vapors, although the latter did downregulate osteoblast interaction with the dental implant material. Overall results suggest the need to consider e-cigarettes as a possible contributor to dental implant failure and/or complications.
There are an estimated billion smokers worldwide, along with millions more who use various oral tobacco products.1 The concern regarding the negative effects of cigarette smoke is linked to the chemicals released during cigarette smoking, such as nitrogen, carbon monoxide, carbon dioxide, ammonia, hydrogen cyanide, benzene, and nicotine, among others.2 Smoking is associated with various human diseases, including coronary heart disease, stroke, subclinical atherosclerosis, chronic obstructive pulmonary disease, pneumonia, and various cancers.3
The oral cavity is the first site to come in contact with cigarette smoke. It has been reported that smoking is associated with a 2- to 8-fold increased risk of periodontitis4,5 and that heavy smokers are at greater periodontal risk than light smokers are.4,5 Smoking appears to be more strongly correlated with the generalized form of aggressive periodontitis than with the localized form.6 These effects are attributed to the chemical components of cigarette smoke (CS), such as, for example, nicotine. Indeed, when smoking, oral soft and hard tissues are exposed to high doses of nicotine,7,8 which binds to tooth root surfaces in smokers.9 This can alter gingival soft tissue by reducing cell attachment and increasing their apoptosis through different mechanisms, including Bax and caspase-3 activity.10,11 Smokers reportedly have a higher number of missing teeth than do nonsmokers, thus emphasizing the deleterious effects of tobacco smoke on bone metabolism.12,13 A strong link between local exposure of peri-implant tissues to tobacco products leading to implant failure in smokers has been suggested.14 The rate of implant failure has been reported to be between 6.5% and 20% compared with that in nonsmokers.15,16 The adverse effects of tobacco smoking on implant outcome may be related to the local biologic route involving bone tissue.17
In recent years, multiple studies have demonstrated the negative effects of cigarette smoke compounds such as nicotine on bone metabolism and osteoblast growth.18 Smoking has a definite negative influence on bone mineral density and volume, as well as on trabecular structure and osteoblast numbers.19 Osteoblasts are key cells that contribute to bone regeneration and interact with dental implants. Dysregulating osteoblast activity may lead to a reduction in bone mineral density in conjunction with an increase in bone resorption, thereby contributing to weakening the bone and, ultimately, to implant failure. To increase dental implant survival in smokers, various protocols have been recommended, including smoke cessation for at least 1 week prior to surgery. Although this cessation contributes to reducing the adverse effects of smoke products,20–22 due to the addiction to nicotine, quitting smoking can be extremely difficult.23,24 Electronic cigarettes (e-cigarettes) have thus been introduced on the market as a “safe alternative” to combustible cigarettes.25,26
The e-cigarette combines a plastic tube, an electronic heating component, and a cartridge containing a liquid solution of propylene glycol and glycerol with or without nicotine.27 When a sensor in the device detects airflow, the heating component in contact with the cartridge is activated, vaporizing the solution and producing a smoke-like aerosol that is subsequently inhaled. Inhaling nicotine through an e-cigarette has been claimed to be a safer alternative to cigarette smoking by eliminating the inhalation of harmful compounds, such as tar and carbon monoxide, found in standard cigarettes. However, following heating, the presence of fine particles has been shown to increase after e-liquid vaping.28.29 The humectants in e-cigarettes are oxidized, leading to the formation of the different aldehydes. Indeed, studies have shown that a certain amount of the e-liquid solvent's molecules convert to formaldehyde, acetaldehyde, and acrolein in e-cigarette vapor.30,31 These chemicals may have serious adverse effects on human health. Following a puff, the e-vapor is delivered into the smoker's mouth, thus in contact with the oral soft and hard tissues, as well as materials used for teeth reconstruction, such as dental implants.
