Friday, September 28, 2018 4:05 PM–5:05 PM abstracts: basic science of spinal fusion

BACKGROUND CONTEXT Cigarette smoking has been shown to inhibit bone healing and increase the risk of pseudarthrosis after spinal fusion. We previously reported that activation of the Aryl Hydrocarbon Receptor (Ahr) by dioxin, a prototype activator of the Ahr, inhibits bone regeneration and spine fusion in rat model. We also reported that the dioxin-mediated inhibition of osteogenic differentiation is mitigated by co-treatment with Ahr antagonists in vitro. PURPOSE Since dozens of dioxin-like Ahr ligands are present in cigarette smoke, we are investigating the downstream mechanisms of cigarette smoke action on osteogenic differentiation to identify potential therapeutic options to mitigate the effects of cigarette smoke on bone. METHODS Particulate phase extract (PPE) was prepared by passing CSE through a 0.1μm PTFE filter and eluting in the DMSO to yield a final concentration of 40mg/mL. Primary rat bone marrow stromal cells (BMSC) were purchased from Cell Biologics (Chicago, IL) and cultured under basal standard or osteogenic media. BMSC were subsequently exposed to either the vehicle control (DMSO) or dioxin or PPE. Some cells received co-treatment with Ahr antagonists, including: a-naphthoflavone (ANF, a synthetic antagonist); resveratrol (Res, a stilbenoid found in grapes and present in red wine), and 3,3’-diindolylmethane (DIM, a breakdown product of the indole-3-carbinol, which is present in cruciferous vegetables). EROD assays were used to evaluate Ahr-mediated CYP1 family induction, and ALP and mineralization assays were used to quantify effects on osteogenic differentiation. Cell proliferation assays and Wnt signaling assays were also performed. RESULTS PPE-treated cells showed increased activity of CYP1A subfamily proteins, demonstrating Ahr activation. PPE treatment inhibited ALP activity, an effect which was at least partially mitigated by co-treatment with each of the Ahr antagonists. A similar response was seen in and mineralization and cell proliferation assays. RNA expression studies showed that PPE down-regulated numerous pro-osteogenic genes, such as ALP, RunX2, OCN, and Phex, whereas co-treatment with Ahr antagonists prevented PPE-mediated inhibition of those RNA. Interestingly, PPE decreased the ratio of active:inactive β-Catenin, an effect which was recapitulated by treatment with dioxin (TCDD). CONCLUSIONS Cigarette smokers are a historically difficult patient population for spine surgeons to treat due to increased rates of pseudarthrosis and complications following spinal fusion procedures. Our results suggest that Ahr hyper-activation may play an important role in the adverse effects of cigarette smoke on bone healing—potentially by perturbation of canonical Wnt signaling—and that Ahr antagonists may be protective in combating cigarette smoke-mediated inhibition of bone regeneration and healing.