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cytes compared to the negative control group (Figure 2).
Normal oral fibroblast and OKF6/TERT-2 keratino- cyte co-culture system showed a significant reduction in cell viability in 10% E-liquid concentration group following short-term exposure. In contrast, medium-
Figure 1. Tissue viability of normal oral fibroblasts and OKF6 keratinocyte monolayer cell cultures exposed to different concentrations of E-liquid as assessed by the PrestoBlue assay.
Figure 2. Tissue viability of TR146 keratinocyte mono- layer cell cultures exposed to different concentrations of E-liquid as assessed by the PrestoBlue assay.
term exposure resulted in significantly lower viability in 5% and 10% concentration groups (P<0.0001) compared to the negative control group (Figure 3). Histologically, the 3D oral mucosal models utilizing the NOFs and TR146 keratinocytes showed an in- crease in the thickness of the cancerous epithelial layer in high E-liquid concentration groups compared to the negative control after short-term and medium- term exposure (Figures 4 and 5).
Figure 6 demonstrates microscopic views of the wound healing at different stages for normal oral fi- broblasts and OKF6/TERT-2 keratinocytes. Table 1 presents the mean values and standard deviations of the total time of wound healing for the control and test groups. There was a statistically significant difference in the wound healing time of both NOF and OKF6/TERT-2 monolayer systems exposed to 1%, 5%, and 10% E-liquid solutions compared to those of the negative control group (P<0.05).
Three different cell types, including NOFs, OKF6/TERT2 keratinocytes, and cancerous TR146 keratinocytes, were used in this study. Normal oral fi- broblasts were selected as they are thought to play a major role in mucosal wound healing; they are also responsible for extracellular matrix synthesis in the connective tissue layer. OKF6/TERT-2 keratinocytes exhibit high reproducibility, avoid batch-to-batch var- iations; they were selected to represent normal oral epithelial cells. Conversely, squamous cell carci- noma-derived TR146 cells were utilized to assess the effects of E-liquid on oral cancer cells.
Figure 3. Tissue viability of normal oral fibroblast and OKF6 keratinocyte 3D co-culture systems exposed to different concentrations of E-liquid as assessed by the PrestoBlue assay.
E-liquid with no added flavors was used to elimi- nate the confounding effects of different additives that are observed with various types of flavoring agents used in E-cigarettes. Different studies have shown that certain flavors, such as menthol, cinnamon, cara- mel, butterscotch, bubble-gum, and coffee, have more cytotoxic effects on cells compared to some other fla- vors.39-41
Four different concentrations (0.1%, 1%, 5%, and 10%) of E-liquid solution were prepared to assess the effects of various concentrations of E-liquid on the cells, covering the range from light vapers to heavy e- cigarette users. The duration of exposure included three days and seven days to simulate short-term and medium-term vaping.
In this study, electronic cigarette liquid exhibited an adverse effect on the viability of normal oral
Figure 4. Histological sections of 3D tissue-engineered oral mucosa models after short-term exposure to (A) 0.1% E- liquid; (B) 1% E-liquid; (C) 5% E-liquid; (D) 10% E-liquid; (E) negative control; (F) positive control (H & E staining, original magnification ×10).
Figure 5. Histological sections of 3D tissue-engineered oral mucosa models after medium-term exposure to (A) 0.1% E-liquid; (B) 1% E-liquid; (C) 5% E-liquid; (D) 10% E-liquid; (E) negative control; (F) positive control (H & E Staining, original magnification ×10).
fibroblasts and OKF6/TERT-2 keratinocytes. These results are consistent with a previous study that tested different types of E-cigarette liquids on the human periodontal ligament fibroblasts and showed a reduc- tion in the viability of cells in the samples that were exposed to E-liquids.39
Conversely, a dose-dependent stimulatory effect of E-liquid on the growth of cancerous TR146 cells was observed in our study, exhibiting increased viability and proliferation of TR146 cells with increasing con- centrations of E-liquid. Similarly, histological evalu- ation of the 3D oral mucosa models showed an in- crease in the thickness of the cancerous epithelial layer exposed to high concentrations of E-liquid. This is the first study reporting the use of a full-thickness
3D tissue-engineered oral mucosal model for the bio- logical evaluation of electronic cigarettes on cancer- ous oral tissues. These findings have not been re- ported previously and might indicate tumor-promot- ing effects of the ingredients of the E-liquid tested in this study.
