Exposure to Secondhand Smoke Impairs Fracture Healing in Rats

Abstract

Background: Nonsmokers may be affected by environmental tobacco smoke (secondhand smoke), but the effects of such exposure on fracture healing have not been well studied.

Questions/purposes: 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.

Methods: 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).

Results: Tobacco exposure resulted in delayed bone callus formation, which is represented by decreased BMD (Control: 0.302 ± 0.008 g/cm² vs Preexposed: 0.199 ± 0.008 g/cm² and Pre- and Postexposed: 0.146 ± 0.009 g/cm²; mean difference = 0.103 g/cm², 95% CI, 0.094-0.112 g/cm²and mean difference = 0.156 g/cm², 95% CI, 0.147-0.167 g/cm²; 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 mm³ vs Preexposed: 17.952 ± 1.600 mm³ and Pre- and Postexposed: 40.410 ± 3.340 mm³; mean difference = -10.30 mm³, 95% CI, -14.12 to 6.471 mm³ and mean difference, -32.75 mm³, 95% CI, -36.58 to 28.93 mm³; 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).

Conclusions: 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.

Clinical relevance: The potential hazardous effects of secondhand smoke on fracture healing in rodents should stimulate future clinical studies in human passive smokers.

Fig. 1

A schematic of the device used to expose the animals to cigarette smoke is shown. The numbers on the schematic refer to the peristaltic pump (1), pipe (2), cigarette (3), spherical container for smoke distribution (4), and cylindrical tubes (5) where the animals were placed for tobacco exposure. The peristaltic pump aspirates the cigarette smoke, drives it to the distribution chamber, and the smoke then is distributed to the individual chambers.

Fig. 2A–B

DXA examination showed that (A) the BMD is decreased in the smoke-exposed animals. (B) The BMC also is reduced in the smoke-exposed animals (Control vs Preexposed p < 0.01; Control vs Pre- and Postexposed p < 0.01).

Fig. 3A–B

(A) Although callus volume was increased in the smoke exposed animals, as determined by µCT analysis, (B) the percentage of woven bone volume was decreased as a result of passive smoking (less mineralized bone callus), underlying the impaired bone healing in the tobacco exposed groups (Control vs Preexposed p < 0.01; Control vs Pre- and Postexposed p < 0.01).

Fig. 4A–C

(A) Control rats showed normal bone callus formation with the presence of newly formed trabecular bone. (B) Rats preexposed to tobacco smoke showed delayed bone healing with bone callus mostly formed by nonosseous tissue. (C) Rats with pre- and postexposure also showed impaired bone healing, but to a greater degree than with preexposure to tobacco smoke. The arrows indicate cortical bone, the asterisks indicate woven bone, and the hashtags indicate immature nonosseous tissue in bone callus.

Fig. 5A–B

(A) Tobacco smoke exposure reduced callus resistance (maximum force). (B) The callus stiffness also was reduced by the smoke exposure (Control vs Preexposed p < 0.01; Control vs Pre- and Postexposed p < 0.01).

Fig. 6A–B

(A) Tobacco smoke exposure reduced bone maximum force. (B) Bone stiffness was also reduced by the smoke exposure (Control vs Preexposed p < 0.01; Control vs Pre- and Postexposed p < 0.01).