Implant dentistry offers successful alternatives to many restorative problems, from replacing missing teeth or entire arches to simply stabilizing a moving denture. Unfortunately, stress factors such as smoking are now known to weaken implant functionalities.14–16 Because e-cigarettes contain compounds (even at low levels) similar to those found in combustible cigarettes, e-cigarette vapor may potentially dysregulate osteoblast interaction with dental implants, thereby affecting implant sustainability. The goals of this study were therefore to investigate the effect of e-cigarette vapor on osteoblast attachment to and growth on dental implant materials. We also evaluated alkaline phosphatase (ALP) activity and the degradation of mineralized tissue following osteoblast growth on implant material exposed to smoke and e-vapors. Finally, we investigated whether smoke/e-vapor promoted osteoblast death through the expression of caspase-3 following osteoblast culture on dental implant material.
Materials and Methods
For the purposes of this study, commercially available pure titanium (Ti6Al4V) disks were used (grade 2; 10 mm diameter, 0.5 mm thickness; Portland Precision Mfg Co, Portland, Ore). The disks were cleaned with 70% ethanol and distilled water, autoclaved, rinsed with sterile phosphate buffered saline (PBS), and stored under sterile conditions at 4°C until use.
E-cigarette devices and e-liquids
eGo one constant temperature (CT) electronic cigarette devices (http://www.joyetech.com) were chosen to deliver the e-vapor. The resistance for CT mode ranged from 0.2 to 1.0 ohm. The eGo one CT electronic cigarette device has a transparent clearomizer tube to display the visual follow-up of the remaining e-liquid. This device is also easy to refill. Disposable e-cigarette liquids with and without nicotine (flavor: Smooth Canadian tobacco, http://shop.juicyejuice.com/juicy-canadian-tobacco-e-liquid.ejuice) were also selected for this study. Nicotine concentration in the e-liquid was 18 mg/mL. The e-cigarette devices and e-liquids were chosen because of their availability to users. The eGo one CT electronic cigarette devices and disposable cartomizer cartridges were purchased from local retailers. For conventional cigarette, we used 1R3F cigarettes being purchased from the Kentucky Tobacco Research & Development Center (Orlando, Fla).
Saos2 osteoblast-like cells, a human osteosarcoma cell line with osteoblastic properties, were used in this study. The cells were cultured in Dulbecco-Vogt modified Eagle's medium with Ham's F-12 (DMEH; Invitrogen Life Technologies, Burlington, ON, Canada) supplemented with 24.3 μg/mL adenine, 10 μg/mL human epidermal growth factor, 0.4 μg/mL hydrocortisone, 5 μg/mL bovine insulin, 5 μg/mL human transferrin, 2 × 10−9 M 3,3′,5′-triiodo-l-thyronine, and 10% fetal calf serum (FCS). Subconfluent cell cultures split 1:10 to maintain cell growth were subsequently incubated at 37°C in a humid 5% CO2 atmosphere until use.
Osteoblast culture on dental implant disks and exposure to either cigarette smoke or e-cigarette vapor
Sterile implant disks were placed into the wells of 12-well plates, with 1 disk per well. Osteoblasts were seeded onto the titanium implant disks at 4 × 104 cells per disk and subsequently cultured in DMEH medium supplemented with 10% FCS and growth factors. Incubation was performed in a humidified atmosphere of air containing 5% CO2 at 37°C for 24 hours. At the end of this culture period, fresh medium (0.5 mL/well) was added to cover each disk with a thin layer of medium, after which time the plates were placed inside a custom-made smoke chamber. The e-cigarette device was linked to one end of a silicone tube while the other end of the tube was linked to the smoke chamber. A peristaltic pump was used to deliver the e-cigarette vapor into the chamber. Cultures were placed inside the chamber under sterile conditions, and the chamber was hermetically sealed prior to activating the peristaltic pump, which activated the e-cigarette device system to produce the e-vapor through the silicone tube. The e-cigarette vapor was then drawn into the exposure chamber (2 puffs every 60 seconds: a 10-second puff followed by a 20-second pause).
