Inhibition of tibial fracture healing in smokers: cellular and molecular aspects

Tobacco smoking has a detrimental impact on fracture healing, and has been implicatedin the non-union and delayed union of bone. Whilst previous studies have concentratedon the clinical manifestations of smoking, little work has been undertaken biochemically.Aim: To study the effects of smoking at the cellular and molecular level in vitro.Methods: Cell-culture assay: Fracture haematomas were collected from anaesthetisedpatients (n=16; 5 smokers vs. 11 non-smokers) that had sustained a tibial fracture. Thesemisolid material was explanted into tissue culture fl asks and allowed to clot. Completeculture media was introduced into the fl asks, which were placed in an incubator (37°C;humidifi ed CO2). Cultured cells were characterised via immunofl uorescence andimmunophenotyping using known mesenchymal stem cell antibody markers (CD29,CD44, and CD166); CD34 was applied as a negative control. A fl ow cytometer was usedto count cell populations at the end of each passage. ELISA assays: Serum and cellsobtained from the fracture haematomas were subjected to an ELISA to compare theamount of VEGF-A (serum) and IL-6 (cells) between smokers and non-smokers.Results: Cell counting showed a reduction in the rate of proliferation of cells in smokersover 3 passages (~-200%). The VEGF-A (serum) and IL-6 (cells) ELISA revealed areduction of these acute phase proteins in patients who were smokers (VEGF-A ~-10%;IL-6 ~-15%).Conclusion: Fracture haematoma mesenchymal stem cells proliferate at a slower ratein vitro in smokers than non-smokers. The amounts of VEGF-A and IL-6 are reduced insmokers’ serum and cell cytoplasm respectively.