AB0074 Influence of cobalt and chromium ions on TGF-BETA expression and mineralization of bone forming cells IN-VITRO

Background Bone homeostasis is regulated at different levels. Transforming growth factor-beta (TGF-β) which exists in three isoforms in human and is stored in the latent form in the extracellular matrix is a major player in the regulation of osteoblast and osteoclast maturation, development, and function. The balance of bone formation and resorption is affected by numerous influences, such as hormone status, inflammation, and mechanical load. In the aging population, supplied increasingly with artificial joint replacements, release of particles or ions from arthroplasty may exert local effects in the periprosthetic tissue. Cobalt and chromium ions are released through corrosion from metal-on-metal arthroplasty and modulate the gene expression level of several cytokines, chemokines and other mediators in bone cells. Objectives It was the aim of the study to analyse the effect of Co2+ and Cr3+ ions on the expression of TGF-β isoforms in bone forming cells and their impact on mineralization. Methods The study investigated the effect of Co2+ and Cr3+ ions on the expression of TGF-β1–3 in the human osteosarcoma cell lines (MG63 and SaOs2) and primary human osteoblasts. Cells (3x105) were seeded in 2 ml DMEM (10%FCS) into 12 well plates and stimulated with CoCl2 and CrCl3 in concentrations between 50–250 μM for 24 hours. Total RNA was extracted and changes of expression levels of TGF-β1–3 were analysed by real-time PCR using sequence-specific primers and probes. For mineralization cells were cultivated for up to 4 weeks in DMEM (10% FCS) supplemented with 0.2mM ascorbic acid, 10nm dexamethasone and 10mM glycerophosphate and Co2+ and Cr3+. Calcium deposits were detected by 1% Alizarin Red S (pH 4.1) staining. Results The osteosarcoma cell lines as well as primary human osteoblasts isolated from bone explants expressed all three TGF-β isoforms, with TGF-β1 as most abundently expressed isoform. A dose dependent reduction of all TGF-β isoforms by Co2+ ions was observed, the strongest effect was found for TGF-β2. In MG63 cells and primary osteoblasts the mRNA levels of TGF-β2 decreased to 15±4% and 17±15% compared to the unstimulated control. The effect was lesser in SaOs2 cells with a reduction to 61±7% compared to control. In contrast to bivalent Co ions, the trivalent Cr ions had no significant effect on the expression of all TGF-β isoforms. While Co2+ in a concentration range of 50–250 μM did not impair the mineralization, Cr3+ exerted a strong inhibitory effect on the mineralization in osteosarcoma cells and primary osteoblasts. In a dose dependent fashion mineralization was influenced already at concentrations of 50 μM CrCl3 and showed the strongest effect at 250 μM. Conclusions The results of our study show that bivalent cobalt ions and trivalent chromium ions have different effects on bone forming cells. While Cr2+ affects the expression of the different TGF-β isoforms in osteoblast-like cells and primary osteoblasts, no inhibitory effect on mineralization in the tested concentrations was seen. Cr3+ however, did not influence the expression of TGF-β but strongly inhibited the mineralization in vitro. Our data implicate that the inhibitory effect of metal ions such as Co and Cr ions on the transcription of the bone regulating cytokines TGF-β1–3 and on bone forming activity may influence bone homeostasis. Acknowledgements Study supported by Stiftung Endoprothetik (S01/16). Disclosure of Interest None declared