Applied Science, Faculty of
Biomedical Engineering, School of
DSpace
UBCV
Eltit Guersetti, Felipe Yasser
Doctor of Philosophy - PhD
University of British Columbia
About two million people receive hip implants annually, to relieve the pain generated by osteoarthritis. Hip implants are composed of a titanium femoral stem and a Co-Cr-Mo head that articulates with a titanium-alloy acetabular cup, covered by a polyethylene liner (MoP). Other designs directly use a metal (Co-Cr-Mo) acetabular articulating surface (MoM). Concerns have arisen due to the elevated numbers of adverse local tissue reactions (ALTRs) to hip implants that generate pain and soft-tissue destruction. Due to the high rates of ALTRs in MoM implants, the corrosion products or wear particles from the metal surfaces are thought to trigger the immunological reactions. This thesis aimed to understand the mechanisms of ALTRs development through three studies: 1) histological analyses and comparison of ALTRs in MoM and MoP implants. 2) analysis of corrosion products in synovial fluid and tissues. 3) mechanistic study based on gene expression analysis of ALTRs, and cell culture of primary synovial fibroblasts that were exposed to Cr and Co. The histological description of ALTRs showed structural similarities between MoM and MoP, with common elements such as tissue necrosis and the presence of perivascular lymphocyte aggregates. Significantly higher levels of metal ions were found in the synovial fluid of ALTR patients, but no differences in the particles present in tissues were found in ALTRs compared to non-ALTRs. The gene expression analysis of lymphocytes aggregates found an identical and non-specific Th1/Th2 reaction in ALTRs in both MoM and MoP, with no evidence of Th17 reaction. Finally, primary synovial fibroblasts responded to concentrations of metal ions observed in the synovial fluid of patients, by releasing pro-inflammatory cytokines. After 24 hours of exposure the secreted cytokines were demonstrated to be chemotactic for human monocytes which is a key processes during inflammation. These results support the hypothesis that metal ions from the hip implants trigger cytokine secretion by synovial fibroblasts, which initiate the immune reaction to hip replacements, providing evidence to reject the hypothesis of hypersensitivity as an etiologic factor of ALTRs.
ROLES OF CORROSION PRODUCTS OF METAL HIP REPLACEMENTS IN THE ETIOLOGY OF ADVERSE LOCAL TISSUE REACTIONS by Felipe Yasser Eltit Guersetti D.D.S., Universidad de Chile, 2003 M.Sc., Universidad de Chile, 2010 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biomedical Engineering) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2018 © Felipe Yasser Eltit Guersetti, 2018 ii Committee members: The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Roles Of Corrosion Products Of Metal Hip Replacements In The Etiology Of Adverse Local Tissue Reactions submitted by Felipe Eltit in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Engineering Examining Committee: Rizhi Wang Co-supervisor Bassam Masri Co-supervisor Michael Cox Supervisory Committee Member Frank Ko Supervisory Committee Member Vincent Duronio University Examiner Karen Cheung University Examiner iii Abstract About two million people receive hip implants annually, to relieve the pain generated by osteoarthritis. Hip implants are composed of a titanium femoral stem and a Co-Cr-Mo head that articulates with a titanium-alloy acetabular cup, covered by a polyethylene liner (MoP). Other designs directly use a metal (Co-Cr-Mo) acetabular articulating surface (MoM). Concerns have arisen due to the elevated numbers of adverse local tissue reactions (ALTRs) to hip implants that generate pain and soft-tissue destruction. Due to the high rates of ALTRs in MoM implants, the corrosion products or wear particles from the metal surfaces are thought to trigger the immunological reactions. This thesis aimed to understand the mechanisms of ALTRs development through three studies: 1) histological analyses and comparison of ALTRs in MoM and MoP implants. 2) analysis of corrosion products in synovial fluid and tissues. 3) mechanistic study based on gene expression analysis of ALTRs, and cell culture of primary synovial fibroblasts that were exposed to Cr and Co. The histological description of ALTRs showed structural similarities between MoM and MoP, with common elements such as tissue necrosis and the presence of perivascular lymphocyte aggregates. Significantly higher levels of metal ions were found in the synovial fluid of ALTR patients, but no differences in the particles present in tissues were found in ALTRs compared to non-ALTRs. The gene expression analysis of lymphocytes aggregates found an identical and non-specific Th1/Th2 reaction in ALTRs in both MoM and MoP, with no evidence of Th17 reaction. Finally, primary synovial fibroblasts responded to concentrations of metal ions observed in the synovial fluid of patients, by releasing pro-inflammatory cytokines. After 24 hours of exposure the secreted cytokines were demonstrated to be chemotactic for human monocytes which is a key processes during inflammation. These results support the hypothesis that metal ions from the hip implants trigger cytokine secretion by synovial fibroblasts, which initiate the immune reaction to hip replacements, providing evidence to reject the hypothesis of hypersensitivity as an etiologic factor of ALTRs. iv Lay Summary Adverse local tissue reactions (ALTRs) are one of the main causes of hip implant failure, yet, the mechanisms of their development remains largely