The exposure to whole conventional cigarette smoke followed the same procedure as to e-vapors. Briefly, a cigarette was linked to one end of a silicone tube while the other end of the tube was linked to the smoke chamber. A peristaltic pump was used to deliver the smoke of half cigarette into the chamber, with about 15 seconds burning time. For all conditions, the exposure time consisted of 15 or 30 min/d for 1, 2, or 3 days. We selected these exposures time based on our previous in vitro work with human fibroblasts exposed to whole cigarette smoke.11 The cell culture plates were subsequently incubated for 1 additional hour prior to medium changing. The cells were then fed fresh medium and cultured for 24 hours prior to being subjected to various analyses. Four conditions were performed: osteoblasts exposed to combustible CS, osteoblasts exposed to nicotine-rich (NR) e-vapor, and osteoblasts exposed to nicotine-free (NF) e-vapor. Osteoblast cultures being placed into a smoke/e-vapor free chamber for the same time as the assay conditions were included in the study as control (Ctrl). It should be noted that each exposure condition (CS, NF, NR) was performed in a separate exposure chamber to avoid culture cross-contamination between CS, NR, and NF.
Effect of cigarette smoke and e-vapor on osteoblast growth
Osteoblast growth analyses were performed after 1, 2, and 3 days of exposure. For this purpose, implant disks seeded with osteoblasts and exposed or not to CS or e-vapor were subjected to an MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide)] assay 24 hours after the final exposure. The culture medium in each well was supplemented with 1% (v/v) MTT solution (5 mg/mL), and the osteoblasts were subsequently incubated for 4 hours, after which time the supernatant was removed and the implant disks were washed with PBS. To lyse the osteoblasts attached to the implant disks, and having integrated the MTT chemical, the implant disks were overlayered with 1 mL isopropanol-HCl (0.04 N) solution, followed by incubation for 15 minutes under agitation. Following this incubation, 200 μL (in triplicate) of each condition was transferred to a 96-well flat-bottom plate. The absorbance of the MTT converted to formazan in the live cells was measured at 550 nm using our plate reader (Model 680; BioRad Laboratories, Mississauga, ON, Canada).
Effect of cigarette smoke and e-vapor on osteoblast distribution on the surface of the implant disks
Osteoblasts (4 × 104 cells) were seeded onto dental implant disks and cultured for 24 hours prior to exposure or not to CS or to NR or NF e-vapor. After 1, 2, or 3 exposures for 15 or 30 minutes each day, they were then cultured for 24 hours. The osteoblasts attached to the implant disks were subjected to Hoechst staining. The samples were first fixed with methanol/glacial acetic acid (75/25) for 3 × 15 minutes and subsequently washed 3 times with PBS. They were then incubated with Hoechst 33342 (H42) (Riedel de Haen, Seele, Germany) (1 μg/mL) in PBS for 15 minutes at room temperature in a dark atmosphere. Following 3 washes with deionized water, the samples were observed and photographed using an epifluorescence light microscope (Axiophot; Zeiss, Oberkochen, Germany).
F-actin filament staining
To investigate the effect of e-vapor and CS on cell attachment, we stained the F-actin filaments expressed by adherent fibroblasts. For this purpose, following exposure or not to CS and NR/NF e-vapor, the cells were cultured overnight. They were then washed with PBS buffer, fixed with 4% paraformaldehyde for 15 minutes, and permeabilized with 0.5% (v/v) Triton X-100 in PBS for 10 minutes prior to staining. Nonspecific binding was blocked by adding 1% (w/v) bovine serum albumin for 30 minutes at room temperature. The implant disks were then washed, and the F-actin fibers were stained with fluorescein isothiocyanate (FITC)-labeled phalloidin (Invitrogen, Molecular Probes, Thermo Fisher Scientific, Saint-Laurent, QC, Canada). Following three washes, the slides were visualized under a fluorescence microscope (Axiophot; Zeiss), and digital images were collected (Coolpix 950; Nikon Canada, Montréal, QC, Canada). Image analysis was executed using ImageJ version 1.44 software (National Institutes of Health, Bethesda, Md). Briefly, JPEG images were imported into the ImageJ software. The original images were converted to 8-bit images and thresholded, and the mean of the fluorescent intensity of each condition was collected.
