Abstract

Tobacco smoking has a detrimental impact on fracture healing, and has been implicated
in the non-union and delayed union of bone. Whilst previous studies have concentrated
on 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 anaesthetised
patients (n=16; 5 smokers vs. 11 non-smokers) that had sustained a tibial fracture. The
semisolid material was explanted into tissue culture fl asks and allowed to clot. Complete
culture 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 and
immunophenotyping using known mesenchymal stem cell antibody markers (CD29,
CD44, and CD166); CD34 was applied as a negative control. A fl ow cytometer was used
to count cell populations at the end of each passage. ELISA assays: Serum and cells
obtained from the fracture haematomas were subjected to an ELISA to compare the
amount 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 smokers
over 3 passages (~-200%). The VEGF-A (serum) and IL-6 (cells) ELISA revealed a
reduction 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 rate
in vitro in smokers than non-smokers. The amounts of VEGF-A and IL-6 are reduced in
smokers’ serum and cell cytoplasm respectively.