Use of Organotypic Gingival Cultures for Nicotine Pouch Assessment

Nicotine pouches (NP) are an emerging class of nicotine-containing products for oral use. Due to the absence of tobacco and combustion, NP are considered by the manufacturers to pose a lower risk to consumer health than combusted tobacco products and other oral smokeless tobacco-containing products. However, only a few studies have investigated the biological effects of NP. In this study, human organotypic gingival cultures were apically exposed to the extracts obtained from five types of NP with different flavor types and nicotine contents, a snus product with nicotine concentrations relevant to consumer use (or higher), and cigarette smoke fractions (total particulate matter combined with the gas vapor phase [TPM-GVP]) for 96 h. At concentrations representative of human use, the test products induced different degrees of morphological alterations, mostly related to increased keratinization and atrophy of the basal layers. The extract from the NP with a high menthol level induced the most pronounced alterations, followed by three other NP, the snus, and finally, the extract from the NP with the lowest nicotine content. We observed a similar product-dependent response in the inflammatory mediator profile, with the high-menthol NP extract inducing the strongest inflammatory response. TPM-GVP exposure had the strongest impact on culture morphology and inflammatory response, even at nicotine concentrations 40 times lower than those of the oral products. In conclusion, the organotypic gingival model is suitable for evaluating the biological responses to NP, snus, and TPM-GVP exposure because of the possibility to differentiate the toxicological response across different product formulations.