Effect of CS and e-vapor on osteoblast ALP activity
ALP activity in the culture medium of osteoblasts cultured on the implant disks and exposed or not to CS or e-vapor was measured. For this purpose, osteoblasts (4 × 104) were seeded onto each implant disk and allowed to adhere overnight prior to exposure or not to the CS or e-vapor. The disks were overlayered with fresh medium (1 mL) and then exposed or not for 15 or 30 minutes to CS, NR e-vapor, or NF e-vapor. The exposure regime was once a day for 1, 2, or 3 days. Following each exposure, the medium containing CS or NR/NF vapor was maintained for 1 hour and replaced thereafter with fresh CS/e-cigarette vapor-free medium. At the end of each exposure period (1, 2, or 3 days), culture medium was collected and centrifuged twice at 3000 rpm for 10 minutes to eliminate cell debris and then used to measure the ALP enzyme activity. ALP reactive solution was prepared with a mixture of diethanolamine substrate buffer (1×) and p-nitrophenyl phosphate disodium salt (final concentration 1 mg/mL) according to the manufacturer's protocol. One hundred microliters of collected culture medium was supplemented with 100 μL ALP reactive solution in a 96-well plate, with the plate subsequently incubated for 1 hour at room temperature. The reaction was stopped by adding 50 μL 2 N NaOH to each well. Optical density was read at 405 nm using an X-Mark microplate spectrophotometer (Bio-Rad, Mississauga, ON, Canada) and translated into ALP enzyme activity using a standard curve generated with p-nitrophenol (Sigma-Aldrich, St Louis, Mo) ranging in concentration from 0 to 20 mM.
Qualitative and quantitative analyses of nodule formation
Tissue culture Petri dishes (35 mm diameter) were used to hold 5 implant disks per dish. Each disk was seeded with 5 × 105 osteoblasts and cultured for 3 weeks. Medium was refreshed every 48 h during the first 2 weeks and then every 24 hour thereafter. At the end of the 3-week culture, the implant disks were exposed or not to CS or NR and NF vapor for either 15 or 30 minutes. Exposure took place once a day for 1, 2, or 3 days. At the end of each exposure period, the disks were washed twice with PBS and stained with Alizarin red S (ARS) solution for 2 minutes prior to being washed 3 times with sodium acetate buffer solution (pH 6.3). Mineral nodules stained with ARS were dissolved with 500 μL cetyl-pyridinium chloride (CPC) (Fisher Scientific, Ottawa, ON, Canada) for 1 hour under gentle agitation. Solutions were collected separately and centrifuged 10 minutes at 3000 rpm to eliminate cell debris. The collected supernatants were diluted v/v with distilled water, with 3 × 100 μL of the CPC from each condition transferred to a 96-well plate. ARS absorbance was determined at 570 nm by means of the X-Mark Microplate Spectrophotometer (Bio-Rad), with n = 8 implant disks in each condition.