unknown. In this thesis, we demonstrated that cobalt and chromium ions released from the hip implants, as consequence of wear or corrosion, affect the cells of the hip joint. The cells exposed to metals release inflammatory factors that are able to trigger changes in blood vessels and cells of the immune system, which lead to the changes observed in ALTRs. Our results provided strong evidence that metal allergies are not involved in the development of ALTRs, as has been widely accepted, but that ALTRs are a consequence of the cell damage generated by metal ions. v Preface This thesis corresponds to a collaborative and multidisciplinary work that involved different departments, research centers and clinical institutions, aiming to contribute in the knowledge of the adverse reactions to hip replacements, with emphasis on the role of the metals elements from the implants in the etiology of the adverse reactions. The results of this work represent a significant contribution of the knowledge in the field, which includes patients’ data, analysis of patient’s tissues, fluids and blood, as well as an experimental study based in a cell culture model. The patient recruitment and sample collection were done in the Vancouver General Hospital, thanks to the collaboration with the surgeons Drs. Clive Duncan, Bassam Masri, Donald Garbuz and Nelson Greidanus. The diagnoses were done in the department of Pathology of the Vancouver General Hospital, in collaboration with Dr. Tony Ng. Histology and microscopy observation were performed at the Centre for Hip Health and Mobility. The particle analysis through SEM and SEM-EDX was carried out in the facilities of the Department of Materials Engineering, University of British Columbia. Synchrotron XRF and XRD were performed in the Canadian Light Source with the collaboration with Mr. Qiong Wang and Dr. Renfei Feng. The cell culture experiments involving synovial fibroblasts and human monocytes were carried out at the laboratory of Prof. Michael Cox in the Vancouver Prostate Centre. Complex statistical analysis (e.g. unsupervised hierarchical clustering) was performed in collaboration with Robert Bell and Anne Haegert, from the Vancouver Prostate Centre. The experiments on human vascular endothelial cells were performed at the laboratory of Professor Felipe Simon at Andres Bello University, in Santiago, Chile. The procedures on human subjects performed in this thesis, were approved by the ethics board of the University of British Columbia (H14-03050 and H09-00215). A version of chapter 3 has been published as: Eltit F, Assiri A, Garbuz DS, Duncan CP, Masri BA, Greidanus N, Bell R, Sharma M, Cox ME, Wang RZ. Adverse reactions to metal on vi polyethylene implants: Highly destructive lesions related to elevated concentration of cobalt and chromium in synovial fluid. J Biomed Mater Res Part A, 2017 Jul;105(7):1876-1886. Part of the literature review (chapter 1) has been accepted for publication as a chapter “Corrosion of Orthopedic Implants”, in the book "Encyclopedia of Biomedical Engineering". Elsevier Science & Technology Books, edited by Roger Narayan. Publication date: October 2018. ISBN: 9780128048290. The candidate (Felipe Eltit), was involved in every step of this project. Starting from the study design and planning, samples collection and processing, experiment design and execution, analysis of the results, clinical data analysis, and manuscript drafting and editing. Below is a list of specific experimental procedures performed by the applicant. 1. Clinical data collection: from clinical files and direct information from the surgeons. 2. Collection of human samples: this was carried out directly in the OR by the candidate in each of the studied patients. 3. Preparation of synovial fluid and serum for analysis: Performed immediately after the surgical procedure. Samples were then frozen and aliquots were sent for analysis. 4. Histology sample preparation: Samples obtained from patients, after being released in the Department of Pathology were fixed, embedded and sectioned by Felipe Eltit. 5. Staining and observation of histological sections: these procedures were performed in all the analyzed cases. 6. Immunohistochemistry. 7. Selection of cases used for gene expression analysis. 8. RNA extraction on paraffin-embedded formalin-fixed tissues: performed in the previously selected samples. 9. Gene expression analysis: The procedure was performed in the automated system developed by Nanostring. (nCounter Prep Station), available in Vancouver General Hospital. The reading was also performed in an automated system, the nCounter Digital Analyzer, available in Vancouver General Hospital. 10. Statistical analysis: general statistical analysis (t-test, ANOVA). vii 11. Primary synovial fibroblasts isolation and culture: FE collected the samples from primary surgeries directly in the OR, processed the tissues and isolated cells. Further culture was also performed by FE. 12. Metal exposure experiment of synovial fibroblasts. Measurement of viability, cell quantification, and DNA content performed by FE, by use of flow cytometry. 13. Processing of conditioned media (concentration and processing for later use). 