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and antiviral defenses in a mouse model.17 In vitro toxicity profiles of elec- tronic and tobacco cigarettes, smokeless tobacco, and nicotine replacement therapy products have been compared in a study using Ames Salmonella muta- genicity testing.18
The nicotine in the ECs is mainly absorbed through the buccal mucosa and pharyngeal mucosa. Although nicotine has been shown to have wound healing and angiogenic properties,19 which can make it a potential therapeutic agent, there are some concerns regarding
its ability to promote lung tumor growth through dif- ferent possible mechanisms, including angiogenesis, cell proliferation, and cell migration.20-21
There are few representative studies that illustrate the negative effects of ECs on the human oral mucosa. Evidence supports that conventional smoking has a negative influence on a patient’s response, as well as their treatment outcome, following non-surgical, sur- gical, and regenerative periodontal treatment. Smok- ing also impairs all wound healing phases (particu- larly the inflammatory and proliferation phases, which lead to delays in wound healing).22-26
Clinical studies of ECs have reported mild harmful effects of vaping on selected cardiovascular27-28 and respiratory functional outcomes29-31 to a considerably less extent when compared with conventional ciga- rette smoking32. However, it is difficult to assess the prognostic implications of these studies, and there is a need for further research in this area.
Clinical research and survey-type studies to date have shown that the most common patient-reported side effects were symptoms such as xerostomia, throat irritation, and cough.33-36
A five-year multicenter cohort study was conducted to assess the long-term efficacy and safety of ECs,37 and a pilot study investigated the oral mucosa perfu- sion of intraoral free flaps in EC users and smokers.38 Several toxicological studies have been conducted on ECs, using monolayer cell culture systems.39-43 However, there is no study published in the literature comparing the effects of E-cigarette liquid on differ- ent normal and cancerous oral mucosa cells, including
3D tissue-engineered models of the human oral mu- cosa. Therefore, this study aimed to evaluate the bio- logical effects of E-cigarette liquid on both full-thick- ness 3D oral mucosa models and monolayer cell cul- ture systems utilizing normal oral fibroblasts, OKF6- TERET2 oral keratinocytes, and cancerous TR146 keratinocytes, following short-term (three days) and medium-term (seven days) exposure to flavorless electronic cigarette liquid. Additionally, this study aimed to investigate the influence of E-cigarette liquid on oral mucosa wound healing, using normal oral fi-
broblasts and OKF6 oral keratinocyte cell cultures.
Normal human oral fibroblast cells were obtained from the stocks stored in liquid nitrogen in the labor- atories of the School of Clinical Dentistry at the Uni- versity of Sheffield. These cells had been previously obtained from healthy patients undergoing oral
surgery at Charles Clifford Dental Hospital with their written informed consent under appropriate ethical approval from the UK National Research Ethics Ser- vices Committee.
The immortalized OKF6/TERT-2 human oral keratinocyte cell line was kindly provided by Brigham and Women's Hospital, Harvard Institute of Medicine, USA.
The cancerous human TR146 cell line was derived from a neck lymph node metastasis originating from a carcinoma of the oral buccal mucosa. These cells were kindly provided by Cancer Research UK.
Five different biological systems were tested in this study, including:
Oral fibroblasts and keratinocytes were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, UK), supplemented with 2% L-glu- tamine (Sigma, UK), 100 IU:100 mg ml-1 Penicil- lin/Streptomycin (Sigma, UK), and 10% fetal calf se- rum (FCS) (Biowest Ltd., UK). Six-well plates were utilized for the monolayer cell cultures, inoculating 10,000 cells per well for NOFs, OKF6/TERT2 cells, and TR146 keratinocytes. Addi- tionally, normal oral fibroblasts and keratinocytes were cultured together using six-well plates.
The cultures were maintained in incubators at 5% CO2 at 37C. The cells were cultured until 80‒100% confluency was achieved.
Full-thickness 3D tissue-engineered oral mucosa models were manufactured by air/liquid interface cul- ture of TR146 keratinocytes seeded onto fibroblast- populated collagen gels. A solution of 10 × DMEM, 8.5% (v/v) FBS, 2 mM L- glutamine, reconstitution buffer (22 mg mL−1 sodium bicarbonate and 20 mM 4-(2-hydroxyethyl)-1-pipera- zineethanesulfonic acid), and 5 mg mL-1 rat tail type I collagen (R & D system, UK) was prepared and neu- tralized by 1-M sodium hydroxide to pH=7.4 in an ice-cold environment by keeping everything on ice. Normal oral fibroblasts were added to the solution at
a concentration of 500,000 cells/model, and 1 mL of the resultant cell-containing collagen mixture was transferred to cell culture transwell inserts (0.4 µm pore size, Millipore), incubated at 37°C for 2 hours until solidified, and then completely submerged in complete DMEM for 3 days. Subsequently, 1×106 keratinocytes were seeded onto each model and kept in submerged culture for three days, after which the oral mucosal models were raised to air/liquid interface and cultured for a further 7 days.
