Nicotine Increases Osteoblast Activity of Induced Bone Marrow Stromal Cells in a Dose-Dependent Manner: An in vitro Cell Culture Experiment

摘要

Previous studies by our group showed that nicotine delivered via a transdermal nicotine patch significantly enhanced posterior spinal fusion rates in rabbits. Nicotine transdermal patches provide a steady serum level; there may be a dose-dependent effect of nicotine on posterior spinal fusion. In an in vitro cell culture model of rabbit bone marrow–derived osteoblast-like cells, cells were exposed to different concentrations of nicotine (0, 20, 40, 80 ng/mL and 10, 100, 250 μg/mL). Wells were stained with an alkaline phosphatase (ALP) staining kit to determine ALP enzyme activity. Cells were stained with Von Kossa for mineralization. A two-way analysis of variance (ANOVA) using dose and time as variables showed significant differences among groups; post hoc analysis showed that the 100-μg/mL dose of nicotine significantly enhanced ALP activity over controls. A one-way ANOVA using dose as the variable showed that the 100- and 250-μg/mL doses had significantly greater mineralization than controls. Dose-response analysis revealed a statistically significant effect of nicotine dose on ALP activity and Von Kossa activity. The effects of nicotine on spinal fusion may be dose-dependent and due to stimulation of osteoblastic activity. Nicotine may not be responsible for the inhibited bone healing observed in smokers. Keywords: nicotine, bone healing, spinal fusion, osteoblasts, smoking Several studies demonstrate that cigarette smoking is detrimental to bone health and impairs bone healing. Gerdhem and Obrant 1 showed in elderly women that smoking significantly reduced hip and total body bone mineral density. Andersen et al 2 reported that smoking resulted in an odds ratio of nonunion of 2.01 in lumbar fusion patients. Ueng et al 3 demonstrated that intermittent exposure to cigarette smoke in rabbits reduced torsional strength of the tibia during tibial lengthening procedures. Laroche et al 4 found that smoking significantly reduced serum osteocalcin levels in males, and Nersessian and Arutyunyan 5 showed that exposure to cigarette smoke increased osteoclastogenic activity in mice. Nicotine has been implicated as the agent in cigarette smoke that is responsible for the ill effects of smoking on bone health. Hollinger et al 6 reported that nicotine adversely affected bone healing in a rat model, and Wing et al 7 showed that chronic exposure to nicotine decreased fusion rates in a rabbit model and that quitting improved fusion rates. Silcox et al 8 found that systemic nicotine had a negative effect on bone fusion in a rabbit model of posterior lumbar fusion. Furthermore, Riebel et al 9 showed that nicotine increased nonunion in a bone graft model in rabbits and correlated it to reduced vascularization in the bone graft. They did, however, notice a varied response to nicotine among different animals, which they attributed to “predisposition” of the individual animals. The mechanism by which nicotine affects bone health and healing is not clearly known. Theiss et al 10 showed that gene expression of type I and II collagen, bone morphogenic protein (BMP)-2, BMP-4, BMP-6, basic fibroblast growth factor, and vascular endothelial growth factor were reduced by systemic administration of nicotine. Nicotine reduced vascularization to autologous cancellous bone grafts in the anterior chamber of the eye in a rabbit model. 11 Feitelson et al 12 reported that nicotine increased the effect of norepinephrine in constricting bone vasculature. To the contrary, however, Heeschen et al 13 found nicotine to be a potent stimulator of angiogenesis in three different animal models not involving bone, and Clouse et al 14 found that results of coronary artery bypass grafts in a canine model were unaffected by nicotine. In a previous animal study designed to evaluate the effects of direct current electrical stimulation on spinal fusion in rabbits exposed to nicotine, we found that both the nicotine control group of animals (fusion?+?nicotine administration) and the direct current stimulation group (fusion?+?nicotine?+?direct current stimulator) had increased fusion rates compared with the negative control group (fusion alone). 15 In light of the above information, we found this intriguing, as this was contrary to what other groups had found. 6 7 8 9 To resolve this issue, the current study was undertaken to see if the effects of nicotine on bone could be dose-dependent and if this could be demonstrated in an in vitro evaluation of osteoblast activity.