IADR Abstract Archives
Components of E-Cigarettes Alter Calvarial Pre-Osteoblast Cell Cycle by Autophagy
Objectives: Use of tobacco products has long been linked to detrimental health effects. Our group has previously interrogated the effects of the nicotine on craniofacial development. These data have suggested alterations in calvarial development and stem cells, and altered jaw development. Here we extend our studied to interrogate the effects of the components in e-cigarettes, specifically JUUL, on calvarial pre-osteoblast cells in vitro to inform the potential health effects on the craniofacial skeleton.
Methods: MC3T3-E1 murine pre-osteoblast cells were cultured in vitro with clinically relevant concentrations of nicotine (50ng/ml), propylene glycol/vegetable glycine (PG/VG at 70%/30% relative concentrations), nicotine+PG/VG, and JUUL (50ng/ml nicotine). Cells were treated for 1 ,3, or 7 days in culture when endpoint assays were performed including MTS (mitochondrial activity), Hoecht fluorescence (nuclei), Apoptosis (Caspase), Autophagy (LC3), and Alkaline Phosphatase (ALP).
Results: Although treatments drove changes to cellular morphology, no gross changed were observed for ALP at any timepoint. 1 day assays suggest increases in autophagy in the nicotine+PG/VG and JUUL treated cells. 3 day assays suggest increases in all groups for Hoecht compared to control suggesting proliferative effects of all treatments with a concomitant decrease in apoptosis. Autophagy was decreased in nicotine+PG/VG and JUUL treatments at 3 days. 7 day data suggests a cessation in proliferation, increased autophagy with no evidence of increased apoptosis for the JUUL treated cells.
Conclusions: These data suggest concomitant treatment with nicotine+PG/VG or JUUL drives alterations in pre-osteoblast cell cycle by effects on proliferation and autophagy. Although we suspected cell stress and well as cytotoxic effects of nicotine+PG/VG and JUUL, no increase in apoptosis was observed. Recent literature has suggested autophagy may be a necessary step in osteoblast differentiation and activity. Future research will interrogate the effects of nicotine+PG/VG and JUUL on osteoblast function (collagen production and calcium phosphate precipitation).
Methods: MC3T3-E1 murine pre-osteoblast cells were cultured in vitro with clinically relevant concentrations of nicotine (50ng/ml), propylene glycol/vegetable glycine (PG/VG at 70%/30% relative concentrations), nicotine+PG/VG, and JUUL (50ng/ml nicotine). Cells were treated for 1 ,3, or 7 days in culture when endpoint assays were performed including MTS (mitochondrial activity), Hoecht fluorescence (nuclei), Apoptosis (Caspase), Autophagy (LC3), and Alkaline Phosphatase (ALP).
Results: Although treatments drove changes to cellular morphology, no gross changed were observed for ALP at any timepoint. 1 day assays suggest increases in autophagy in the nicotine+PG/VG and JUUL treated cells. 3 day assays suggest increases in all groups for Hoecht compared to control suggesting proliferative effects of all treatments with a concomitant decrease in apoptosis. Autophagy was decreased in nicotine+PG/VG and JUUL treatments at 3 days. 7 day data suggests a cessation in proliferation, increased autophagy with no evidence of increased apoptosis for the JUUL treated cells.
Conclusions: These data suggest concomitant treatment with nicotine+PG/VG or JUUL drives alterations in pre-osteoblast cell cycle by effects on proliferation and autophagy. Although we suspected cell stress and well as cytotoxic effects of nicotine+PG/VG and JUUL, no increase in apoptosis was observed. Recent literature has suggested autophagy may be a necessary step in osteoblast differentiation and activity. Future research will interrogate the effects of nicotine+PG/VG and JUUL on osteoblast function (collagen production and calcium phosphate precipitation).