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Immune System Involvement in Metal Hip Implant Failure

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Immune System Involvement in Metal Hip Implant Failure UNIVERSITY OF EAST ANGLIA FACULTY OF MEDICINE AND HEALTH SCIENCES Immune System Involvement in Metal Hip Implant Failure Submitted in accordance with the requirements for the degree of Doctor of Philosophy by MUNISE PINAR BLOWERS May 2015 i Acknowledgments First of all, I would like to express my gratitude to Professor Simon Carding for giving me this opportunity to undertake my PhD study in his lab. I also would like to extend my appreciation to past and present members of his group. A few names I must mention here in particular; I would like to thank to Dr Louise Wakenshaw for helping me to get up and running in the lab, to Dr Isabelle Hautefort for general scientific advice and discussions over morning coffees we shared together, to Dr Elisabeth Bassity for critical review of some written work and Dr Kamal Ivory for providing me with invaluable scientific advice and in general for being an awesome friend over the years. To the several other students of the lab who came and gone, too many to mention here; thanks for their contributions and their support to the project. I would like to thank Mrs Anna Mathews for providing me administrative support with great enthusiasm, she was always happy to help, it did not matter what the question might be! I would like to express my sincere gratefulness to Professor Simon Donell for his advice and support throughout the study. I have massive admiration for his work and it has been great to see his friendly face throughout, especially when things did not go according to the plan. I need to extend my thanks to his staff members especially Ms Tracey Potter for looking after my samples when I was late (only occasionally though!), and Mrs Laura Watson for helping me with ethics applications which I am still trying to learn to do properly... Mr Darren Ebreo deserves his own paragraph of appreciation for getting up very early to collect clinical samples and also doing his best to explain the study to patients, from which he had a very high consent rate. This study would not be possible without his commitment and hard work. I would like to acknowledge Dr Stefano Caserta, especially for stimulating discussions and for sharing his passion for immunology with me. At last but not least I would like to thank my dear husband Lee for standing by me through thick and thin of it. He managed to pretend to understand and even like (it’s a strong word, I know) my science over the years for that reason alone he deserves a medal. He survived ii through my moans, many late nights and weekend working. His patience and encouragement has been invaluable. I will love him forever. Statement of Declaration - This study was funded by DePuy Synthes UK, sponsored by Norfolk Norwich University Hospital (NNUH) and hosted by Institute of Food Research (IFR), Norwich, UK. - Stipend for PhD maintenance was paid by charity Action Arthritis, Norwich, UK. - HLA Typing was conducted for a fee-for-service basis by National Health Service Blood and Transplant (NHSBT) Colindale, UK. - Patient consents and clinical sample collection were conducted by Mr Darren Ebreo. - Illustrations were created by Paul Pople at Creative Designs, Norwich, UK. - Statistical advice was provided by Dr Marianne Defernez (Analytical Science Unit, IFR, UK). - TEM images were acquired by Mrs Kathryn Cross at the Norwich Research Park Bioimaging Centre, UK. - Laser ablation was processed by Particular GmbH, Germany. iii Abstract Immune system involvement in metal hip implant failure Osteoarthritis (OA) is the most common debilitating disease, especially in the elderly. Total Hip Replacement (THR) is a last resort treatment for hip OA and Metal-on-Metal (MoM) THR was used commonly for its durability. While this is an effective treatment for many, around 12% of hip implants were revised in 2012. MoM articulations had poor implant survival compared with non-MoM, displaying four times higher failure rate. Between 1997 and 2004, 652 hip replacement surgeries were undertaken in Norfolk using the Ultima TPS MoM THR system (DePuy) which resulted in a high rate of early implant failure (27.4% at year 7). One of the proposed reasons for MoM THR failure is the adverse reaction to metal wear debris and consequent immune system mediated osteolysis resulting in peri-prosthetic loosening. To understand the immune system involvement in metal hip implant failure, a cohort of OA patients were recruited with and without an Ultima implant. This study investigated; The Human leukocyte antigen (HLA) allelic variation and implant failure association. Determined the composition and frequency of immune cells and inflammatory markers in Norfolk cohort. And tested the metal particle impact on immune cells. These experimental approaches were utilised to elucidate whether the metal implant failure is a result of an inflammatory process. The genetic disposition to metal hip implant failure was tested by conducting HLA typing for 25 different alleles across three MHC class II loci which revealed a protective haplotype against implant failure being DQA1*01:02 - DQB1*06. The analysis of immune parameters showed that all patient groups had normal levels of immune cell composition and cytokine levels apart from the Ultima Asymptomatic group, suggesting a regulatory mechanism in place for metal hip implant survival. Analysis of metal particle effect on immune cells demonstrated that these particles are immune-reactive and results in macrophage-initiated and lymphocyte-mediated failure mechanisms. iv Table of Contents 1 CHAPTER 1: Introduction ............................................................................................. 1 1.1 Definition and burden of Osteoarthritis ................................................................... 1 1.2 Hip Joint Structure and Pathophysiology ................................................................ 2 1.3 OA diagnosis and therapeutic options ..................................................................... 5 1.4 Metal/Metal-on-Metal hip implant failure .............................................................. 8 1.5 Chemistry of Co-Cr-Mo alloys .............................................................................. 15 1.6 Immunnological failure mechanism ...................................................................... 18 1.6.1 Innate recognition of metals ........................................................................... 20 1.6.2 Adaptive recognition of metals ...................................................................... 24 1.7 Hypothesis and Aims ............................................................................................. 30 2 CHAPTER 2: Study Protocol .................................................................................... 32 2.1 Obtaining ethical approval .................................................................................... 32 2.2 Patient Recruitment ............................................................................................... 33 2.3 Patient recruitment number and study power ........................................................ 34 2.4 Patient demographics ............................................................................................ 35 2.5 Human Tissue Collection ...................................................................................... 37 3 CHAPTER 3: Role of HLA molecules in immune responsiveness to metal hip implants ................................................................................................................................ 38 3.1 Introduction ........................................................................................................... 38 3.1.1 MHC Genetic organisation and structure ....................................................... 41 3.1.2 HLA Polymorphisms ..................................................................................... 44 3.1.3 HLA Nomenclature ........................................................................................ 45 3.1.4 HLA expression, antigen processing and presentation .................................. 47 3.1.5 MHC-Peptide-TCR Interaction ...................................................................... 48 3.1.6 HLA and Disease ........................................................................................... 50 3.2 Materials and Methods .......................................................................................... 53 3.3 Results ................................................................................................................... 57 3.4 Discussion ............................................................................................................. 64 3.5 Summary ............................................................................................................... 69 4 CHAPTER 4: Investigation of immune parameters in metal hip implant patients ...... 70 4.1 Introduction ........................................................................................................... 70 v 4.2 Material and Methods ............................................................................................ 77 4.2.1 Immunophenotyping by Polychromatic Flow Cytometry.............................. 77 4.2.2 Measurement of serum and SF cytokines ...................................................... 86 4.3 Measurement of serum metal ions ......................................................................... 88 4.4 Results ..................................................................................................................
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