14. Chemotaxis assay of THP-1 cells: both experiments on chemotaxis, to synovial fluid and conditioned media performed by FE. Experiments with HUVECs cells were performed in collaboration with Prof. Felipe Simon, Universidad Andres Bello, Chile. Felipe Eltit was involved in the design of the experiment and data analysis. viii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ....................................................................................................................... viii List of Tables .............................................................................................................................. xiii List of Figures ............................................................................................................................. xiv List of Abbreviations ................................................................................................................. xvi Glossary .................................................................................................................................... xviii Acknowledgements ......................................................................................................................xx Dedication .................................................................................................................................. xxii Chapter 1: Introduction. ...............................................................................................................1 1.1 Epidemiology of hip replacements ................................................................................. 1 1.2 Materials and designs in arthroplasty systems ................................................................ 2 1.2.1 Development of hip replacement systems; articulating surfaces and modular components. ............................................................................................................................ 3 1.2.2 Degradation of materials: wear of the articular surfaces. ....................................... 7 1.2.3 Degradation of materials: corrosion at the tapered modular junctions. .................. 9 1.3 Products of material degradation in human tissues....................................................... 10 1.3.1 Metal particles:...................................................................................................... 11 1.3.2 Metal ions: ............................................................................................................ 12 1.4 Adverse local tissue reactions to metal implants .......................................................... 14 1.4.1 Nomenclature: ....................................................................................................... 15 ix 1.4.2 Histological description of ALTRs: ...................................................................... 16 1.4.3 Etiology and pathogenesis of ALTRs: .................................................................. 17 1.4.4 Hypersensitivity as an event in ALTRs: ............................................................... 18 1.4.5 Tertiary lymphoid structures and lymphocyte activation in ALTRs: ................... 20 1.4.6 Etiology of ALTRs, the effect of Co and Cr in cell populations: ......................... 21 1.5 Research questions ........................................................................................................ 23 Chapter 2: Hypothesis and aims. ................................................................................................24 Chapter 3: Histological description of ALTRs in Metal on Metal and Metal on polyethylene hip replacement systems. .............................................................................................................26 3.1 Rationale: ...................................................................................................................... 26 3.2 Patients and Methods .................................................................................................... 26 3.2.1 Histology and scoring. .......................................................................................... 31 3.2.2 Cellularity: ............................................................................................................ 32 3.2.3 Immunohistochemistry. ........................................................................................ 33 3.2.4 Cytokine microarrays. ........................................................................................... 33 3.3 Results ........................................................................................................................... 36 3.3.1 MoP ALTRs are fibro-necrotic lesions, while MoM ALTRs are cystic-inflammatory. ........................................................................................................................ 36 3.3.2 MoM and MoP ALTRs exhibit common morphological elements. ..................... 41 3.3.3 Degenerative changes in blood vessel’s walls associated to MoP ALTRs. .......... 43 3.3.4 ALTRs showed a distinctive cytokine pattern in the periprosthetic tissues. ........ 45 3.4 Discussion ..................................................................................................................... 49 3.4.1 MoP ALTRs are highly destructive lesions. ......................................................... 49 x 3.4.2 Cytokines could play a key role in ALTR’s etiology and pathogenesis. .............. 50 Chapter 4: Degradation of materials: ions and metal particles in periprosthetic tissues. ....52 4.1 Rationale ....................................................................................................................... 52 4.2 Methods......................................................................................................................... 52 4.2.1 Analysis of chromium and cobalt ions concentrations in serum and synovial fluid................................................................................................................................ 