gms | German Medical Science

Objectives: Cigarette smoking has negative effects on bone integrity, and is associated with increased incidence of fractures, delayed fracture healing and osteoporosis. A statistical study in the BG Trauma Clinic Tübingen showed that smokers have longer hospital stays and more complications than non-smokers.

Previous results demonstrated that high concentrations of cigarette smoke extract (CSE) significantly reduced human osteoblast viability by accumulating free radicals. However, the long term effects of CSE on osteogenic differentiation and underlying mechanisms are not extensively studied. Our aim was to investigate the effect of CSE exposure on primary cilia structure during osteogenic differentiation of mesenchymal stem cells (MSCs) as well as to evaluate the protective effect of antioxidants.

Methods: Human immortalized MSCs (SCP-1) were osteogenically differentiated in vitro with CSE (0.1%- 10%) and resveratrol (1 µM) as antioxidant treatment strategy. The CSE concentrations correspond to exposures associated with smoking up to 20 cigarettes/day. Cell viability and function were analyzed by mitochondrial activity and live dead staining, alkaline phosphatase (AP) activity and alizarin red staining. Morphology of primary cilia was subsequently examined by immunofluorescent staining for acetylated α tubulin. Free radical production was evaluated by DCFH-DA assay. Gene analysis was performed by semi-quantitative RT-PCR. Data were analyzed using Kruskal-Wallis H-test followed by a Dunn's test for comparisons between multiple groups.

Results and conclusion: Our study showed that 5% and 10% CSE exposure caused a significant reduction in the length of primary cilia and the number of ciliated cells (p<0.001). Disruption of primary cilia by chloral hydrate significantly reduced AP activity (p<0.05). Co-incubation of CSE with resveratrol significantly reduced ROS accumulation (p<0.001) and protected the primary cilia structure. Additionally, resveratrol treatment significantly enhanced the AP activity (p<0.001) and improved matrix mineralization (p<0.05). Moreover, resveratrol upregulated gene expression of osteogenic transcription factors and osteoblast markers (RUNX2, GLI2, OPG, BMP2).

In conclusion, CSE mediated inhibition of osteogenic differentiation, was closely associated with the abrogation of primary cilia by oxidative stress. Antioxidant treatment reduced free radical generation, maintained primary cilia integrity and promoted osteogenic differentiation of MSCs exposed to CSE. Thus, resveratrol can be proposed as a potential therapeutic to improve bone function and enhance fracture healing in smokers.