Assessment of the Effects of Acute and Continuous Exposure to Tobacco Heating System 2.4 and Cigarette Smoke Total Particulate Matter on a Direct 2D Osteoblast/Osteoclast Coculture Model

Heated tobacco products are potential reduced-risk alternatives to cigarette smoke (CS). The effects of these products on bone cells are, however, not well known. We aim to develop a 2D bone coculture in vitro system suitable for investigating the osteoporotic mechanisms of CS and evaluating the impact of reduced-risk products and responsiveness of the system to anti-osteoporotic drugs.

Coculture was generated by using SCP-1 cells (a human immortalized stem cell line) as an osteoblast model and THP-1 cells as osteoclast precursors in a ratio 1:8. A decellularized (via heat treatment at 47°C) Saos-2 cell matrix was used as platform for the coculture. The effects of total particular matter extracts from reference cigarette smoke (1R6F) or Tobacco Heating System (THS) 2.4 (Philip Morris Products S.A.) aerosol on bone cell viability and function were tested. After an acute 24-h exposure to various concentrations of 1R6F or THS (corresponding to 10, 2 or 0.4 μg/mL nicotine for both test items). To confirm the appropriate response of the coculture to anti-osteoporotic drugs, the system was treated continuously with 1R6F (2, 0.4 or 0.08 μg/mL nicotine) in combination with zoledronate/alendronate (50 μM) for 14 days. At 3, 7, and 14 days of culture, viability (mitochondrial activity [resazurin conversion], adenosine triphosphate, and lactate dehydrogenase [LDH] content), functionality and morphology were assessed.

Cocultures exposed to 10 μg/mL 1R6F showed a dose-dependent significant decrease in ATP content and mitochondrial activity at days 3, 7, and 14 following exposure, relative to cocultures exposed to vehicle controls. LDH release and morphology analysis confirmed these results. 1R6F exposure at 10 μg/mL significantly reduced osteoclast function and the number of osteoclastand osteoblast-like cells, relative to vehicle control and THS exposure at comparable concentrations. However, CS exposure did not affect osteoblast activity. The anti-osteoporotic drugs reduced the detrimental effects of daily exposure to 0.4 μg/mL 1R6F for 14 days on coculture cell viability and function.

We demonstrated that acute exposure to 10 μg/mL THS had less impact on the viability and functionality of the 2D bone coculture than 1R6F at a comparable nicotine concentration. Furthermore, anti-osteoporotic drug treatment reduced the pro-osteoclastic effects of continuous exposure to 0.4 μg/mL 1R6F, confirming the relevance of our coculture system in relation to in vivo exposure.