53 4.2.2 Analysis of particles in the tissues: ....................................................................... 54 4.3 Results ........................................................................................................................... 56 4.3.1 Significantly higher concentrations of chromium and cobalt in synovial fluid of patients with ALTRs. ............................................................................................................ 56 4.3.2 Elevated concentration of metal ions in serum of patients with ALTRs in MoP. 59 4.3.3 Similar amount of metal particles in ALTR and non-ALTR joints. ..................... 61 4.3.3.1 Wear particles and corrosion products have different chemical composition and both are found in MoM and MoP ALTRs. ....................................................................... 67 4.3.3.2 Corrosion products observed as amorphous chromium-rich particles. ............. 70 4.4 Discussion: .................................................................................................................... 72 4.4.1 Metal ions in blood as predictors of function of MoP implants. .......................... 73 4.4.2 Local concentration of metals as a pathogenic element in ALTRs. ..................... 73 4.4.3 Particles in periprosthetic tissues: no association with pathologic events in the hip................................................................................................................................ 74 Chapter 5: Biological effects of Cobalt and Chromium on the periprosthetic tissues of the hip. .................................................................................................................................................77 5.1 Rationale: ...................................................................................................................... 77 xi 5.2 Study design and patients.............................................................................................. 78 5.2.1 Gene expression analyses by use of Nanostring n-counter system: ..................... 79 5.2.2 Determination of cytokines in synovial fluid and serum. ..................................... 81 5.2.3 Biomarkers in patient’s serum. ............................................................................. 82 5.2.4 Cell culture of human primary synovial fibroblasts. ............................................ 83 5.2.5 Analysis of cell cycle and cytokine release by metal exposed fibroblasts. .......... 83 5.2.6 NF-kb activation ................................................................................................... 84 5.2.7 Preparation of synovial fluid and conditioned media concentrates. ..................... 84 5.2.8 Expression of V-CAM and P-selectin in human endothelial cells. ...................... 84 5.2.9 Chemotaxis of THP-1 cells. .................................................................................. 85 5.3 Results ........................................................................................................................... 87 5.3.1 Th1/Th2 immune reaction is characteristic of ALTRs. ........................................ 87 5.3.2 Elevated concentration of inflammatory cytokines in synovial fluid of ALTRs affected joints. ....................................................................................................................... 96 5.3.3 Cytokines that are highly concentrated in synovial fluid are not expressed in TLOs. ............................................................................................................................. 104 5.3.4 Cytokines highly concentrated in synovial fluid showed no differences in serum.............................................................................................................................. 105 5.3.5 Chemotaxis of THP-1 cells is induced by ALTRs synovial fluid. ..................... 105 5.3.6 Synovial fibroblasts release inflammatory cytokines when exposed to concentrations of Co and Cr observed in ALTRs. .............................................................. 107 5.3.7 Cobalt and Chromium ions induce activation of NF-B in synovial fibroblasts:............................................................................................................................. 112 xii 5.3.8 Conditioned media of metal-exposed fibroblast induce chemotaxis in human monocytes and activates human endothelial cells. ............................................................. 114 5.4 Discussion: .................................................................................................................. 117 5.4.1 ALTRs in MoM and MoP hip replacements correspond to a single immunological entity. ............................................................................................................................. 117 5.4.2 Hypersensitivity may not play a role in ALTRs etiology. .................................. 117 5.4.3 Th1-Th2 reaction in ALTRs. .............................................................................. 118 5.4.4 Presence of cytokines in synovial fluid. ............................................................. 119 5.4.5 Effects of metals in cytokine secretion of synovial fibroblasts. ......................... 120 Chapter 6: Conclusion ...............................................................................................................122 6.1 Limitations. ................................................................................................................. 