Effects of ‘Halo Purity’ E-Juices on Proliferation and Toxicity of Dental Pulp Stem Cells (DPSC) Alexandria Frazier and Dr. Jack Windsor Indiana University School of Dentistry Indiana University-Purdue University Indianapolis Abstract Current Status of Research The use of electronic cigarettes (EC) worldwide has increased tremendously. There is limited information on the effects of the EC nicotine liquids (e-juices) on cells in the oral cavity that are the first ones exposed to the vaporized e-juices. There are multiple brands of e-juices available with different concentrations of nicotine. The contents of the e-juices contain propylene glycol, vegetable glycerin, natural and artificial flavorings. Therefore, the purpose of this study is to evaluate one brand of e-juice with and without nicotine on dental pulp stem cells (DPSC) to determine cell toxicity and its effects on cell proliferation, as well as its ability at a non-toxic concentration to alter the inflammatory cytokine expression pattern of these cells. The e-juice chosen was Halo Purity, which is a popular brand in the Indianapolis vapor shops. DPSC were grown in cell culture media with serum and then plated in six well plates (75000 cells per well). After allowing 24 hours of cell attachment through incubation, the media was removed and media without serum and with nicotine containing and not e-juices were added at different concentrations. After 3 days, assays for cell proliferation and cell toxicity were performed. Preliminary results from the assays show that approximately 180 g/ml of nicotine e-juice was the highest concentration that did not affect cell proliferation or that was not cytotoxic. Cytokine protein arrays will used to examine if this concentration will induce inflammation by altering the expression of inflammatory cytokines as occurs when cells are exposed to tobacco cigarette smoke condensate since tobacco cigarette smoking is at risk for periodontal disease, an inflammatory disease. Measurement of human gingival fibroblast proliferation using the water soluble tetrazolium-1 assay Measurement of cigarette smoke condensate cytotoxicity using lactate dehydrogenase assay Methods Cell Culture Human gingival fibroblast were cultured from explants of clinically healthy gingival connective tissue removed from a patient undergoing crown-lengthening surgery, with Institutional Review Board approval at the Indiana University School of Dentistry. We grew stem-pulp cells out of the explants while subculturing and maintaining them throughout our experiments. Measurement of human gingival fibroblast proliferation using the water-soluble tetrazolium-1 assay The purpose of this experiment was to determine how many live cells were in the human gingival fibroblasts before the exposure of nicotine. I exposed my cells to the e-juice brand, ‘Halo Purity’. I used a nicotine and non-nicotine version of the brand. After exposing them, I proceeded through this instructional procedure. This procedure was repeated several times and the mean value was calculated. The absorbance values of each sample were to compared with the untreated cell control, by percentage, using the following equation: Cell proliferation (%)= (absorbance value of cigarette smoke condensate treatment/ absorbance value of no cigarette smoke condensate treatment) X 100% Measurement of cigarette smoke condensate cytotoxicity using lactate dehydrogenase assay The purpose of this experiment was to determine how toxic my nicotine brand, ‘Halo Purity’, was and how many human gingival fibroblasts were killed because of it. This procedure was repeated several times and the mean value was calculated. The percentage release of lactate dehydrogenase from the treated cells was calculated by comparing it to the maximum release of lactate dehydrogenase achieved by the lysis solution used in the control cells. To determine the cytotoxicity, the absorbance values of the background were subtracted from that of the experimental samples and the cytotoxicity was calculated using the following equation: Cytotoxicity (%)= (experiment value-low control)/ (high control-low control) X 100% Introduction/ Review of Literature Tobacco cigarette smoke condensate increases cytotoxicity in human gingival fibroblasts. The functions of the human gingival fibroblasts are important to periodontal health because they are the main cellular component of periodontal connective tissues. The development of periodontal disease causes collagen degradation will begin to occur. Collagen is a extracellular matrix-degrading component of the gingiva. The major extracellular matrix degrading enzymes are the matrix metalloproteinase (MMP). These MMP’s have shown to play a role in periodontal tissue destruction. The balance of MMP’s is believed to be important to periodontal health and nicotine may affect this balance. The purpose of this study was to examine the effects of the electronic cigarettes nicotine liquids (e-juices) to the first cells exposed in the oral cavity which are stem-pulp cells. Due to the lack of factual evidence over the toxicology data, there has not been a thorough assessment of the effects caused by electronic cigarettes. References Zhang W1, Song F, Windsor LJ. Cigarette smoke condensate affects the collagen-degrading ability of human gingival fibroblasts. J Periodontal Res. 2009 Dec;44(6):704-13 Allam E1, Zhang W, Al-Shibani N, Sun J, Labban N, Song F, Windsor LJ. Effects of cigarette smoke condensate on oral squamous cell carcinoma cells. Arch Oral Biol. 2011 Oct;56(10):1154-61.