Date of Award


Document Type


Degree Name

Master of Science (MS)



Committee Member

Hai Yao

Committee Member

Ann Foley

Committee Member

Vincent D Pellegrini, Jr.

Committee Member

William Barfield


Fractures that fail to heal (nonunions) substantially contribute to patient morbidity and overall healthcare costs. When nonunions occur in the absence of a critical size defect it is the result of impaired healing via one of the two distinct bone healing pathways: primary (intramembranous) and secondary (endochondral) ossification. Furthermore, the recent characterization of circulating osteoprogenitor cells (COPs) presents yet another important mechanism by which fractures may heal. Clinically, patients who smoke cigarettes are at increased risk of suffering from a nonunion; however, the mechanism by which this occurs has still not yet been defined. We sought to determine whether cigarette smoking exerts a differential effect on two distinct bone healing pathways with an aim to define the optimal treatment approach for patients who are active smokers and to explore whether smoking cigarettes affects the production of COPs. To do this, we aimed to (1) Establish a reproducible smoke-exposure protocol for use in vivo with the Sprague-Dawley rat, (2) Develop a bilateral femur fracture model for measuring the effects of cigarette smoke on both primary and secondary healing pathways, (3) Determine whether cigarette smoke exerts a preferential inhibitory effect on cartilage production in the endochondral ossification pathway and (4) Measure the production of COPs and compare their induction under the condition of smoke exposure.

Forty-six male Sprague-Dawley rats were obtained, half (n = 23) were exposed to daily one-hour cigarette smoke exposures, 12 sessions per week, for one month preoperatively and one month postoperatively. A goal of 200mg/m3 total particulate matter (TPM) was set for each smoke exposure. All animals received bilateral femur fracture surgery, one side treated with an intramedullary nail and the opposite treated with a compression plating technique. COPs were measured by identifying CD34, osteocalcin double positive cells via flow cytometry. Animals were harvested at 10 days, 1 month, 3 months and 6 months postoperatively. Endochondral ossification was assessed using Safranin-O histomorphometry and microCT assessment of calcified callus volume. Fracture healing strength was assessed via 4-point bending at 3 months and 6 months postoperatively. Statistical analyses were performed using two-way ANOVA with Fisher’s least significant difference for post-hoc comparisons. A p-value less than 0.050 was considered statistically significant.

Smoke-exposed animals received an average an average TPM exposure of 200.6 ± 73.0 mg/m3 (95% CI: 193.9 - 207.3, Range: 61 - 704), without any difference in average exposures between animals (one-way ANOVA, p = 0.061). All animals exhibited fracture healing postoperatively via plain X-ray radiographs without apparent impairment in gait. Animals lost a significant amount of bodyweight after 10 days of cigarette smoke exposure (mean difference: 50.1, 95% CI: 19.4, 80.8, p = 0.002) and this difference was maintained following one month of smoking cessation (mean difference: 50.2, 95% CI: 26.3, 74.2; p

This study represents the successful establishment of a smoke-exposure protocol in a bilateral femur fracture rodent model. Smoke exposure exerted an anorexigenic effect and subsequent weight loss; however nutritional impairment was not evident. Endochondral ossification was substantially impaired as evidenced by the reduced cartilage production in the nailed specimen. Early COP production was also found to be impaired in response to smoke exposure, but enhanced at four weeks. These results offer a unique understanding of how cigarette smoke plays a role in fracture biology, specifically in its inhibitory effect on secondary bone healing and on early COP recruitment. These results imply that choice of the optimal fracture fixation method may be dictated by the differential effects of smoking on the two fracture healing pathways. Future work will investigate the effects of smoke exposure on production of the calcified callus and acquisition of mechanical strength through each of the fracture healing pathways.



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