Use of Organotypic Gingival Cultures for the Assessment of Nicotine Pouches

      Zanetti, F.; Alriquet, M.; Ortega Torres, L.; Neau, L.; Maranzano, F.; Pak, C.; Schmutz, A.; Mathis, C.; Gunduz, N.; Chua, J.

      Conference date
      Sep 27, 2022
      Conference name
      EUSAAT Congress 2022

      Nicotine pouches (NP) are an emerging class of nicotine products for oral use. The appearance and usage of NP are similar to those of the Swedish snus, but they do not contain tobacco. They consist of a nicotine-containing cellulose matrix inside a fiber sachet, with flavor-imparting and other ingredients, such as fillers, stabilizers, pH adjusters, and noncaloric sweeteners. NP with different nicotine strengths and flavors are available in different markets worldwide. Owing to the absence of tobacco and combustion, NP are considered by the manufacturers to pose a lower risk to consumer health than smokeless and combusted tobacco products. However, few studies have investigated the biological effects of NP.

      In this study, we used human organotypic gingival epithelial cultures to assess the biological effects of NP. We optimized a methodology to extract NP in an aqueous solution in a reproducible and controlled manner. We also assessed nicotine yield and stability of the key components of the NP extract over time. Organotypic gingival cultures were apically exposed to the extracts obtained from five types of NP (with different nicotine and flavor contents) and one snus product (two nicotine concentrations: one relevant to consumer use and a higher concentration) for 96 h. Liquid fractions from cigarette smoke (total particulate matter combined with the gas vapor phase) were also tested. At the end of the exposure period, we collected the basolateral medium for the analysis of a panel of 12 secreted inflammatory mediators using Luminex technology, and the morphology of the exposed organotypic tissue cultures was evaluated by a histopathologist after hematoxylin and eosin staining.

      We observed that the extracts from two types of NP and the snus had a similar mild impact on the morphology of the gingival cultures at nicotine concentrations relevant to human exposure. The extract from the NP containing a high level of menthol induced a more pronounced effect on gingival morphology, whereas the NP with the lowest nicotine content had the lowest effect. A similar product-dependent effect was observed in case of inflammatory mediator profiling, wherein the high-menthol NP extract was observed to induce the strongest inflammatory response. Exposure to the cigarette smoke fraction had the maximum impact on culture morphology and inflammatory response, even at nicotine concentrations 40 times lower than those of the oral products.

      Therefore, the biological effects of NP may depend on their formulation. The impact of NP and snus extracts on gingival organotypic cultures was lower than that of the cigarette smoke fractions, even at higher nicotine concentrations. The use of the human organotypic gingival model, in addition to being compliant with the reduce-replace-refine (3R) approach to reduce the use of animals in research, proved to be a suitable system to differentiate the effects of exposure to NP, snus, and cigarette smoke fractions.