Caspase-3 analyses, Western blot
Osteoblasts (5 × 105) were seeded onto each implant disk and incubated overnight to allow for cell adhesion. The cells were then exposed to CS, NR e-vapor, or NF e-vapor for either 15 or 30 min/d for 1, 2, or 3 days. After each exposure, the medium containing either smoke or e-cigarette vapor was maintained for 1 hour and then replaced with fresh medium. Following 24 hours of culture, proteins were extracted from each condition using lysis buffer (25 mM of Tris–HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 10% glycerol, 0.1% SDS, 0.05% sodium deoxycholate, and 1% Triton X-100). The lysis buffer was supplemented with 2 μL protease and phosphatase inhibitors (Sigma-Aldrich). Protein concentration was determined by the Bradford assay using Coomassie brilliant blue G-250 stain. The protein was denatured by adding a reducing buffer (61.5 mM Tris, 2% SDS, 10% glycerol, and 100 mM dithiothreitol) and boiling for 5 minutes. The protein (20 μg) was then separated by SDS-PAGE by means of a Bio-Rad Mini-PROTEAN II electrophoresis system in a vertical slap gel apparatus using 10% separating gel and 4% stacking gel. The gel with a refrigerated Tris–glycine transfer buffer (25 mM Tris, 19.2 mM glycine, 20% methanol, and 100 mM Na3VO4) was then transferred to a poly-(vinylidene difluoride) membrane for 1 hour at 100 V. The membrane was then incubated with 5% nonfat milk in 0.1% tween-20–tris-buffered saline (TTBS) for 1 hour and subsequently incubated overnight with anti–caspase-3 primary antibody, after which time the membrane was washed 4 × 10 minutes with TTBS buffer. The peroxidase-conjugated secondary antibodies were then applied onto the blot for 1 hour, and the membrane was subsequently washed 4 × 10 minutes with TTBS buffer. Finally, detection was performed using the chemiluminescence-based horseradish peroxidase substrate system (Millipore, Billerica, Mass) according to the manufacturer's instructions. Visualization was carried out using the VersaDoc MP 5000 System (Bio-Rad Laboratories Ltd, Mississauga, ON, Canada). The public domain National Institutes of Health Image program was used to quantitatively analyze the intensity of the caspase-3 bands (n = 6).
Continuous variables were expressed using mean ± SD. Data were analyzed using a 2-way ANOVA. CS, NF, and NR conditions were merged with 15 and 30 minutes to define six conditions as the control conditions were the same for both times (15 and 30 minutes) to create the first factor with seven levels. The second factor refers to the different exposure (1, 2, and 3 exposures). All statistical analyses have a significant interaction factor (P < .0001) and have heterogeneous variances. The Satterthwaite's degree of freedom statement was added for unequal variance structures. Comparisons among the different conditions at different days were performed by partitioning interactions. The normality assumption was verified with the Shapiro-Wilk tests, after a Cholesky factorization on residuals. The results were considered significant with P ≤ .05. All analyses were conducted using the statistical package SAS, version 9.4 (SAS Institute Inc, Cary, NC) and R (R Core Team 2016, Foundation for Statistical Computing, Vienna, Austria).
Whole cigarette smoke and nicotine-rich e-cigarette vapor reduced osteoblast growth following culture on dental implant disks
Following single or repeated exposures to whole CS, a significant (P < .01) decrease of viable osteoblasts still attached to the dental implant material was observed (Figure 1). Indeed, whole CS reduced osteoblast growth regardless of exposure time (15 or 30 minutes). The reduction in osteoblast growth was greater at 2 and 3 days of exposure to CS than at 1 day of exposure. Osteoblast growth inhibition was also observed with the nicotine-rich e-vapor. As shown in Figure 1, following a single exposure of either 15 or 30 minutes, osteoblast absorbance dropped from 0.34 ± 0.015 in the control to 0.23 ± 0.03 with a 15-minute exposure and to 0.14 ± 0.05 with a 30-minute exposure to NR e-vapor. After 2 or 3 exposures, the osteoblast growth inhibition with the NR e-vapor was greater than that observed in the single exposure (Figure 1). However, the effect of the NR e-vapor was lower than that recorded with the whole CS. Exposure of osteoblast-cultured dental implant disks to NF e-vapor for 15 and 30 minutes showed a slight yet significant reduction in osteoblast growth (basically after 2 and 3 days). The effect of the NF e-vapor was, however, lower than that observed with the NR e-vapor and the whole CS. Osteoblast growth inhibition was thus classified as follows: CS > NR > NF.