Neutral E-Cigarette Liquid (Vype-UK) with no added flavors, containing medium nicotine level of 12 mg/mL, was used in this experiment. Four different concentrations (0.1%, 1%, 5%, and 10%) of E-ciga- rette liquid were prepared by diluting the E-liquid with DMEM.
The monolayer cell culture systems and the 3D oral mucosa model’s epithelial surfaces were exposed to four different concentrations of E-cigarette liquid (0.1%, 1%, 5%, and 10%), to the negative control (DMEM) and the positive control (70% ethanol). The groups consisted of six samples each (n=6).
All the samples were exposed to their respective re- agents for one hour daily for three continuous days and incubated at 5% CO2 and 37ºC. Following the testing for the short-term exposure, the samples were washed twice with phosphate-buffered saline (PBS) (Sigma, UK), and the medium-term exposure contin- ued for four more days by exposing the samples to the reagents for one hour daily, totaling seven days.
Following the short-term and medium-term exposures, tissue viability test was carried out using the Presto- Blue assay. The PrestoBlue reagent (Biosource, Cam- arillo, CA) was added to the samples at a ratio of 9:1 (volume of cells and culture medium: volume of Pres- toBlue reagent). The plates were then incubated for 60 minutes at 37°C and 5% CO2. Following incubation, triplicate 200-L samples were placed into the wells of a 96-well plate, and the fluorescence intensity of each well was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm us- ing a fluorescent plate reader (Infinite 200 PRO TECAN, Switzerland).
The oral mucosa model samples were first fixed in 10% formalin solution for 24 hours; the samples were then mounted in Optimal Cutting Temperature (OCT) em- bedding compound, followed by freezing at -20 to -
80°C. Afterward, the samples were sectioned at a thickness of 10‒30 μm using a cryostat machine. The sections were then mounted on the histological slides. This was followed by drying the slides for around 30 minutes at room temperature. The slides were then stored in the freezer at -80°C until the pro- cessing for hematoxylin and eosin (H&E) staining.
The slides were examined under a light microscope by more than one histopathology expert to assess the changes in the connective tissue and the epithelial lay- ers of the model following the exposure to test agents. Assessment criteria included the continuity and thick- ness of the epithelium, cell morphology, presence or absence of pyknotic nuclei, presence of a distinct in- terface between the epithelium and the connective tis- sue layer.
Monolayer cultures of normal oral fibroblasts and OKF6/TERT-2 keratinocytes were developed in 6- well tissue culture plates and divided into negative control (DMEM), positive control (70% ethanol), and test groups with various E-Cigarette concentrations (0.1%, 1%, 5%, and 10%) (n=6). The wounds were produced vertically in the middle of the surface of the monolayer systems using a disposable cell scraper. The test groups were exposed to the culture media containing E-cigarette liquid immediately before and continued daily after creating a wound, and then the wounds were monitored until complete healing had occurred. Microscopic images were obtained pre- and post-wound creation daily to assess the healing time in all the groups.
SPSS 20 was used for statistical analysis. The normal- ity of the data was analyzed using the normality test (Shapiro-Wilk test). Means of the samples were com- pared with ANOVA, followed by multiple compari- sons using post hoc Tukey tests to determine the dif- ferences between the different groups. The level of significance for all the statistical tests was set at
=0.05.
The results of the PrestoBlue assay for normal oral fi- broblasts and OKF6/TERT-2 keratinocyte monolayer cell cultures exposed to different concentrations of the E-Liquid for three days and seven days are shown in Figure 1. Short-term exposure to 10% E-liquid solu- tion caused a statistically significant reduction in the viability of NOFs (P<0.0001) compared to the nega- tive control group. Following medium-term exposure,
all the E-liquid concentration groups exhibited signif- icantly lower viability compared to the negative con- trol group.
OKF6/TERT-2 keratinocyte monolayers showed significantly lower viability after short-term exposure to all the E-liquid concentrations, whereas medium- term exposure resulted in significantly lower viability in all the groups except for 1% concentration group compared to the negative control group.
