Exposure to Secondhand Smoke Impairs Fracture Healing in Rats

Hildemberg A. R. Santiago, Ariane Zamarioli, Manoel D. Sousa Neto, Jose B. Volpon

Nonsmokers may be affected by environmental tobacco smoke (secondhand smoke), but the effects of such exposure on fracture healing have not been well studied. To explore the possible effects of passive inhalation of tobacco smoke on the healing of a diaphyseal fracture in femurs of rats. We hypothesized that secondhand exposure to tobacco smoke adversely affects fracture healing. A mid-diaphyseal fracture was created in the femur of 41 female Wistar rats and fixed with an intramedullary metallic pin; 14 rats were excluded (nine for inadequate fractures and five for K wire extrusion). Tobacco exposure was provided by a smoking machine on a daily basis of four cigarettes a day. Each cigarette yielded 10 mg tar and 0.8 mg nicotine, and was puffed by alternating injections of fresh air for 30 seconds and smoke air for 15 seconds. The smoke exposure was previously adjusted to provide serum levels of cotinine similar to human secondhand tobacco exposure. Cotinine is a predominant catabolite of nicotine that is used as a biological biomarker for exposure to tobacco smoke. In one group (n = 11), the animals were intermittently exposed to tobacco smoke before sustaining the fracture but not afterward. In another group (n = 7), the exposure occurred before and after the fracture. The control group (n = 9) was sham-exposed before and after the fracture. We evaluated the specimens 28 days after bone fracture. The callus quality was measured by dual-energy x-ray absorptiometry (bone mineral density [BMD], bone mineral content [BMC], and callus area), μCT (callus volume and woven bone fraction), and mechanical bending (maximum force and stiffness). Tobacco exposure resulted in delayed bone callus formation, which is represented by decreased BMD (Control: 0.302 ± 0.008 g/cm(2) vs Preexposed: 0.199 ± 0.008 g/cm(2) and Pre- and Postexposed: 0.146 ± 0.009 g/cm(2); mean difference = 0.103 g/cm(2), 95% CI, 0.094-0.112 g/cm(2)and mean difference = 0.156 g/cm(2), 95% CI, 0.147-0.167 g/cm(2); p < 0.01), BMC (Control: 0.133 ± 0.005 g vs Preexposed: 0.085 ± 0.0034 g and Pre- and Postexposed: 0.048 ± 0.003 g; mean difference = 0.048 g, 95% CI, 0.045-0.052 g and mean difference = 0.085 g, 95% CI, 0.088-0.090 g; p < 0.01), callus volume (Control: 7.656 ± 1.963 mm(3) vs Preexposed: 17.952 ± 1.600 mm(3) and Pre- and Postexposed: 40.410 ± 3.340 mm(3); mean difference = -10.30 mm(3), 95% CI, -14.12 to 6.471 mm(3) and mean difference, -32.75 mm(3), 95% CI, -36.58 to 28.93 mm(3); p < 0.01), woven bone fraction (Control: 42.076 ± 3.877% vs Preexposed: 16.655 ± 3.021% and Pre- and Postexposed: 8.015 ± 1.565%, mean difference = 0.103%, 95% CI, 0.094-0.112% and mean difference = 0.156%, 95% CI, 0.147-0.166%; p < 0.01), maximum force (Control: 427.122 ± 63.952 N.mm vs Preexposed: 149.230 ± 67.189 N.mm and Pre- and Postexposed: 123.130 ± 38.206 N.mm, mean difference = 277.9 N.mm, 95% CI, 201.1-354.7 N.mm and mean difference = 304 N.mm, 95% CI, 213.2-394.8 N.mm; p < 0.01) and stiffness (Control: 491.397 ± 96.444 N.mm/mm vs Preexposed: 73.157 ± 36.511 N.mm/mm and Pre- and Postexposed: 154.049 ± 134.939 N.mm/mm, mean difference = 418.2 N.mm/mm, 95% CI, 306.3-530.1 N.mm/mm and mean difference = 337.3 N.mm/mm, 95% CI, 188.8-485.9 N.mm/mm; p < 0. 01). Rats exposed to tobacco smoke showed delayed fracture healing and callus that was characterized by decreased maturity, density, and mechanical resistance, which was confirmed by all assessment methods of this study. Such effects were more evident when animals were exposed to tobacco smoke before and after the fracture. Future studies should be done in human passive smokers to confirm or refute our findings on fracture callus formation. The potential hazardous effects of secondhand smoke on fracture healing in rodents should stimulate future clinical studies in human passive smokers.