Assessment of repeated THS2.2 aerosol exposure in in vitro Human gingival epithelial cultures

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Periodontal diseases are inflammatory disorders associated with the accumulation of a bacterial biofilm and characterized by the destruction of periodontal tissues. Although classified as bacterial infections, epidemiological studies have revealed that cigarette smoke (CS) is one of the major lifestyle-related risk factors for periodontal disease. CS can alter the epithelial structure of the gingival mucosa, leading to pathologies such as increased loss of attachment, development and progression of periodontal inflammation, increased gingival recession, and cancer 1,2.

While stopping smoking would clearly avoid further smoke-related challenge and damage to the gingival epithelium as well as to the respiratory tract and other organ systems such as the blood vessels and the heart, not all adult smokers are able or willing to quit. For these smokers, Modified Risk Tobacco Products (MRTP), i.e. products that present, are likely to present, or have the potential to present less risk of harm to smokers who switch to these products versus continued smoking, are developed. In particular the Tobacco Heating System (THS) 2.2 is such a candidate MRTP, which is based on heating, rather than burning, specifically designed tobacco sticks 3. Because of the absence of combustion, the THS2.2 aerosol contains greatly reduced levels of the harmful and potentially harmful constituents (HPHCs) found in CS 4,5. It is therefore important to assess the relative biological impact of exposure to these products compared with exposure to cigarettes.

Systems Toxicology , such as illustrated in the video below, allow to adapt to new toxicity-assessment paradigms of environmental exposures proposed by the 21st Century Toxicology framework. This framework recommended that animal use should be minimized (see 3Rs below) and mechanistic data should be acquired using human cellular-based in vitro systems 6-9. A recent review outlines technological advances in in vitro assessment of tobacco products 6.

In line with the 3Rs principle (replacement, reduction, and refinement) 10-12 aimed at reducing the use of laboratory animals, in a Systems Toxicology approach, the objective of this study was to assess the biological impact of an aerosol generated from a candidate MRTP, the tobacco heating system 2.2 (THS2.2), compared with smoke generated from 3R4F reference cigarette, on a human organotypic gingival epithelial tissue culture model.


The recently established three dimensional (3D) organotypic gingival epithelial culture models are composed by normal human gingival keratinocytes cultured in serum-free medium to form 3D differentiated tissue at the air-liquid interface (ALI) (EpiGingival™; MatTek, Ashland, MA, USA) 13.

This culture model recapitulates many of the features of the native human gum: the thickness and morphology of this tissue are similar to those in the gingival tissues in vivo 13,14. It resembles the in vivo paradigm under cytomegalovirus infection 13, it shows good reproducibility of the human situation for carcinogenic studies 15,16, and it is suitable for oral care product testing 17.

Interestingly, the organotypic cultured cells have been shown to retain their ability to release proinflammatory mediators (e.g., cytokines, chemokines, and growth factors) and reactive oxygen species, allowing researchers to investigate the potential mechanisms underlying the local and potentially systemic effects of the exposure 18-22. Moreover, the organotypic cultured cells express several members of the cytochrome P450 system, such as CYP1A1 and CYP1B1, whose expression and activity can be monitored following exposure to CS or other toxicants 22.

Click on the play button to play the video.

A systems toxicology study design

A Systems Toxicology approach was applied to elucidate the biological impact of the exposure. The endpoints of our systems toxicology approach included cytotoxicity, histopathology, activity of cytochrome P450 (CYP) 1A1/1B1, secretion of inflammatory mediators, and molecular investigations using transcriptomics (mRNA and miRNA) and metabolomics, complemented by computational network biology analyses.

The study can be divided into three sections:

  1. PBS Pilot. To be closer to the physiological situation and mimic the presence of saliva in the oral cavity, the gingival organotypic cultures were exposed to PBS on the apical side over the duration of the experiments. We selected PBS because it shares a similar composition but does not contain the additives normally present in artificial saliva 23. Moreover, many different types of artificial saliva are available and their composition vary largely, not meeting the biophysical properties of real saliva; some may even induce an inflammatory condition in various cell types 24-26. In this experimental phase the effects of PBS apical exposure to gingival tissue over a 96-h time frame have been assessed.
  2. Dose Range Finding (DRF) to evaluate the concentrations of 3R4F CS and THS2.2 aerosol at which toxicity or morphological changes were observed. Organotypic gingival cultures were exposed for 3 consecutive days to a broad range of 3R4F CS or THS2.2 aerosol concentrations for 28 min. Before each exposure, the basolateral medium was collected for different assays (AK and MAP) and replaced with fresh medium; apical PBS was also replaced before each exposure. Different endpoints were analyzed at the indicated time-points post-exposure.
  3. Main Phases (MPs) + Metabolomics. Organotypic gingival cultures were exposed for 3 consecutive days to different concentrations of 3R4F CS or THS2.2 aerosol selected from the DRF but with comparable levels of nicotine. Different endpoints were analyzed at the indicated time-points to determine the biological impact of THS2.2 aerosol and combustible 3R4F CS exposure. The MP was repeated three times. For the metabolomics phase, only the high concentrations were applied (84.6 and 100.4 mg/L for 3R4F CS and THS2.2 aerosol, respectively).

A more detailed view of the study design is given in the figure below.

Smoke from the reference item (3R4F) was generated using a 30-port carousel smoking machine (SM-2000). Aerosol from the test item (THS2.2) was generated using a modified 30-port carousel smoking machine (SM-2000 THS2.2). Each of the smoking machines was connected to a Vitrocell® 24/48 exposure system (Vitrocell Systems GmbH), where the culture inserts were exposed.

The Vitrocell® exposure system is equipped with a dilution system. To achieve the desired concentrations of nicotine, the smoke and aerosol were diluted with fresh air, as indicated by the following figure.


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  3. Smith, M. R. et al. Evaluation of the Tobacco Heating System 2.2. Part 1: Description of the system and the scientific assessment program. Regul Toxicol Pharmacol 81, S2, S17–S26 (2016)
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Organotypic gingival (THS2.2) Results

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Publications and Reports

Zanetti et al. Food and Chemical Toxicology (2017)

Comparative systems toxicology analysis of cigarette smoke and aerosol from a candidate modified risk tobacco product in organotypic human gingival epithelial cultures: A 3-day repeated exposure study

Smoking is one of the major lifestyle-related risk factors for periodontal diseases. Modified risk tobacco products (MRTP) offer a promising alternative in the harm reduction strategy for adult smokers unable to quit. Using a systems toxicology approach, we investigated and compared the exposure effects of a reference cigarette (3R4F) and a heat-not-burn technology-based candidate MRTP, the Tobacco Heating System (THS) 2.2. Human gingival epithelial organotypic cultures were repeatedly exposed (3 days) for 28 min at two matching concentrations of cigarette smoke (CS) or THS2.2 aerosol. Results showed only minor histopathological alterations and minimal cytotoxicity upon THS2.2 aerosol exposure compared to CS (1% for THS2.2 aerosol vs. 30% for CS, at the high concentration). Among the 14 proinflammatory mediators analyzed, only 5 exhibited significant alterations with THS2.2 exposure compared with 11 upon CS exposure. Transcriptomic and metabolomic analysis indicated a general reduction of the impact in THS2.2 aerosol-exposed samples with respect to CS (~79% lower biological impact for the high THS2.2 aerosol concentration compared to CS, and 13 metabolites significantly perturbed for THS2.2 vs. 181 for CS). This study indicates that exposure to THS2.2 aerosol had a lower impact on the pathophysiology of human gingival organotypic cultures than CS.