Previous studies have raised some concerns regard- ing the potential effects of nicotine on promoting tu- mors in the lungs through various possible mecha- nisms, such as cell migration, proliferation and angi- ogenesis.20-21
The influence of nicotine on dysplastic oral keratinocyte cell line and precancerous lesions of the mouse tongue has been investigated previously, showing an inhibitory effect on apoptosis and a
Figure 6. Microscopic views of the wound healing assay showing (A) the center of the wound on day 1 of fibroblast culture; (B) central wound on day 3 of fibroblast culture; (C) completely healed fibroblast cultures; (D) the center of the wound on day 1 of keratinocyte culture; (E) central wound on day 3 of keratinocyte culture; and (F) completely healed keratinocyte cultures.
Table 1. Mean values and standard deviations of the to- tal time of wound healing for the control and test groups
stimulatory effect on the growth of oral precancerous lesions.44 Similarly, Chernyavsky et al42 assessed the tumor-promoting effects of nicotine on oral and lung cancer cells. Nicotine exhibited resistance to apopto- sis, increasing the counts of both lung and oral cancer cells.
Some other studies have used monolayer cell cul- ture systems to assess the cytotoxicity of electronic cigarettes.39-43 Different types of cell lines have been exposed to either electronic cigarette aerosols or E- liquid solutions. These studies have also confirmed the adverse effects of E-cigarettes, with some cell lines being more sensitive than the others.
Recently, studies have been conducted to assess the effects of electronic cigarettes on oral mucosal cells. In a study by Yu et al,43 electronic cigarette exposure reduced cell viability along with high levels of apop- tosis and necrosis, with alterations in augmented DNA strands, in normal epithelial and squamous cell carcinoma (head and neck) cell lines. However, Gut- tenplan et al44 observed a stimulatory effect of E-cig- arette exposure on the proliferation of human oral leu- koplakia cells. Conversely, Willershausen et al37 re- ported a reduction in cell proliferation of periodontal ligament fibroblasts after exposure to various E-liq- uids.
Sundar et al45 utilized human periodontal ligament fibroblasts and a 3D gingival epithelium-only tissue model to assess the effects of electronic cigarettes, re- porting an increase in pro-inflammatory cytokines and an elevated oxidative/carbonyl stress resulting in the production of high levels of cyclooxygenase-2 and prostaglandin E2. Although the 3D split-thickness tis- sue model used in this study was more clinically rele- vant than monolayer cell culture systems, it lacked the connective tissue component.
A study by Sancilio et al46 raised concerns regarding E-cigarette’s role in the pathogenesis of oral diseases. Human gingival fibroblasts exposed to E-Liquids showed decreased production of collagen I and in- creased levels of lactate dehydrogenase.
There is a lack of research on the effects of E-ciga- rettes on human oral mucosa wound healing. It has been indicated that exposure to E-cigarettes reduces the viability of cells and compromises cell migra- tion.46 Therefore, this study aimed to investigate the effects of E-cigarette liquids with different concentra- tions on oral mucosa wound healing. Two wound healing models were used in this study based on nor- mal oral fibroblasts to represent connective tissue wound healing and OKF6/TERT2 keratinocytes to simulate epithelial wound healing. The results of this in vitro assay were consistent with the results of the cytotoxicity tests and indicated that the E-liquid tested in this study might have potential dose-dependent ad- verse effects on oral mucosa wound healing, prolong- ing the healing times both in the epithelial and the connective tissue layers. In a recent study,47 human gingival fibroblasts were exposed to three different groups (cigarette smoke condensate, nicotine-free or nicotine-rich electronic cigarette vapor condensates), and the results showed that both cigarette smoke and electronic cigarette vapors affected the proliferation and migration of fibroblasts. Additionally, the cell scratch test revealed delayed wound healing, con- sistent with the findings of this study.
There were certain limitations in our study; firstly, this was an in-vitro study, and the results may not be extrapolated to an in-vivo situation. Secondly, only E- liquids were tested in this study rather than the E-cig- arette aerosols. Ideally, the cytotoxic effects of E-cig- arettes should be assessed in both liquid and vapor form, as the E-cigarette vapors come into direct con- tact with oral mucosa.
Hence, further research is required to overcome these limitations by assessing E-cigarettes of different flavors and various concentrations of nicotine. The liquid, as well as the vapor form of E-cigarettes, can be tested and compared with the conventional ciga- rette smoke. Furthermore, evaluating the long-term effects of E-cigarettes would further add to our under- standing of the biological effects of E-cigarettes on human oral tissues.