The TR146 keratinocyte monolayers did not exhibit any statistically significant difference in the viability between different E-liquid concentration groups com- pared to the negative control group after short-term exposure. However, medium-term exposure to 5% and 10% E-liquid solution caused a statistically sig- nificant increase in the viability of TR146 keratino-
The nicotine in the ECs is mainly absorbed through the buccal mucosa and pharyngeal mucosa. Although nicotine has been shown to have wound healing and angiogenic properties,19 which can make it a potential therapeutic agent, there are some concerns regarding
its ability to promote lung tumor growth through dif- ferent possible mechanisms, including angiogenesis, cell proliferation, and cell migration.20-21
There are few representative studies that illustrate the negative effects of ECs on the human oral mucosa. Evidence supports that conventional smoking has a negative influence on a patient’s response, as well as their treatment outcome, following non-surgical, sur- gical, and regenerative periodontal treatment. Smok- ing also impairs all wound healing phases (particu- larly the inflammatory and proliferation phases, which lead to delays in wound healing).22-26
Clinical studies of ECs have reported mild harmful effects of vaping on selected cardiovascular27-28 and respiratory functional outcomes29-31 to a considerably less extent when compared with conventional ciga- rette smoking32. However, it is difficult to assess the prognostic implications of these studies, and there is a need for further research in this area.
Clinical research and survey-type studies to date have shown that the most common patient-reported side effects were symptoms such as xerostomia, throat irritation, and cough.33-36
A five-year multicenter cohort study was conducted to assess the long-term efficacy and safety of ECs,37 and a pilot study investigated the oral mucosa perfu- sion of intraoral free flaps in EC users and smokers.38 Several toxicological studies have been conducted on ECs, using monolayer cell culture systems.39-43 However, there is no study published in the literature comparing the effects of E-cigarette liquid on differ- ent normal and cancerous oral mucosa cells, including
3D tissue-engineered models of the human oral mu- cosa. Therefore, this study aimed to evaluate the bio- logical effects of E-cigarette liquid on both full-thick- ness 3D oral mucosa models and monolayer cell cul- ture systems utilizing normal oral fibroblasts, OKF6- TERET2 oral keratinocytes, and cancerous TR146 keratinocytes, following short-term (three days) and medium-term (seven days) exposure to flavorless electronic cigarette liquid. Additionally, this study aimed to investigate the influence of E-cigarette liquid on oral mucosa wound healing, using normal oral fi-
broblasts and OKF6 oral keratinocyte cell cultures.
Methods
Cell Source and Biological Systems
Normal human oral fibroblast cells were obtained from the stocks stored in liquid nitrogen in the labor- atories of the School of Clinical Dentistry at the Uni- versity of Sheffield. These cells had been previously obtained from healthy patients undergoing oral
surgery at Charles Clifford Dental Hospital with their written informed consent under appropriate ethical approval from the UK National Research Ethics Ser- vices Committee.
The immortalized OKF6/TERT-2 human oral keratinocyte cell line was kindly provided by Brigham and Women's Hospital, Harvard Institute of Medicine, USA.
The cancerous human TR146 cell line was derived from a neck lymph node metastasis originating from a carcinoma of the oral buccal mucosa. These cells were kindly provided by Cancer Research UK.
Five different biological systems were tested in this study, including:
-
Monolayer cultures of normal oral fibroblasts (NOF) -
Monolayer cultures of the immortalized oral keratinocyte cell line (OKF6/TERT-2) -
Monolayer cultures of cancerous TR146 keratino- cytes -
Co-cultures of NOF and OKF6/TERT-2 cells -
3D tissue-engineered oral mucosa models using TR146 keratinocytes and NOFs
Cell Culture
Oral fibroblasts and keratinocytes were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, UK), supplemented with 2% L-glu- tamine (Sigma, UK), 100 IU:100 mg ml-1 Penicil- lin/Streptomycin (Sigma, UK), and 10% fetal calf se- rum (FCS) (Biowest Ltd., UK). Six-well plates were utilized for the monolayer cell cultures, inoculating 10,000 cells per well for NOFs, OKF6/TERT2 cells, and TR146 keratinocytes. Addi- tionally, normal oral fibroblasts and keratinocytes were cultured together using six-well plates.
The cultures were maintained in incubators at 5% CO2 at 37C. The cells were cultured until 80‒100% confluency was achieved.
Tissue-engineered 3D Oral Mucosa Models
Full-thickness 3D tissue-engineered oral mucosa models were manufactured by air/liquid interface cul- ture of TR146 keratinocytes seeded onto fibroblast- populated collagen gels. A solution of 10 × DMEM, 8.5% (v/v) FBS, 2 mM L- glutamine, reconstitution buffer (22 mg mL−1 sodium bicarbonate and 20 mM 4-(2-hydroxyethyl)-1-pipera- zineethanesulfonic acid), and 5 mg mL-1 rat tail type I collagen (R & D system, UK) was prepared and neu- tralized by 1-M sodium hydroxide to pH=7.4 in an ice-cold environment by keeping everything on ice. Normal oral fibroblasts were added to the solution at
a concentration of 500,000 cells/model, and 1 mL of the resultant cell-containing collagen mixture was transferred to cell culture transwell inserts (0.4 µm pore size, Millipore), incubated at 37°C for 2 hours until solidified, and then completely submerged in complete DMEM for 3 days. Subsequently, 1×106 keratinocytes were seeded onto each model and kept in submerged culture for three days, after which the oral mucosal models were raised to air/liquid interface and cultured for a further 7 days.
