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

Abstract

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.

Keywords: Bone; Cigarette smoke; Electronic nicotine delivery systems; Mesenchymal stem cells; Primary human osteoblast; Tobacco heating system.

Conflict of interest statement

Conflict-of-interest statement: The authors have not conflict of interest.

©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.

Figures

Figure 1

Tobacco heating systems and conventional cigarette aqueous extract characterization. A: Time to consume one unit [cigarette or tobacco heating systems (THS) stick] measured in minutes; B: pH from the aqueous extract (AE) generated with conventional cigarettes or THS; C: Absorbance at 320 nm from the different fractions produced from conventional cigarette or THS; D: Representative picture of gas washing bottle after conventional cigarette or THS AE generation, respectively. Each measure was conducted with six independent AE for each condition in triplicates. Data were analyzed using the nonparametric Mann Whitney test or the Kruskal-Wallis H test followed by Dunn’s post-hoc tests. Data are presented as mean ± SE, and P < 0.001 for the comparison. THS: Tobacco heating systems.

Figure 2

High concentrations of aqueous extract from tobacco heating systems has minor effects on bone cells viability after a single exposure than aqueous extract from conventional cigarettes. SCP-1 cells and primary human osteoblasts were treated with increasing concentrations of aqueous extract (AE) from conventional cigarettes and tobacco heating systems. Cell viability was evaluated by resazurin conversion (mitochondrial activity) in SCP-1 cells (A) and primary osteoblast (B) after 48 h of treatment; C: Representative live staining pictures from SCP-1 cells using calcein-AM (green) and nuclear staining using Hoechst 33342 (blue) was shown after 48 h of exposure to AE (scale bar 400 µm). Each measure was conducted at least three independent times in triplicates. Data were analyzed using the Kruskal-Wallis H test followed by Dunn’s post-hoc tests. Data are presented as mean ± SE, and P values indicated as bP < 0.01 and dP < 0.001 for comparisons with untreated cells within the same AE type. AE: Aqueous extract.

Figure 3

Tobacco heating systems is less toxic than conventional cigarettes after chronic exposure. SCP-1 cells and primary human osteoblasts were osteogenically differentiated with increasing concentrations of aqueous extract (AE) from conventional cigarette and tobacco heating systems (THS) for 21 d. Cell viability was evaluated by resazurin conversion (mitochondrial activity) in SCP-1 cells (A, B) and primary human osteoblast (C, D) after 14 (A, C) and 21 d (B, D). Each measurement was conducted at least three independent times in triplicates. Data were analyzed using the Kruskal-Wallis H test followed by Dunn’s post-hoc test. Data are presented as mean ± SE. P values are classified as aP < 0.05, bP < 0.01, cP < 0.001 for comparisons with untreated cells within AE type and as eP < 0.05, fP < 0.01, gP < 0.001 for comparisons of conventional cigarette with THS within the same concentration. AE: Aqueous extract.

Figure 4

Tobacco heating systems has a lower impact on bone cells function than conventional cigarettes. SCP-1 cells and primary human osteoblasts were osteogenically differentiated with increasing concentrations of aqueous extract (AE) from conventional cigarettes and tobacco heating systems (THS) for 21 d. Cell function was evaluated by alkaline phosphatase activity (early differentiation marker) (A, C) after 14 d. Calcium deposition (late marker of differentiation) was evaluated by Alizarin red staining (B, D) after 21 d (B, D). Each measurement was conducted at least three independent times in triplicates. Data were analyzed using the Kruskal-Wallis H test followed by Dunn’s post-hoc test. Data are represented as mean ± SE, and P values are classified as aP < 0.05, bP < 0.01, cP < 0.001 for comparisons with untreated cells within the same AE type and fP < 0.01, gP < 0.001 for comparisons conventional cigarette with THS within the same concentration. AE: Aqueous extract.

Figure 5

Tobacco heating systems induces less oxidative stress and causes less damage to primary cilia structures on bone cells precursor than conventional cigarettes. SCP-1 cells were treated with increasing concentrations of aqueous extract (AE) from conventional cigarettes and tobacco heating systems (THS). ROS production was evaluated by DCFH-DA assay in SCP-1 cells. 0.01%v/v H2O2 was used as a positive control (A). Primary cilium length was quantified on day 14 by acetylated α-tubulin (green), and nuclei (blue) immunostaining (B). Representative immunostaining images of SCP-1 cells primary cilia after 14 days of chronic exposure to THS or conventional cigarettes AE (C) (scale bar 100 µm). Each measurement was conducted at least three independent times in triplicates. Data were analyzed using the Kruskal-Wallis H test followed by Dunn’s post-hoc tests. Data are represented as mean ± SE, and P values are classified as aP < 0.05, bP < 0.01, cP < 0.001 for comparisons with untreated cells within AE type. AE: Aqueous extract.

Figure 6

No differential variation in cytotoxicity between different tobacco heating systems flavors on bone cells precursor. SCP-1 cells were treated with increasing concentrations of aqueous extract (AE) from tobacco heating systems bronce, amber and yellow flavor. Cell cytotoxicity was evaluated by resazurin conversion (mitochondrial activity) in SCP-1 cells after 48 h of exposure. Each measurement was conducted at least three independent times in triplicates. Data were analyzed using the Kruskal-Wallis H test followed by Dunn’s post-hoc tests. Data are presented as mean ± SE, and P values are classified as bP < 0.01, cP < 0.001 for comparisons with untreated cells within AE type. AE: Aqueous extract.