This in vitro study revealed that short-term and me- dium-term exposure to the electronic cigarette liquid had dose-dependent cytotoxic effects on normal hu- man oral fibroblasts and OKF6/TERT-2 oral keratino- cytes. However, E-liquid exposure had a cumulative stimulatory effect on the growth of cancerous TR146 keratinocytes using both monolayer and 3D cell cul- ture systems.
The full-thickness 3D tissue-engineered human oral
mucosal model has the potential to be used as a clini- cally relevant biological test system for the evaluation of electronic cigarettes.
In addition, E-liquid exposure prolonged the wound healing of both normal oral fibroblasts and OKF6/TERT-2 epithelial cells.
Competing Interests
The authors declare no conflict(s) of interest related to the publication of this work.
Authors’ Contributions
Conceptualization, Z.S., A.A., T.A., K.F., L.T. and K.M.; Data curation, Z.S. and A.A.; Investigation, Z.S., A.A., T.A., K.F., L.T. and K.M.; Methodology, Z.S., A.A., T.A.
and K.F.; Supervision, L.T. and K.M.; Validation, Z.S., and A.A.; Writing—original draft, Z.S. and A.A.; Writing—re- view and editing, T.A., K.F., L.T. and K.M.
Acknowledgments
The authors would like to acknowledge Cancer Research UK for kindly providing the TR146 cell line and Brigham and Women's Hospital, Harvard Institute of Medicine, for providing the immortalized OKF6/TERT-2 human oral keratinocyte cell line.
Funding
This research received no external funding.
Ethics Approval
This study has been approved by National Research Ethics Service, NRES Committee London—Hampstead. Re- search Ethics Committee (REC) Reference number: 15/LO/0116; date of approval: 21/01/2015.
spectrometric detection. Journal of chromatography A. 2010 Nov 26;1217(48):7547-55.
82.
Vardavas CI, Anagnostopoulos N, Kougias M, Evangelopou- lou V, Connolly GN, Behrakis PK. Short-term pulmonary ef- fects of using an electronic cigarette: impact on respiratory flow resistance, impedance, and exhaled nitric oxide. Chest. 2012 Jun 1;141(6):1400-6.
G. Multicentric cohort study on the long-term efficacy and safety of electronic cigarettes: study design and methodology.
BMC public health. 2013 Dec;13(1):883.
Normal oral fibroblast and OKF6/TERT-2 keratino- cyte co-culture system showed a significant reduction in cell viability in 10% E-liquid concentration group following short-term exposure. In contrast, medium-
Figure 1. Tissue viability of normal oral fibroblasts and OKF6 keratinocyte monolayer cell cultures exposed to different concentrations of E-liquid as assessed by the PrestoBlue assay.
Figure 2. Tissue viability of TR146 keratinocyte mono- layer cell cultures exposed to different concentrations of E-liquid as assessed by the PrestoBlue assay.
term exposure resulted in significantly lower viability in 5% and 10% concentration groups (P<0.0001) compared to the negative control group (Figure 3). Histologically, the 3D oral mucosal models utilizing the NOFs and TR146 keratinocytes showed an in- crease in the thickness of the cancerous epithelial layer in high E-liquid concentration groups compared to the negative control after short-term and medium- term exposure (Figures 4 and 5).
Figure 6 demonstrates microscopic views of the wound healing at different stages for normal oral fi- broblasts and OKF6/TERT-2 keratinocytes. Table 1 presents the mean values and standard deviations of the total time of wound healing for the control and test groups. There was a statistically significant difference in the wound healing time of both NOF and OKF6/TERT-2 monolayer systems exposed to 1%, 5%, and 10% E-liquid solutions compared to those of the negative control group (P<0.05).
1 ... 4 5 6 7 8 9 10 11 12
Discussion
Three different cell types, including NOFs, OKF6/TERT2 keratinocytes, and cancerous TR146 keratinocytes, were used in this study. Normal oral fi- broblasts were selected as they are thought to play a major role in mucosal wound healing; they are also responsible for extracellular matrix synthesis in the connective tissue layer. OKF6/TERT-2 keratinocytes exhibit high reproducibility, avoid batch-to-batch var- iations; they were selected to represent normal oral epithelial cells. Conversely, squamous cell carci- noma-derived TR146 cells were utilized to assess the effects of E-liquid on oral cancer cells.
Figure 3. Tissue viability of normal oral fibroblast and OKF6 keratinocyte 3D co-culture systems exposed to different concentrations of E-liquid as assessed by the PrestoBlue assay.