1 2 3 4 5 6 7 8 9 ... 12
Exposure Protocol
Neutral E-Cigarette Liquid (Vype-UK) with no added flavors, containing medium nicotine level of 12 mg/mL, was used in this experiment. Four different concentrations (0.1%, 1%, 5%, and 10%) of E-ciga- rette liquid were prepared by diluting the E-liquid with DMEM.
The monolayer cell culture systems and the 3D oral mucosa model’s epithelial surfaces were exposed to four different concentrations of E-cigarette liquid (0.1%, 1%, 5%, and 10%), to the negative control (DMEM) and the positive control (70% ethanol). The groups consisted of six samples each (n=6).
All the samples were exposed to their respective re- agents for one hour daily for three continuous days and incubated at 5% CO2 and 37ºC. Following the testing for the short-term exposure, the samples were washed twice with phosphate-buffered saline (PBS) (Sigma, UK), and the medium-term exposure contin- ued for four more days by exposing the samples to the reagents for one hour daily, totaling seven days.
Cell Viability Assay
Following the short-term and medium-term exposures, tissue viability test was carried out using the Presto- Blue assay. The PrestoBlue reagent (Biosource, Cam- arillo, CA) was added to the samples at a ratio of 9:1 (volume of cells and culture medium: volume of Pres- toBlue reagent). The plates were then incubated for 60 minutes at 37°C and 5% CO2. Following incubation, triplicate 200-L samples were placed into the wells of a 96-well plate, and the fluorescence intensity of each well was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm us- ing a fluorescent plate reader (Infinite 200 PRO TECAN, Switzerland).
Histological and Morphological Assessment
The oral mucosa model samples were first fixed in 10% formalin solution for 24 hours; the samples were then mounted in Optimal Cutting Temperature (OCT) em- bedding compound, followed by freezing at -20 to -
80°C. Afterward, the samples were sectioned at a thickness of 10‒30 μm using a cryostat machine. The sections were then mounted on the histological slides. This was followed by drying the slides for around 30 minutes at room temperature. The slides were then stored in the freezer at -80°C until the pro- cessing for hematoxylin and eosin (H&E) staining.
The slides were examined under a light microscope by more than one histopathology expert to assess the changes in the connective tissue and the epithelial lay- ers of the model following the exposure to test agents. Assessment criteria included the continuity and thick- ness of the epithelium, cell morphology, presence or absence of pyknotic nuclei, presence of a distinct in- terface between the epithelium and the connective tis- sue layer.
1 ... 4 5 6 7 8 9 10 11 12
Wound Healing Assay
Monolayer cultures of normal oral fibroblasts and OKF6/TERT-2 keratinocytes were developed in 6- well tissue culture plates and divided into negative control (DMEM), positive control (70% ethanol), and test groups with various E-Cigarette concentrations (0.1%, 1%, 5%, and 10%) (n=6). The wounds were produced vertically in the middle of the surface of the monolayer systems using a disposable cell scraper. The test groups were exposed to the culture media containing E-cigarette liquid immediately before and continued daily after creating a wound, and then the wounds were monitored until complete healing had occurred. Microscopic images were obtained pre- and post-wound creation daily to assess the healing time in all the groups.
Statistical Analysis
SPSS 20 was used for statistical analysis. The normal- ity of the data was analyzed using the normality test (Shapiro-Wilk test). Means of the samples were com- pared with ANOVA, followed by multiple compari- sons using post hoc Tukey tests to determine the dif- ferences between the different groups. The level of significance for all the statistical tests was set at
=0.05.
Results
The results of the PrestoBlue assay for normal oral fi- broblasts and OKF6/TERT-2 keratinocyte monolayer cell cultures exposed to different concentrations of the E-Liquid for three days and seven days are shown in Figure 1. Short-term exposure to 10% E-liquid solu- tion caused a statistically significant reduction in the viability of NOFs (P<0.0001) compared to the nega- tive control group. Following medium-term exposure,
all the E-liquid concentration groups exhibited signif- icantly lower viability compared to the negative con- trol group.
OKF6/TERT-2 keratinocyte monolayers showed significantly lower viability after short-term exposure to all the E-liquid concentrations, whereas medium- term exposure resulted in significantly lower viability in all the groups except for 1% concentration group compared to the negative control group.
The TR146 keratinocyte monolayers did not exhibit any statistically significant difference in the viability between different E-liquid concentration groups com- pared to the negative control group after short-term exposure. However, medium-term exposure to 5% and 10% E-liquid solution caused a statistically sig- nificant increase in the viability of TR146 keratino-