Cigarette Smoke as a Risk Factor for Delayed Fracture Healing: Role of Oxidative Stress Impairs Primary Cilia on Osteogenic Differentiation

Abstract

Since 1976, several studies have demonstrated the correlation between cigarette smoking, reduced bone mass, and impaired fracture healing. Cigarette smoking can affect bone homeostasis directly through harmful effects in bone-forming and bone-resorbing cells or indirectly via disturbed hormonal and immune responses. Following orthopedic surgery, smokers frequently show an increased complication rate, which results in augmented health system costs. As a main risk factor for osteoporosis, the underlying mechanisms behind impaired bone remodeling in smokers remain unclear. The purpose of this dissertation was to link the delayed fracture healing observed in orthopedic patients who smoke with the effects of cigarette smoke–induced oxidative stress on primary cilia structure with regard to osteoprogenitor cells signaling and differentiation. Our study cohort confirmed cigarette consumption as a hazard for developing complications compared to malnutrition and daily alcohol consume. Interestingly, smokers were 5.4 years younger compared with non-smokers but with comparable levels of comorbidities. Additionally, the complication rate positively correlated with the number of cigarette packs consumed per years. Subsequently, smokers' hospitalizations were on average 3 days longer compared with non-smokers. Analysis of blood plasma levels of bone formation markers showed downregulation in smokers. However, bone resorption markers were not affected, and oxidative stress markers were upregulated in comparison with non-smokers. Thus, these results support the impaired fracture healing observed in orthopedic patients who smoke. Regarding the increased complications observed in smokers, blood plasma levels of immune modulator cytokines demonstrated an immunosuppressive status for smokers. Therefore, cigarette consumption negatively affects bone-forming cell function and inhibits the immune response in orthopedic patients. Consequently, investigating treatment strategies that stimulate bone-forming cell function, decrease oxidative stress, or boost the immune system are mandatory to improve the outcome of smoker orthopedic patients. We developed an in vitro system that represents the clinical condition observed in smokers to investigate the underlying mechanisms behind the deleterious effects of cigarette smoking on bone-forming cells. In addition to delayed MSC osteogenic differentiation, cigarette smoke exposure increased oxidative stress and affected the integrity of the cellular sensor (primary cilia). Interesting, we out results have shown that cigarette smoke exposure impairs MSC differentiation to bone forming cells was not associated with nicotine as well as its principal metabolite cotinine. As a protective approach, resveratrol prevented free radical production, decreased oxidative stress levels, preserved the primary cilia integrity, and improved osteogenic differentiation in response to cigarette smoke exposure. Accordingly, cigarette smoke–induced oxidative stress via impaired primary cilia structure negatively affected osteoprogenitor cell osteogenic differentiation and function. Moreover, we demonstrated that cigarette smoke–induced oxidative stress by an accumulation of superoxide radicals and reduced levels of intracellular glutathione. While nicotine and cotinine did not increase oxidative stress, these substances inhibited the function of antioxidative enzymes, contributing indirectly to the adverse outcome observed on MSCs by cigarette smoke exposure. Furthermore, treatment with N-acetylcysteine and L-ascorbate reversed the compromised osteogenic differentiation generated by cigarette smoke via upregulation of the cellular antioxidative system and free radical scavenging abilities. TGF-β is a crucial cytokine that promotes osteoprogenitor cell motility, growth, and suitable differentiation to the bone injury area. There were lower levels of TGF-β in smokers along with orthopedic patients with disturbed fracture repair. Additionally, our results demonstrated that the nuclear import of downstream TGF-β signaling activated effector complex was negatively affected by cigarette smoke, namely due to abrogated primary cilia. Furthermore, the abolition of primary cilia with CH and the protection of primary cilia structure with resveratrol supported the link between TGF-β signaling and primary cilia. Besides, cigarette smoke-blocking TGF-β signaling perturbed osteoprogenitor cell motility, growth, and chondrogenic differentiation. TGF-β treatment did not improve osteoprogenitor cell proliferation and chondrogenic differentiation due to the lack of functional TGF-β signaling. For the first time, we showed the detrimental effects of cigarette smoke–induced oxidative stress on the primary cilium structure and associated signaling pathways. Antioxidant treatment provided scavenging properties, activated the cellular antioxidative defense system, reduced oxidative stress levels, protected primary cilia structure, and reestablished TGF-β signaling. Consequently, osteoprogenitor cell migration, proliferation, and differentiation are improved. These results suggest that preserving the primary cilia structure may represent a therapeutic goal to support the reparative and remodeling phase of fracture healing in smokers.