0817 - Bone Volume Fraction Measured via Micro-CT Imaging is a Predictor for Hydraulic Permeability and Elastic Modulus of the Trabecular Bone of Human Lumbar Vertebral Bodies

INTRODUCTION: Tungsten has been increasing in demand for use in manufacturing and recently, medical devices, as it imparts flexibility, strength, and conductance of metal alloys. Given the surge in tungsten use, our population may be subjected to elevated exposures. For instance, embolism coils made of tungsten have been shown to degrade in some patients. In a cohort of breast cancer patients who received tungsten-based shielding for intraoperative radiotherapy, urinary tungsten levels remained over tenfold higher 20 months post-surgery. In an in vivo mouse model, tungsten exposure increased tumor metastasis by altering the tumor microenvironment. In a recent report, tungsten was shown to rapidly accumulate in bone and enhance the adipogenesis of bone marrow-derived mesenchymal stem cells while inhibiting osteogenesis. Whether tungsten accumulates in other tissues and affects viability and/or function remains unknown. We recently determined that when mice are exposed to tungsten [15 ug/mL] in their drinking water, it bioaccumulates in the intervertebral disc [~3 ppm], equivalent to what is observed in bone. This study was performed to determine the effects of tungsten on the intervertebral disc. METHODS: Mice (C57BL/6) were given tap water or water supplemented with 15 ppm sodium tungstate for 4 weeks. Animals were sacrificed and spines were removed for histology. Discs were excised and analyzed for proteoglycan and collegen content. For gene expression studies bovine nucleus pulposus (bNP) and annulus fibrosus (bAF) cells were isolated from bovine caudal tails. Cells were expanded and prepared for 3D culturing in alginate beads at a density of 2*10^6 cells/mL. Beads were cultured in PrimeGrowthu00ae Disc Cell medium supplemented with increasing tungsten concentrations in the form of sodium tungstate [0, 0.5, 5, 15 ug/mL] for 12 days. A modified GAG assay was performed on the beads to determine proteoglycan content and the hydroxyproline assay was performed for collagen synthesis. Gene expression of matrix proteins (ACAN, COL1A1, COL2A1), catabolic enzymes (ADAMTS4, ADAMTS5, MMP3, MMP13), and markers of inflammation (TNFalpha, IL-1beta) and pain (NGF, BDNF) were analyzed by qPCR and verified by IHC on in vivo discs. RESULTS SECTION: Tungsten dose-dependently decreased the synthesis of proteoglycan in both NP and AF cells reaching greatest significance in NP cells at 15 ppm (3.3 ug/ug DNA to 2.3 ug/ug DNA; p < 0.01; n = 4). Decreases in proteoglycan content was also observed in mouse IVDs (30.3 ug/mg to 19.5 ug/mg tissue; p < 0.001; n = 10). Furthermore, tungsten increased the synthesis of collagen in both discs cells and in vivo (12.4 ug/mg to 25.3 ug/mg; p < 0.01; n = 10). These changes in matrix protein content was also reflected by significant decreases in disc height. Interestingly, in disc cells synthesis of COL2A1 was downregulated but COL1A1 was increased by 6-fold (p<0.0001, n=4). This increase in Col I was also observed in vivo. Upregulation of the catabolic enzymes ADAMTS4 and -5 was also observed both in disc cells and in vivo as determined by IHC. Although inflammatory cytokines were not upregulated in disc cells following tungsten exposure, NGF and BDNF were upregulated by approximately 10-fold (p<0.05, n=4) and 3-fold (p<0.05, n=4) in NP and AF cells, respectively. Upregulation of NGF was confirmed upon histological examination of the discs of tungsten exposed mice (Figure 1). Interestingly, in the endplates of tungsten exposed animals, significant increases in the detection of the neuronal marker PGP9.5 and CGRP were observed.DISCUSSION: We provide evidence that tungsten alters matrix protein synthesis in the IVD by enhancing disc fibrosis and increases markers of pain.SIGNIFICANCE/CLINICAL RELEVANCE: Prolonged exposure to tungsten may contribute to the development of chronic back pain. REFERENCES: Bolt A et al. Toxicol. Sci. 2015, 143:165-177; Bolt A et al. Toxicol. Sci. 2016, 150(2):333-346ACKNOWLEDGEMENTS: This research is supported by the Canadian Institute of Health Research (CIHR).

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