Whole cigarette smoke and nicotine-rich e-cigarette vapor downregulated osteoblast attachment to dental implant disks
Exposure to whole cigarette smoke for 1 or 2 days showed a reduced cell density of the osteoblasts still attached to the dental implant disks (data not shown), whereas repeated exposures to whole CS for 3 days resulted in a visible reduction in osteoblast attachment (Figure 2). Exposure for 15 or 30 minutes contributed to the decrease of Hoechst-stained cell density, compared with that observed in the control. Osteoblast exposure to NR e-vapor for 3 days showed a higher density of cells still attached to the dental implant disks compared with that of cells exposed to the whole CS. However, this NR e-vapor–exposed osteoblast density was lower than that in the control (nonexposed osteoblasts). When osteoblasts attached to the implant disks were exposed to NF e-vapor, the cell density was greater than that observed in the NR e-vapor–exposed culture. These data confirm that both CS and NR e-vapor were able to decrease cell proliferation and interaction (attachment) with the implant disks. This dysregulation of osteoblast attachment could take place through the inhibition of adhesion protein production (such as F-actin) by osteoblasts.
Whole cigarette smoke and nicotine-rich e-cigarette vapor decreased the expression of F-actin by osteoblasts following culture on dental implant disks
The effect of CS, as well as of NR and NR e-vapor, on osteoblast attachment was further confirmed by F-actin cytoskeleton staining. Figure 3 shows attached cells expressing dense F-actin staining in the Ctrl culture. Indeed, the staining intensity of the F-actin filaments was strong over the entire culture. An almost complete inhibition of F-actin production by the osteoblasts was found in whole CS-exposed culture (Figure 3a and b). The inhibitory effect on F-actin expression was greater in the 30-minute exposure than in the 15-minute exposure to whole CS. Exposure to NR e-vapor led to a decrease of F-actin intensity compared with that observed in the control. However, F-actin expression was greater with NR e-vapor than with whole CS (Figure 3a and b). Finally, NF e-vapor showed a slight decrease in the F-actin intensity by the osteoblast-cultured dental implant disks. Thus, the dysregulating effect of whole CS and NR e-vapor on osteoblast interaction with dental implants may occur through adhesion protein (F-actin) inhibition/degradation.
Whole cigarette smoke and nicotine-rich e-cigarette vapor decreased osteoblast ALP activity following culture on dental implant disks
The effect of CS, NR e-vapor, and NF e-vapor on the terminal differentiation of osteoblasts was determined by tissue-nonspecific ALP activity. Exposure for 24 hours had no effects on ALP activity (data not shown). However, longer exposure time showed ALP activity changing. As shown in Figure 4, the nontreated osteoblasts produced a high level of ALP (3.5 ± 0.002 mM). Exposure for 15 minutes to CS once a day for 2 or 3 days induced a significant reduction in ALP protein production compared with that observed in the control. When the osteoblast cultures were exposed to NR e-vapor, low levels of ALP protein were observed compared with what was observed in the control. Of interest is that the ALP protein levels obtained with NR e-vapor were higher than those obtained with CS (Figure 4). Finally, exposure to NF e-vapor resulted in a slight yet significant decrease in ALP protein activity by the osteoblasts compared with that observed in the control.
Whole cigarette smoke and nicotine-rich e-cigarette vapor decreased cell mineralization following culture on dental implant disks
Osteoblasts in mineralization medium were cultured for 3 weeks and then exposed or not to CS, NR e-vapor, or NF e-vapor once a day for 30 minutes for 1, 2, or 3 days. Cultures were then used to examine mineralized tissue. As shown in Figure 5, CS significantly (P < .001) reduced alizarin red staining intensity, even after 1 exposure. NR e-vapor but not NF e-vapor also significantly (P < .01) decreased mineralized tissue after a single exposure. After 2 exposures, CS, NR e-vapor, and NF e-vapor all led to a significant decrease in the amount of mineralized tissue, with mineralization levels going from 1.7 ± 0.06 in the control to 1.35 ± 0.04 with exposure to NR e-vapor and to 1.57 ± 0.08 with NF e-vapor. The reduction in tissue mineralization was confirmed in the 3 exposure periods. This decrease was highest with CS, followed by NR e-vapor and NF e-vapor compared with the control.