E-liquid with no added flavors was used to elimi- nate the confounding effects of different additives that are observed with various types of flavoring agents used in E-cigarettes. Different studies have shown that certain flavors, such as menthol, cinnamon, cara- mel, butterscotch, bubble-gum, and coffee, have more cytotoxic effects on cells compared to some other fla- vors.39-41
Four different concentrations (0.1%, 1%, 5%, and 10%) of E-liquid solution were prepared to assess the effects of various concentrations of E-liquid on the cells, covering the range from light vapers to heavy e- cigarette users. The duration of exposure included three days and seven days to simulate short-term and medium-term vaping.
In this study, electronic cigarette liquid exhibited an adverse effect on the viability of normal oral
Figure 4. Histological sections of 3D tissue-engineered oral mucosa models after short-term exposure to (A) 0.1% E- liquid; (B) 1% E-liquid; (C) 5% E-liquid; (D) 10% E-liquid; (E) negative control; (F) positive control (H & E staining, original magnification ×10).
Figure 5. Histological sections of 3D tissue-engineered oral mucosa models after medium-term exposure to (A) 0.1% E-liquid; (B) 1% E-liquid; (C) 5% E-liquid; (D) 10% E-liquid; (E) negative control; (F) positive control (H & E Staining, original magnification ×10).
fibroblasts and OKF6/TERT-2 keratinocytes. These results are consistent with a previous study that tested different types of E-cigarette liquids on the human periodontal ligament fibroblasts and showed a reduc- tion in the viability of cells in the samples that were exposed to E-liquids.39
Conversely, a dose-dependent stimulatory effect of E-liquid on the growth of cancerous TR146 cells was observed in our study, exhibiting increased viability and proliferation of TR146 cells with increasing con- centrations of E-liquid. Similarly, histological evalu- ation of the 3D oral mucosa models showed an in- crease in the thickness of the cancerous epithelial layer exposed to high concentrations of E-liquid. This is the first study reporting the use of a full-thickness
3D tissue-engineered oral mucosal model for the bio- logical evaluation of electronic cigarettes on cancer- ous oral tissues. These findings have not been re- ported previously and might indicate tumor-promot- ing effects of the ingredients of the E-liquid tested in this study.
Previous studies have raised some concerns regard- ing the potential effects of nicotine on promoting tu- mors in the lungs through various possible mecha- nisms, such as cell migration, proliferation and angi- ogenesis.20-21
The influence of nicotine on dysplastic oral keratinocyte cell line and precancerous lesions of the mouse tongue has been investigated previously, showing an inhibitory effect on apoptosis and a
Figure 6. Microscopic views of the wound healing assay showing (A) the center of the wound on day 1 of fibroblast culture; (B) central wound on day 3 of fibroblast culture; (C) completely healed fibroblast cultures; (D) the center of the wound on day 1 of keratinocyte culture; (E) central wound on day 3 of keratinocyte culture; and (F) completely healed keratinocyte cultures.
Table 1. Mean values and standard deviations of the to- tal time of wound healing for the control and test groups
| Cells | Groups | Mean (days) | SD |
| NOF | 0.1% E-liquid | 6.17 | 0.75 |
| 1% E-liquid | 7.33 | 0.51 | |
| 5% E-liquid | 9.167 | 0.75 | |
| 10% E-liquid | 12.33 | 1.03 | |
| Control (DMEM) | 5.17 | 0.41 | |
| OKF6/ TERT2 | 0.1% E-liquid | 6.00 | 0.63 |
| 1% E-liquid | 6.67 | 0.52 | |
| 5% E-liquid | 7.67 | 0.52 | |
| 10% E-liquid | 10.50 | 0.84 | |
| Control (DMEM) | 5.05 | 0.63 |
stimulatory effect on the growth of oral precancerous lesions.44 Similarly, Chernyavsky et al42 assessed the tumor-promoting effects of nicotine on oral and lung cancer cells. Nicotine exhibited resistance to apopto- sis, increasing the counts of both lung and oral cancer cells.
Some other studies have used monolayer cell cul- ture systems to assess the cytotoxicity of electronic cigarettes.39-43 Different types of cell lines have been exposed to either electronic cigarette aerosols or E- liquid solutions. These studies have also confirmed the adverse effects of E-cigarettes, with some cell lines being more sensitive than the others.
Recently, studies have been conducted to assess the effects of electronic cigarettes on oral mucosal cells. In a study by Yu et al,43 electronic cigarette exposure reduced cell viability along with high levels of apop- tosis and necrosis, with alterations in augmented DNA strands, in normal epithelial and squamous cell carcinoma (head and neck) cell lines. However, Gut- tenplan et al44 observed a stimulatory effect of E-cig- arette exposure on the proliferation of human oral leu- koplakia cells. Conversely, Willershausen et al37 re- ported a reduction in cell proliferation of periodontal ligament fibroblasts after exposure to various E-liq- uids.
