The effect of nicotine on dental pulp stem cells survival

Authors

Bianca Amy Lau, University of the PacificFollow
Nan (Tori) Xiao, University of the PacificFollow
Der Thor, University of the PacificFollow

Lead Author Affiliation

Doctor of Dental Surgery

Lead Author Program & Year

DDS Year 2

Presentation Category

Research

Introduction/Context/Diagnosis

Consumption of nicotine, a parasympathomimetic alkaloid that is a major part of tobacco, poses as a contributing factor for periodontal disease. Research has shown that nicotine affects blood flow, cytokine production, and periodontal tissue regeneration. This study aims to analyze the effect of different concentrations of nicotine on the survival of dental pulp stem cells.

Methods/Treatment Plan

Cell Culture

Human DPSCs were purchased from Lonza. Cells before passage 10 were used for experiments. DPSCs were cultured as previously reported. Briefly, DPSCs were cultured in DMEM (Gibco) supplemented with 10% FBS, antibiotics and ascorbic acids.

Survival assay

6 x 10^4 or 6 x 10^3 cells were seeded in 6 well culture plates in triplicates. Cells were cultured with nicotine (Sigma) at different concentrations for up to 10 days. Medium with nicotine was not changed throughout the experiment. Cells fixed with ETOH and stained with 0.5% crystal violet. Images were taken with Leica DMi1 inverted microscope.

Image Quantification

Quantification was done with ImageJ. After converting the image to grayscale, the upper and lower thresholds were manually adjusted to illustrate the cells. 825 pixels was the known distance, and the scale was set to micrometers. The area, mean gray area, and % area were measured. Survival of the cells were evaluated based on the percentage of the stained area of cells per images.

Statistics

Data were expressed as SD. Statistical ANOVA and Student’s t-tests (2-tailed) were used to compare the data. P ≤ 0.05 was considered to be significant.

Results/Outcome

For the 6 x 10^4 cell experiment, the average % area of cells in the control, ETOH, 100nM, 1uM, 10uM, 100uM, 1mM, 10mM were 41.66, 37.52, 40.28, 38.52, 37.10, 26.85, 11.78, and 0.04 respectively. For the 6 x 10^3 cell experiment, the variables were used and the average % area of cells were 7.89, 5.35, 4.38, 5.11, 2.55, 1.68, 0.42, and 0.01 respectively. At 10mM, there were virtually no cells present for both experiments.

Significance/Conclusions

Through this experiment, we were able to confirm that nicotine decreases the amount of human dental pulp stem cells. There was a steady decline in the number of cells as the concentration of nicotine increased; however, there was a significant decrease in the number of cells at 100uM for the 6 x10^4 cell culture and at 10uM for the 6 x 10^3 culture. The experiment demonstrates that as little as 100nM of nicotine can have a negative effect on dental pulp cells while greater concentrations of nicotine will have an even greater effect on the cells. A future long term study would be to compare the survival of DPSCs harvested from extracted teeth of smokers and non-smokers. Another follow-up experiment would be to explore if nicotine also has time-dependent effects on DPSC’s in addition to its dose-dependent effects.

Format

Event