degradation. REFERENCES: 1 L. Wu, B. Luthringer, L. L.. Grünherz, N.Wojtas, U. Hopfner, F. Feyerabend,. R. Willumeit, A. F. Schilling (2013) Osteologie. 2013 ...
European Cells and Materials Vol. 26. Suppl. 5, 2013 (page 35)
ISSN 1473-2262
In vitro strategies to mimic in vivo events after osseous implantation BJC Luthringer1, L Wu1; 2, M Costantino1, A Burmester1, AF Schilling2, F Feyerabend1, R Willumeit1 1
Department for Structural Research on Macromolecules, Institute of Materials Research, Helmholtz-Centre Geesthacht, Geesthacht, Germany
2
Klinik und Poliklinik für Plastische Chirurgie und Handchirurgie, Technische Universität München, Munich, Germany
INTRODUCTION: At least three important types of cells are interplaying before and after implantation of osseous medical devices: macrophages, osteoblasts, and osteoclasts. Even if the canonical cascade of events is understood, the effects of degradation products from magnesiumbased implants on this scene remain unclear. To unveil these mechanisms, different in vitro models considering different aspects of interactions were developed. The successful implementation may decrease costs and avoid ethical issues of in vivo experiments while maximising experimental convenience. METHODS: For osteoblasts, several cell lines (Saos-2, U2OS, and MG63) as well as primary cells (human bone derived cells (HBDC) and human umbilical cord perivascular (HUCPV)) were investigated. The U937 cell line (promonocytes) and peripheral blood mononuclear cells (PBMC) were selected as macrophage and osteoclast models, respectively1. The cell models were differentiated and the effect of magnesium (MgCl2 and degradation products from magnesium implant material (“magnesium extract”)2) on this process was investigated. Model-specific markers were followed by biochemical means (e.g., real time PCR, Elisa tests, and microscopy). RESULTS: The first issue was to find the most suitable cell model for the different stages of interaction. For osteoblasts only Saos-2 exhibit a similar protein and gene expression profile than primary cells, MG-63 the least comparable. Furthermore, a more drastic effect of magnesium extract than MgCl2 on bone specific markers (e.g., osteocalcin) was measured on HBDC supplemented with factors promoting osteogenic differentiation. For osteoclasts and macrophages, the attainableness of differentiated cells was another obstacle. To approbate the cell model, selection was performed on their ability to exhibit closed patterns to what has been reported in vivo via i.e., specific osteoclast tartrate resistant acid
phosphatase and cathepsin K or macrophage tumour necrosis alpha (TNFα) interleukin 6 & 1 alpha (IL6 & IL1β) activity /expression. Osteoclast (PBMC) and osteoclast / osteoblast models were successfully established and effect of magnesium on cell differentiation was observed1. While osteoclast activity was increasing with higher MgCl2 concentrations, magnesium extract had a two-phase effect: first induced by low magnesium concentration and then reduced with higher concentrations. Similar results were obtained in co-culture model. Similarly, macrophages were obtained and characterised. The co-culture macrophage / osteoblast remains under set-up and the effect of magnesium is currently investigated. DISCUSSION & CONCLUSIONS: More complex models were successfully established and characterised to investigate the intricate in vivo processes (inflammation / repair / remodelling) which are taking place during implantation. Magnesium extracts show in general different results as compared to Mg salt which is implying that other compounds/mechanisms are interplaying during in vivo magnesium-based implant degradation. REFERENCES: 1 L. Wu, B. Luthringer, L. L. Grünherz, N.Wojtas, U. Hopfner, F. Feyerabend, R. Willumeit, A. F. Schilling (2013) Osteologie 2013, Weimar, Germany. 2 F. Feyerabend, H. Drücker, D. Laipple, C. Vogt, M. Stekker, N. Hort, R. Willumeit (2012) J Mater Sci: Mater Med 23:9–24. ACKNOWLEDGEMENTS: This research project has been supported by the Marie Curie Initial Training Network MagnIM (Grant agreement no.: 289163).
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