Whole cigarette smoke and nicotine-rich e-cigarette vapor increased caspase-3 protein in osteoblasts cultured on dental implant disks
As shown in Figure 1, the most significant effects were obtained after 30 minutes of exposure to CS/e-vapor than after 15 minutes of exposure; thus, the effect of CS/e-vapor on caspase-3 following a 30-minute exposure was investigated. Using Western blot analysis, we found (Figure 6) a significant increase of caspase-3 protein following osteoblast exposure for 30 min/d for 1 or 2 days to CS/e-vapor. Caspase-3 was indeed overproduced in the CS-exposed cultures compared with that observed in the control, even after a single exposure. A similar result was obtained with the NR e-vapor, showing a high level of caspase-3 protein after a single exposure to NR e-vapor. Caspase-3 expression was also analyzed following osteoblast exposure for 2 days. It should be noted that cell exposure once a day for 2 or 3 days to CS resulted in a very low level of proteins; thus, the Western blot analyses could not be performed. However, under NR and NF e-vapor exposure conditions, we were able to collect more protein and perform Western blot analysis, even after 2 exposure times. Results show an increase of caspase-3 protein in both the NR and NF e-vapor conditions compared with that observed in the control. Of interest is that the caspase-3 protein levels were higher with the NR e-vapor than with the NF e-vapor. Quantitative analyses using ImageJ scanning revealed (Figure 6b) that the ratio of caspase-3/b-actin ranged between 0.15 ± 0.002 in the control and 0.21 ± 0.09 in the NF e-vapor-exposed conditions, 0.31 ± 0.009 in the NR e-vapor-exposed cultures, and 0.21 ± 0.79 in the CS-exposed cells after 1 exposure. After 2 exposures, the ratio of caspase-3/b-actin increased to 1.21 ± 0.003 with the NF e-vapor and 8.45 ± 0.3 with the NR e-vapor compared with that observed in the control (0.24 ± 0.002). These findings may suggest the involvement of the caspase-3 pathway in cell apoptosis following osteoblast exposure to CS and NR e-vapor and to a lesser extent to NF e-vapor. Further studies are mandatory to shed light on the mechanisms being involved in such cell apoptosis.
E-cigarettes are becoming more and more popular among smokers who believe that e-cigarette aerosols represent a safer alternative to smoking conventional cigarettes.32 Studies have, however, reported the presence of substantial levels of nanoscale particles, as well as detectable levels of toxic materials (eg, aluminum, copper, magnesium, zinc, lead, chromium, manganese, and nickel) in e-cigarette aerosols.33 These particles may reach the upper and lower airways in smokers, weakening cell defenses and promoting disease.34
The upper airway, specifically the oral cavity, is the first direct target of e-cigarettes on vaping. In the oral cavity, e-vapor comes in contact with different structures and compounds such as dental implants. As a result, this contact may dysregulate the bone–tissue interaction with these implant surfaces. E-vapor can affect osteoblast behavior and ultimately promote implant failure or complications. This hypothesis is indeed confirmed in the present study by the reduced osteoblast attachment observed to dental implant material through a decrease of focal adhesion proteins, such as F-actin. Cell adhesion is not only a critical process in the interaction of cells with materials but also controls cell survival, migration, proliferation, and differentiation.35 Adhesion formation is an integrin-dependent process, which mediates cell substrate signaling through the activation of intracellular focal adhesion kinase and the phosphatase signal to trigger downstream biochemical signaling pathways.36 A previous study showed that focal adhesion formation is essential for osteoblastic markers and thus bone formation.37 The downregulation of F-actin protein by CS and e-cigarette vapor may explain the growth inhibition of the osteoblasts cultured on the dental implant disks. Further studies are needed to see whether other adhesion proteins (integrins) are also downregulated and what signaling mechanism is involved. Cell adhesion was also reported to be related to the implant surface topography. Indeed, the implant surface topography can either promote or decrease cell adhesion.38 The investigation of the effects of the implant topography and the e-vapor on the osteoblast adhesion will be performed in the future.
