Effects of electronic cigarette liquid on monolayer and 3D tissue-engineered models of human gingival mucosa

Abstract

Background. There is limited data available on potential biological effects of E-cigarettes on human oral tissues. The aim of this study was to evaluate the effects of E-cigarette liquid on the proliferation of normal and cancerous monolayer and 3D models of human oral mucosa and oral wound healing after short-term and medium-term exposure. Methods. Normal human oral fibroblasts (NOF), immortalized OKF6-TERET-2 human oral keratinocytes, and cancerous TR146 keratinocyte monolayer cultures and 3D tissue engineered oral mucosal models were exposed to different concentrations (0.1%, 1%, 5% and 10%) of E-cigarette liquid (12 mg/ml nicotine) for 1 hour daily for three days and for 7 days. Tissue viability was monitored using the PrestoBlue assay. Wounds were also produced in the middle surface of the monolayer systems vertically using a disposable cell scraper. The alterations in the cell morphology and wound healing were visualized using light microscopy and histological examination. Results. Statistical analysis showed medium-term exposure of TR146 keratinocytes to 5% and 10% E-liquid concentrations significantly increased the viability of the cancer cells compared to the negative control. Short-term exposure of NOFs to 10% E-liquid significantly reduced the cell viability, whereas medium-term exposure to all E-liquid concentrations significantly reduced the NOF cells’ viability. OKF6 cells exhibited significantly lower viability following short-term and mediumterm exposure to all E-cigarette concentrations compared to the negative control. 3D oral mucosal model containing normal oral fibroblasts and keratinocytes showed significant reduction in tissue viability after exposure to 10% E-liquid, whereas medium-term exposure resulted in significantly lower viability in 5% and 10% concentration groups compared to the negative control. There was a statistically significant difference in wound healing times of both NOF and OKF6 cells after exposure to 1%, 5% and 10% E-cigarette liquid. Conclusion. Medium-term exposure to high concentrations of the E-cigarette liquid had cytotoxic effects on normal human oral fibroblasts and OKF6 keratinocytes, but a stimulatory cumulative effect on the growth of cancerous TR146 keratinocyte cells as assessed by the PrestoBlue assay and histological evaluation of 3D oral mucosal models. In addition, E-liquid exposure prolonged the wound healing of NOF and OKF6 oral mucosa cells.

Comparison of Culture Media for <em>in-vitro</em> Expansion of Oral Epithelial Keratinocytes: Implications for Testing E-liquid Flavors

“…Our protocol includes a mixture of DMEM/F12 and KSFM, which results in a set of nutrients that yield faster growth and wound-healing recovery compared to DMEM/F12 alone. Shaikh and coworkers [84] also tested the effects of E-liquids on OKF6/TERT-2 cells and reported a decrease in viability after treatments, which agree with our results in figure 7B. Alanazi et al [85] show that the pathogenesis of yeast Candida albicans on a human gingival epithelial carcinoma cell line (grown in RPMI + 10% FBS) is increased after the microbe is exposed to tobacco flavored aerosol.…”

“…In addition, the same cell line, cultured in Roswell Park Medical Institute (RPMI) medium + 10% FBS recovered by 24 hours after onset of the scratch in the wound-healing assay [83]. In a study by Shaikh et al, using DMEM/F12 + 10% FBS, full recovery of OKF6/TERT-2 cells took over three days [84]. This study demonstrates that cell growth with DMEM/F12 alone, even when supplied with FBS, is not as effective as KSFM or DFK.…”

Nicotine Exerts Cytotoxic Effects in a Panel of Healthy Cell Lines and Strong Irritating Potential on Blood Vessels

“…Similarly, Shaikh et al studied the effects of liquids found in electronic cigarettes on oral fibroblasts and oral keratinocytes. The authors reported that e-liquids had cytotoxic effects that depended on the concentration when cells were exposed for short to medium periods of time [ 29 ]. There are several possible mechanisms by which nicotine could cause toxicity in human keratinocytes, including the stimulation of nicotine-like acetylcholine receptors (nAChRs), thereby altering the skin’s barrier function [ 30 ].…”

Comparison of Culture Media for <em>in-vitro</em> Expansion of Oral Epithelial Keratinocytes: Implications for Testing E-liquid Flavors

“…In addition, the same cell line, cultured in Roswell Park Medical Institute (RPMI) medium + 10% FBS recovered by 24 hours after onset of the scratch in the wound-healing assay [86]. In a study by Shaikh et al, using DMEM/F12 + 10% FBS, full recovery of OKF6/TERT-2 cells took over three days [87]. In our hands, cells in cultured in complete DMEM/F12 without FBS (see materials and methods section 4.1), achieved full recovery within two days (Fig 3).…”