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The use of M-FISH to analysis the presence of genome instability in cells exposed to orthopaedic implant wear debris

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The use of M-FISH to analysis the presence of genome instability in cells exposed to orthopaedic implant wear debris Presented by: Martin Figgitt – PowerPoint PPT presentation

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Title: The use of M-FISH to analysis the presence of genome instability in cells exposed to orthopaedic implant wear debris


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
ASR
Birmingham
Hip Resurfacing Orthopaedic Implants
6
Outline of procedure
X-ray-Implant in situ
Hip Resurfacing- Principle
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
Experimental Outline
  • Fibroblasts (BJ cell line) seeded at 2.5x 105 per
    75 cm2 culture flask
  • 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
METAL ION EXPOSURE 24 HOURS
CoCl2
1.3,25,50 ppb
Cr III
HARVESTING AND PASSAGING
2,20,40 ppb
DAY 1,5,10,15---------30
Cr VI
2,20,40 ppb
Cr IIICo
1.3/2,25/20,50/40 ppb
Cr VICo
1.3/2,25/20,50/40 ppb
M-FISH ANALYSIS
Control
Experimental Schematic
10
True Colour
Labelling Scheme
Pseudo Colours
M-FISH Metaphase
11
Translocation
Deletion
Fragment
Aneuploidy
M-FISH Chromosomal Aberrations
12
Chromosome aberration levels
13
Do these aberration levels change overtime?
What is the composition of the aberrations, is
there a pattern?
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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
BIRC
Image Associates M-FISH technology
Grant Sponsors ARC
21
Thank your for attention
Any questions?
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