Sundar et al45 utilized human periodontal ligament fibroblasts and a 3D gingival epithelium-only tissue model to assess the effects of electronic cigarettes, re- porting an increase in pro-inflammatory cytokines and an elevated oxidative/carbonyl stress resulting in the production of high levels of cyclooxygenase-2 and prostaglandin E2. Although the 3D split-thickness tis- sue model used in this study was more clinically rele- vant than monolayer cell culture systems, it lacked the connective tissue component.
A study by Sancilio et al46 raised concerns regarding E-cigarette’s role in the pathogenesis of oral diseases. Human gingival fibroblasts exposed to E-Liquids showed decreased production of collagen I and in- creased levels of lactate dehydrogenase.
There is a lack of research on the effects of E-ciga- rettes on human oral mucosa wound healing. It has been indicated that exposure to E-cigarettes reduces the viability of cells and compromises cell migra- tion.46 Therefore, this study aimed to investigate the effects of E-cigarette liquids with different concentra- tions on oral mucosa wound healing. Two wound healing models were used in this study based on nor- mal oral fibroblasts to represent connective tissue wound healing and OKF6/TERT2 keratinocytes to simulate epithelial wound healing. The results of this in vitro assay were consistent with the results of the cytotoxicity tests and indicated that the E-liquid tested in this study might have potential dose-dependent ad- verse effects on oral mucosa wound healing, prolong- ing the healing times both in the epithelial and the connective tissue layers. In a recent study,47 human gingival fibroblasts were exposed to three different groups (cigarette smoke condensate, nicotine-free or nicotine-rich electronic cigarette vapor condensates), and the results showed that both cigarette smoke and electronic cigarette vapors affected the proliferation and migration of fibroblasts. Additionally, the cell scratch test revealed delayed wound healing, con- sistent with the findings of this study.
There were certain limitations in our study; firstly, this was an in-vitro study, and the results may not be extrapolated to an in-vivo situation. Secondly, only E- liquids were tested in this study rather than the E-cig- arette aerosols. Ideally, the cytotoxic effects of E-cig- arettes should be assessed in both liquid and vapor form, as the E-cigarette vapors come into direct con- tact with oral mucosa.
Hence, further research is required to overcome these limitations by assessing E-cigarettes of different flavors and various concentrations of nicotine. The liquid, as well as the vapor form of E-cigarettes, can be tested and compared with the conventional ciga- rette smoke. Furthermore, evaluating the long-term effects of E-cigarettes would further add to our under- standing of the biological effects of E-cigarettes on human oral tissues.
1 ... 4 5 6 7 8 9 10 11 12
Conclusion
This in vitro study revealed that short-term and me- dium-term exposure to the electronic cigarette liquid had dose-dependent cytotoxic effects on normal hu- man oral fibroblasts and OKF6/TERT-2 oral keratino- cytes. However, E-liquid exposure had a cumulative stimulatory effect on the growth of cancerous TR146 keratinocytes using both monolayer and 3D cell cul- ture systems.
The full-thickness 3D tissue-engineered human oral
mucosal model has the potential to be used as a clini- cally relevant biological test system for the evaluation of electronic cigarettes.
In addition, E-liquid exposure prolonged the wound healing of both normal oral fibroblasts and OKF6/TERT-2 epithelial cells.
Competing Interests
The authors declare no conflict(s) of interest related to the publication of this work.
Authors’ Contributions
Conceptualization, Z.S., A.A., T.A., K.F., L.T. and K.M.; Data curation, Z.S. and A.A.; Investigation, Z.S., A.A., T.A., K.F., L.T. and K.M.; Methodology, Z.S., A.A., T.A.
and K.F.; Supervision, L.T. and K.M.; Validation, Z.S., and A.A.; Writing—original draft, Z.S. and A.A.; Writing—re- view and editing, T.A., K.F., L.T. and K.M.
Acknowledgments
The authors would like to acknowledge Cancer Research UK for kindly providing the TR146 cell line and Brigham and Women's Hospital, Harvard Institute of Medicine, for providing the immortalized OKF6/TERT-2 human oral keratinocyte cell line.
Funding
This research received no external funding.
Ethics Approval
This study has been approved by National Research Ethics Service, NRES Committee London—Hampstead. Re- search Ethics Committee (REC) Reference number: 15/LO/0116; date of approval: 21/01/2015.
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