Presenting Author:

Chawon Yun

Principal Investigator:

Erin Hsu, Ph.D.

Department:

Orthopaedic Surgery

Keywords:

aryl hydrocarbon receptor, dioxin, cigarette smoke, spinal fusion, osteogenic differentiation

Location:

Third Floor, Feinberg Pavilion, Northwestern Memorial Hospital

B123 - Basic Science

Ahr Antagonists Protect Against Cigarette Smoke Inhibition of Osteogenesic Differentiation

Cigarette smoking significantly impairs bone regeneration and is associated with higher rates of pseudarthrosis after spine fusion procedures. However, the molecular mechanisms underlying these effects are unclear. Numerous toxic ligands for the Aryl hydrocarbon receptor (Ahr) are present in cigarette smoke, and recent work has implicated the Ahr in mediating the inhibition of osteogenic differentiation. Our previous work with dioxin, a minor constituent of cigarette smoke and ligand of the Ahr, has shown that dioxin-induced Ahr activation inhibits bone regeneration and spine fusion in vivo. In this study we sought to elucidate the downstream mechanisms underlying the adverse effects of cigarette smoke extract (CSE), including dioxin, on bone regeneration and if these adverse effects are reversible with co-administration of an Ahr antagonist. Bone marrow stromal cells (BMSC) were harvested from Long-Evans rats and cultured under standard or osteogenic conditions. CSE was prepared by drawing smoke from reference cigarettes through a 0.1 μm PTFE membrane filter and washing the filter in dimethyl sulfoxide (DMSO) overnight to yield a final concentration of 40 mg/mL. Factors critical to osteogenesis were then evaluated after BMSC were exposed to DMSO vehicle, 10 nM dioxin, 10 ug/mL CSE, or co-treated with Ahr antagonists (4 μM Resveratrol, Res; 2 μM α-Naphthoflavone, ANF; 10 μM 3’3-Diindolylmethane, DIM). Cell viability (MTS assay), ALP activity, mineralization, and gene and protein expression of targets relevant to osteogenic differentiation were assessed. CYP1A1 mRNA was induced in CSE-treated BMSC which is a marker for Ahr activation. CSE reduced cell proliferation and ALP activity, and inhibited mineral deposition relative to vehicle control. CSE also led to reduced expression of ALP, OCN, RUNX2, CXCL12, PHEX, and OPN mRNA and protein expression. Co-treatment with each of the Ahr antagonists generally mitigated CSE effects of mineralization and ALP activity. Our results suggest that Ahr activation may play a critical role in the adverse effects cigarette smoke has on bone healing, and that these effects may be reduced with Ahr antagonist co-treatment. Administration of natural and synthetic Ahr antagonists should be investigated as a therapeutic option to block these inhibitory effects. The phytochemical-based Ahr antagonists used in this study are available over-the-counter as dietary supplements or found in foods, and are currently under investigation in clinical trials for chemoprotective effects. The identification of safe Ahr antagonists that provide a protective effect against the negative impact of the dioxin-like compounds found in cigarette smoke on bone could serve as therapeutic that protects against the deleterious effects of smoking on bone healing. This could provide surgeons, who are currently limited in their ability to reduce risks in patients who smoke, with a treatment that could lead to improved surgical outcomes.