Abstract

Introduction: Clinical and demographic studies have shown that tobacco smoking is a major contributor to non- and delayed-union in fracture healing. The cellular and molecular basis for this is poorly understood, and few studies in human fractures have been undertaken.
Aims: To analyse the in vitro biological effects of tobacco smoking at the cellular level within the human fracture microenvironment, with specific regard to mesenchymal stem cell (MSC) proliferation and to ascertain whether the application of bone morphogenetic factor-2 (BMP-2) could be used therapeutically to improve fracture healing.
Methods: Fracture haematomas (n=10) were collected from anaesthetised, non-smoking patients who had sustained a tibial fracture, and who were undergoing surgical operative fixation. The semi-solid material was dissected and explanted into tissue culture flasks. Complete culture media was introduced, and cultures were incubated at 37oC in a humidified 5% CO2 environment. Cigarette smoke extract (CSE) was produced and infused into the cell cultures to establish an in vitro smoking environment. A control group (n=10) was set-up and left  ntreated by CSE. Harvested, spindle-shaped adherent cells were characterised by immunocytochemistry. Cell populations were counted via flow cytometry to assess and compare proliferation rates between CSE-treated and untreated cell cultures. BMP-2 concentrations (10 and 100 ng/mL (an additional dose of 500 ng/mL in CSE- reated cells)) were infused into cell cultures to enhance in vitro cellular viability, which was analysed by means of the MTT assay.
Results: There was a significant reduction in the rate of proliferation of osteoblastic MSCs in CSE-treated cells after 5 days of culture (p < 0.05). At a dose of 100 ng/mL, BMP-2 augmented cellular growth and improved cellular viability in cultures not treated with CSE (p < 0.0001). No significant improvement was seen in CSE-treated cell cultures.
Summary: The effect of smoking on bone fracture healing appears to contribute to the inhibition of osteoblast proliferation, which may not be reversible with the therapeutic use of exogenous BMP-2. Moreover, the improvement seen in non-smokers does strengthen the case for smokers to cease using tobacco in the perioperative setting in order that such treatments are rendered more effective.