1. The use of M-FISH to analysis the presence of genome instability in cells exposed to orthopaedic implant wear debris Presented by: Martin Figgitt Bristol Implant Research Centre Southmead Hospital Bristol

2. Contents Introduction – orthopaedic background, implant types etc The Bristol Study In vitro studies-metal ions Results Discussion Future work Acknowledgements

3. Introduction Hip and knee orthopaedic implants the second most common elective operation in UK More orthopaedic implants in younger eg 40 years old Implants produce wear debris in both particulate and metal ion form ( eg cobalt and chromium) Does any of this wear debris have any detrimental effects upon patients eg genotoxicity

4. The Bristol Study A comparison of the genotoxicity of two hip resurfacing implants: Birmingham and ASR The study is comprised of three components: 1)Patients; M-FISH performed on pre-operative and post- operative blood samples (6 months, 1 year and 2 year) 2)In-vitro study of nanoparticle wear debris 3)In-vitro study of metal ions associated with wear debris

5. Hip Resurfacing Orthopaedic Implants ASRBirmingham

6. Hip Resurfacing- Principle Outline of procedureX-ray-Implant in situ

7. The metal ions in vitro model Use primary human fibroblasts (BJ) Expose cells to metal ions (Chromium, Cobalt) physiological concentrations Culture and harvest cells over a 30 day period M-FISH analysis (whole genome to be visualised)- to investigate genome instability

8. Fibroblasts (BJ cell line) seeded at 2.5x 10 5 per 75 cm 2 culture flask Experimental Outline Cells left overnight to adhere, before exposure metal ions Pulse exposure to cobalt, chromium III and chromium VI ions One set of cultures exposed to the ions separately Second set with chromium III and chromium VI ions in combination with cobalt ions M-FISH analysis of fibroblast metaphases

9. CoCl 2 Cr III Cr VI Cr III+Co Cr VI+Co Control HARVESTING AND PASSAGING DAY 1,5,10,15---------30 METAL ION EXPOSURE 24 HOURS M-FISH ANALYSIS Experimental Schematic 1.3,25,50 ppb 2,20,40 ppb 1.3/2,25/20,50/40 ppb

10. M-FISH Metaphase True Colour Labelling Scheme Pseudo Colours

11. M-FISH Chromosomal Aberrations TranslocationDeletion FragmentAneuploidy

12. Chromosome aberration levels

13. Do these aberration levels change overtime? What is the composition of the aberrations, is there a pattern?

18. Summary Initial exposure to the metal ions induce chromosomal aberrations Over the time period analysis metal ion induced chromosomal aberrations regresses Both CrIII and Cr VI induce simple and complex aneuploidy The combination of Cobalt with CrIII and CrVI results in higher levels of chromosomal aberrations Cr VI displays a higher incidence and persistence of complex aneuploidy overtime with and without the presence of cobalt ions

19. Future Work Compare metal ion results with nanoparticle experiments Investigate possible mechanisms causing aneuploidy eg tubulin disruption Prolonged exposure experiments Investigations with cell lines defective in DNA repair

20. Acknowledgements Dr CP Case Mr A Blom Professor I Learmonth Dr R Newson Grant Sponsors: ARC BIRC Image Associates: M-FISH technology

21. Thank your for attention Any questions?