Assessment of tobacco heating system 2.4 on osteogenic differentiation of mesenchymal stem cells and primary human osteoblasts compared to conventional cigarettes

BACKGROUND: Cigarette smoking (CS) is the most common method of consuming tobacco. Deleterious effects on bone integrity, increased incidence of fractures, and delayed fracture healing are all associated with CS. Over 150 of the 6500 molecular species contained in cigarette smoke and identified as toxic compounds are inhaled by CS and, via the bloodstream, reach the skeletal system. New technologies designed to develop a reduced-risk alternative for smokers are based on electronic nicotine delivery systems, such as e-cigarettes and tobacco heating systems (THS). THS are designed to heat tobacco instead of burning it, thereby reducing the levels of harmful toxic compounds released. AIM: To examine the effects of THS on osteoprogenitor cell viability and function compared to conventional CS. METHODS: Human immortalized mesenchymal stem cells (n = 3) and primary human pre-osteoblasts isolated from cancellous bone samples from BG Unfall Klinik Tübingen (n = 5) were osteogenically differentiated in vitro with aqueous extracts generated from either the THS 2.4 “IQOS” or conventional “Marlboro” cigarettes for up to 21 d. Cell viability was analyzed using resazurin conversion assay (mitochondrial activity) and calcein-AM staining (esterase activity). Osteogenic differentiation and bone cell function were evaluated using alkaline phosphatase (AP) activity, while matrix formation was analyzed through alizarin red staining. Primary cilia structure was examined by acetylated α-tubulin immunofluorescent staining. Free radical production was evaluated with 2’,7’-dichlorofluorescein-diacetate assay. RESULTS: Our data clearly show that THS is significantly less toxic to bone cells than CS when analyzed by mitochondrial and esterase activity (P < 0.001). No significant differences in cytotoxicity between the diverse flavors of THS were observed. Harmful effects from THS on bone cell function were observed only at very high, non-physiological concentrations. In contrast, extracts from conventional cigarettes significantly reduced the AP activity (by two-fold) and matrix mineralization (four-fold) at low concentrations. Additionally, morphologic analysis of primary cilia revealed no significant changes in the length of the organelle involved in osteogenesis of osteoprogenitor cells, nor in the number of ciliated cells following THS treatment. Assessment of free radical production demonstrated that THS induced significantly less oxidative stress than conventional CS in osteoprogenitor cells. CONCLUSION: THS was significantly less harmful to osteoprogenitor cells during osteogenesis than conventional CS. Additional studies are required to confirm whether THS is a better alternative for smokers to improve delays in bone healing following fracture.