Cigarette Smoke and E-Cigarette Vapor Dysregulate Osteoblast Interaction With Titanium Dental Implant Surface
The purpose of this study was to determine the possible deleterious effects of e-cigarette vapor on osteoblast interaction with dental implant material. Osteoblasts were cultured onto Ti6Al4V titanium implant disks and were then exposed or not to whole cigarette smoke (CS), as well as to nicotine-rich (NR) or nicotine-free (NF) e-vapor for 15 or 30 minutes once a day for 1, 2, or 3 days, after which time various analyses were performed. Osteoblast growth on the titanium implant disks was found to be significantly (P < .001) reduced following exposure to CS and to the NR and NF e-vapors. Osteoblast attachment to the dental implant material was also dysregulated by CS and the NR and NF e-vapors through a decreased production of adhesion proteins such as F-actin. The effects of CS and e-cigarette vapor on osteoblast growth and attachment were confirmed by reduced alkaline phosphatase (ALP) activity and tissue mineralization. The adverse effects of CS and the NR and NF e-vapors on osteoblast interaction with dental implant material also involved the caspase-3 pathway, as the caspase-3 protein level increased following exposure of the osteoblasts to CS or e-vapor. It should be noted that the adverse effects of CS on osteoblast growth, attachment, ALP, and mineralized degradation were greater than those of the NR and NF e-vapors, although the latter did downregulate osteoblast interaction with the dental implant material. Overall results suggest the need to consider e-cigarettes as a possible contributor to dental implant failure and/or complications.

Cigarette smoke (CS) and e-cigarette vapor decreased osteoblast growth on dental implant material. Osteoblasts were seeded onto dental implant disks, cultured for 24 hours, and then exposed or not to CS, or to nicotine (NF) or nicotine rich (NR) e-vapor for 1, 2, or 3 days. At the end of each period, a MTT assay was performed to determine cell growth. ***P < .0001 compared with the control. Results are presented as the mean ± SD (n = 16 implant disks, with 2 implants in each experiment). Ctrl indicates control; Expo, exposure; OD, optical density.

Cigarette (CS) smoke and e-cigarette vapor decreased osteoblast attachment to dental implant material. Cells were seeded onto the dental implant disks and cultured for 24 hours prior to exposure 3 times, once a day. Following exposure, cells attached to the implants were subjected to Hoechst staining. Representative photos of 12 implant disks, with 2 implants in each experiment. Scale bar = 50 μm. NR indicates nicotine rich; NF, nicotine free; ctrl, control.

F-actin expression by osteoblasts following exposure to cigarette smoke (CS) or e-cigarette vapor. Osteoblasts were seeded onto dental implant disks then exposed or not to CS or e-vapor 3 times, once a day. F-actin filaments were stained with phalloidin-FITC. Note the well-organized, dense filamentous actin cytoskeleton (arrows) in the control (ctrl; nonexposed cells) cells vs the CS and e-vapor–exposed cultures. Representative images are from 12 different implant disks (a). Scale bars = 50 μm. Fluorescence intensity was quantified by ImageJ and plotted (b). Results are presented as the mean ± SD (n = 16 implant disks, with 2 implants in each experiment). A significant difference was observed when comparing the cells exposed to CS or to nicotine rich (NR) and nicotine free (NF) e-vapor and those of the ctrl (nonexposed cells). ***P < .001; **P < .01; *P < .05.

Decrease of alkaline phosphatase (ALP) activity following exposure of osteoblasts to either cigarette smoke or e-cigarette vapor. Cells were seeded onto dental implant disks and cultured for 24 hours prior to exposure or not to cigarette smoke (CS) or e-vapor. Exposure time was 15 or 30 minutes once a day for 2 or 3 days. At the end of each exposure period, ALP activity was assessed by means of alkaline phosphatase substrate p-NPP using cultured supernatant. Data are presented as the mean ± SD (n = 16 implant disks, with 2 implants in each experiment). ***P < .0001; **P < .001 compared with the control (ctrl). NR indicates nicotine rich; NF, nicotine free; Expo, exposure.

Cigarette smoke (CS) and e-cigarette vapor degraded mineralized tissue. Osteoblasts were first cultured onto dental implant disks for 3 weeks and were exposed 30 min/d to either CS or e-vapor for 1, 2, or 3 days, after which time cells were fixed and stained with 0.5% alizarin red. The alizarin red stain was then eluted with cetylpyridinium chloride and quantified. Data represent mean ± SD (n = 16 implant disks, with 2 implants in each experiment). ***P < .001; **P < .01; *P < .05 compared with the control. Ctrl indicates control; NR, nicotine rich; NF, nicotine free.

Cigarette smoke (CS) and e-cigarette vapor increased caspase-3 protein expression by osteoblasts cultured onto dental implant material. Osteoblasts were cultured onto dental implant disks and then exposed to CS or to nicotine rich (NR) or nicotine free (NF) e-vapor during 30 minutes once a day for 1 or 2 days. After each period, the total protein extracts were prepared and stained with specific antibody to caspase-3. (a) Caspase-3 expression as determined by Western blot (representative gel of 12 dental implant disks). (b) Protein bands scanned by National Institutes of Health ImageJ software. The caspase-3/β-actin ratio was calculated and plotted. ***P < .001; **P < .01 compared with the control. Ctrl indicates control; Expo, exposure.
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