The deleterious effects of e-vapor on human cell adhesion and growth were previously reported with human gingival epithelial cells,39 pulmonary epithelial cells,40 and fibroblasts,41 among others. Interestingly, CS produced a much higher adverse effect on osteoblast attachment and growth than did the e-vapor with or without nicotine. This observation confirms previous reports with myocardial and fibroblast cells.5,40 Overall data highlight (1) the low adverse effect of nicotine-rich e-cigarettes compared with that of CS and that (2) the e-cigarette is not as safe as it is claimed to be, as it was shown to not only hamper the attachment of osteoblasts to dental implant material but also decrease cell growth compared with that observed with nonexposed cells.
Because CS and e-cigarette vapor downregulated the attachment and growth of osteoblasts to dental implant disks, we proceeded to examine ALP activity and found that CS significantly (P < .001) decreased osteoblast osteogenic activity, as previously reported.42 We also demonstrated that both NR e-vapor and NF e-vapor led to a decrease in ALP activity. It should be noted that the effect of CS was greater than that recorded with e-cigarette vapor. These observations suggest for the first time that e-cigarettes may produce an antiosteogenic effect. Further investigations on the antiosteogenic activity of e-cigarettes should provide insight regarding their effect on the bone/dental implant interaction to better control dental implant failure, as with conventional CS.43
The decreased pattern in ALP activity following osteoblast exposure to CS or e-cigarette vapor may lead to mineralization dysregulation. Indeed, this study shows that when osteoblasts were cultured for 3 weeks under mineralization conditions, the alizarin staining was high in the control groups; however, following exposure to CS and to NR e-vapor, a significant decrease of mineralized tissue was observed. It should be noted that this diminished mineralization was greater in the presence of CS than of NR e-vapor. NF e-vapor also led to a decrease of mineralized tissue compared with that recorded in the control. These observations are in accordance with previous works showing reduced bone density around titanium implants in smokers.44 The novelty of this study section is the demonstration of the decrease of mineralized tissue even after osteoblast exposure to NR or NF e-vapor. As both CS and e-vapor were found to negatively affect osteoblast attachment, growth, and differentiation/mineralization, these effects may occur through the apoptotic pathway. Apoptosis onset is associated with the proteolytic activation of caspases, such as caspase-3.44 Caspase-3 was shown to increase in cells undergoing apoptosis.46 In this study, we demonstrated that osteoblasts cultured on dental implant material and exposed to either CS or e-vapor exhibited high protein levels of caspase-3. Similar observations were reported using conventional CS47 and e-cigarettes46 with primary gingival cells. Thus, both nicotine-free and nicotine-rich e-cigarette vapor may downregulate osteoblast interaction with dental implant material through the apoptosis pathway involving caspase-3, as did conventional CS, but to a lesser extent. This difference could be attributed to the low chemical present in the e-vapor compared with the CS.
Clinical studies have confirmed that smokers present higher rates of implant failure than nonsmokers48,49 and a greater detrimental effect around successfully integrated implants,50,51 including a higher incidence of peri-implantitis and an increased marginal bone loss.50,52 Smoke cessation was reported to be the most effective strategy to reduce implant failures.21
With the objective of reducing the negative impact of cigarette smoke on smoker's health including implant outcomes, e-cigarettes were proposed as a safe way to quit.32 However, the recent studies showed that e-cigarettes attract more people than pushing them to quit.25,26 Because e-cigarettes contain certain chemicals found in the conventional cigarette,30,31 smokers and clinicians should be aware of the adverse effect of e-cigarettes on the smoker's oral health including dental implant failure.
We demonstrated that exposure to conventional cigarette smoke or to e-cigarette vapor significantly reduced osteoblast attachment to and growth on dental implant material. Furthermore, CS and e-vapor were shown to decrease ALP but increase the degradation of mineralized bone tissue. The mechanism leading to dysregulating osteoblast interaction with the dental implant material involved the caspase-3 pathway. Overall, findings suggest that e-cigarettes could be a factor contributing to dental implant failure.
Alizarin red S
Dulbecco-Vogt modified eagle's medium with Ham's F-12
fetal calf serum
phosphate buffered serum
This study was financed by funds from the American Academy of Implant Dentistry Foundation and the Fonds Émile-Beaulieu, Université Laval.
The authors declare no conflicts of interest.