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.

A literature review of bioactive substances for the treatment of periodontitis: In vitro, in vivo and clinical studies

“…Kaempferol is a yellow bioactive flavonoid that is widely used in traditional medicine due to its numerous therapeutic activities, such as antioxidant, anti-inflammatory, antiobesity, antiaging, antiallergic, antitumor, antimicrobial, and antidiabetic activities [ [138] , [139] , [140] ]. Michelle and B. Michael investigated the anti-inflammatory effect of kaempferol on HGF stimulated with TNF-α (10 ng/ml) [ 48 ]. Kaempferol suppressed IL-8 secretion in HGF without cytotoxicity under 50 μM, and induced cell migration, which is an essential process of wound healing.…”

Comparative effect of anthocyanin on proliferation and migration of human gingival fibroblasts in the absence or presence of nicotine

“…21 In another study, HGFs were exposed to 0.025 to 32 μg/ mL of nicotine, stimulating and inhibiting cell migration in low and high concentrations, respectively. 10 Also, the results of a study in 2014 by Wheater and Mouabbi 32 showed that nicotine at concentrations > 2 mM (400-400 μg/mL) was toxic to oral cells in a cell culture medium and Qualitative rate of migration of HGF cells alone (control: without nicotine and cyanidin chloride), in the presence of nicotine alone (2.5 mM), cyanidin chloride alone (10, 25, and 50 mM), and nicotine with cyanidin chloride in zero time (the moment of scratching and exposure to the studied materials) and 24 hours after exposure. In the group exposed to 2.5-mM nicotine (without CC), the wound (space between the two edges of the scratch) opened more than (wider) zero time, and cytotoxicity was observed (red borderline) reduced viability, adhesion, and migration of HGFs.…”

“… 21 In another study, HGFs were exposed to 0.025 to 32 μg/mL of nicotine, stimulating and inhibiting cell migration in low and high concentrations, respectively. 10 Also, the results of a study in 2014 by Wheater and Mouabbi 32 showed that nicotine at concentrations > 2 mM (400-400 μg/mL) was toxic to oral cells in a cell culture medium and reduced viability, adhesion, and migration of HGFs.…”

Efficacy of vitamins E and C for reversing the cytotoxic effects of nicotine and cotinine

“…These findings are in line with the results of previous studies reporting that cigarette smoking and tobacco use can negatively affect wound healing. For example, it has been reported that nicotine (16–400 μ g ml −1 ) affects the signaling pathway of Rac (a subfamily of the Rho family of GTPases) and inhibits the migration of HGFs . Another study showed that nicotine (1 mM) inhibits transforming growth factor‐ β (TGF‐ β )‐induced myofibroblast differentiation in HGFs and slows the speed of gingival wound healing .…”