Effects of Cotinine on Human Gingival Fibroblast Migration

Abstract

Nicotine has a deleterious impact on gingival fibroblast cell viability, adhesion, and
migration. Less is known regarding the effect of cotinine, the main metabolite of nicotine, on
such processes. The objective of this study was to determine if cotinine affects the adhesion or
migration of Human Gingival Fibroblasts (HGF) in culture. HGF were treated with nicotine
or cotinine at several concentrations and dose-response cytotoxicity was determined by MTT
assay. The effects of nicotine and cotinine on HGF adhesion were measured colorimetrically
and cell migration was determined using the scratch wound assay. The number of HGF oriented
parallel to the wound edge at 24 hours was counted using phase contrast images. Data were
analyzed using ANOVA and Dunnet’s multiple comparison post-test. At the highest concentrations
of cotinine (640 ng/ml) and nicotine (400 μg/ml) both HGF survival and cell adhesion
were significantly inhibited (p<0.01). By scratch wound assay HGF migration from the wound
edge at 24 hours was significantly inhibited by 320 ng/ml (p<0.001) and 640 ng/ml (p<0.001)
cotinine, and by 400 μg/ml (p<0.01) nicotine. HGF migration was significantly inhibited by
80 ng/ml (p<0.05), 320 ng/ml (p<0.01) and 640 ng/ml (p<0.001) cotinine and by 100 μg/ml
(p<0.01), 200 μg/ml (p<0.01) and 400 μg/ml (p<0.001) nicotine at 48 hours. Significantly more
HGF were oriented parallel to wound edge with pre treatment of 10 ng/ml cotinine or 50 μg/ml
nicotine before wounding (p<0.001). In HGF exposed to nicotine (400 μg/ml) or cotinine (640
ng/ml), cell survival, cell adhesion, and migration were significantly decreased, but cell polarity
was not affected. These concentrations are within ranges of serum levels in smokers, providing
evidence that multiple cellular aspects of wound healing are compromised in tobacco users.