124 6.2 Future work ................................................................................................................. 126 6.3 Contributions............................................................................................................... 128 Bibliography ...............................................................................................................................130 Appendix A Histological images of MoM ALTRs..................................................................... 155 Appendix B Histological images of MoP ALTRs. ..................................................................... 156 Appendix C. Histological images of Control cases. ................................................................... 156 Appendix D Microscopic imaes of collagen structure in ALTRs: ............................................. 152 Appendix E concentration of cytokines in tissue homogenates. ................................................ 153 Appendix F Fold change and p-value of genes in TLOs from MoM and MoP ALTRs. ............ 156 Appendix G Rank changes of genes in TLS in Control MoM and MoP.. .................................. 158 Appendix H Cells scores obtained from the gene expression analysis in ALTR TLOs. ............ 166 xiii List of Tables Table 1.1. Metal particles observed in ALTR tissue sections. ......................................................19 Table 3.1 ........................................................................................................................................28 Table 3.2 Chief complaint, diagnostic images and volume of ALTR in MoM and MoP total hip replacements. ................................................................................................................................ 29 Table 3.3 ALVAL score for periprosthetic tissue. ........................................................................ 32 Table 3.5. Cellularity values for MoM and MoP ALTRs in the tissues depth. ............................ 40 Table 3.6. ALVAL assessment of the pseudotumors in MoM and MoP hip replacement. .......... 40 Table 3.7. Cytokines and biomarkers that exhibit significant higher values in the MoM group than the Control group.. ................................................................................................................ 46 Table 4.1. Chromium and cobalt concentration y synovial fluid. Expressed in g / L. ............... 57 Table 4.2. Metal particles observed in ALTR tissue sections. ..................................................... 67 Table 5.1 comparison of gene expression in TLOs according to clinical variables. .................... 87 xiv List of Figures Figure 1.1 Types of hip replacements: ............................................................................................ 5 Figure 3.1 Characteristic ultrasound images of ALTRs. .............................................................. 30 Figure 3.2 Macroscopic representative sections of ALTRs. ......................................................... 37 Figure 3.3 Measurement of cellularity.. ........................................................................................ 38 Figure 3.4 Histological structure of ALTRs in MoM and MoP implants. .................................... 42 Figure 3.5 Immunohistochemistry of perivascular lymphocyte aggregates in MoP ALTRs. ...... 43 Figure 3.6 Low magnification images of synovial surface in cases of MoM ALTRs. ................. 44 Figure 3.7 Low magnification images of synovial surface in cases of MoP ALTRs. .................. 44 Figure 3.8 Low magnification images of synovial surface in Control cases. ............................... 44 Figure 3.9 Structural changes in the vascular wall of blood vessels in ALTRs. .......................... 44 Figure 3.10 Microscopic images of collagen structure in ALTRs. ............................................... 45 Figure 3.11 Unsupervised hierarchical clustering of cytokines in MoM and MoP ALTRs ......... 48 Figure 4.1 Synovial fluid of ALTR patients. ................................................................................ 57 Figure 4.2 Dot plot of the log-2 concentration of Co and Cr in synovial fluid. ........................... 58 Figure 4.3 Concentration of cobalt and chromium in serum. ....................................................... 60 Figure 4.4 Microphotographs of wear particles in periprosthetic tissues. .................................... 62 Figure 4.5 Corrosion particles in periprosthetic tissues. ............................................................... 63 Figure 4.6 Iron rich vesicles in macrophages ............................................................................... 66 Figure 4.7 Average composition of wear particles in periprosthetic tissues. ............................... 68 Figure 4.8 Average composition of corrosion products in periprosthetic tissues. ........................ 69 Figure 4.9 Synchrotron-XRF mapping of corrosion particles in periprosthetic tissue. ................ 71 Figure 4.10 Synchrotron-XRD analysis of corrosion products in periprosthetic tissue. .............. 72 Figure 5.1 Low magnification images of synovial surface in cases of MoP ALTRs. .................. 80 Figure 5.2 Venn diagram of genes differentially expressed ......................................................... 88 Figure 5.3 Volcano plots of the comparison of gene expression in ALTRs ................................. 90 Figure 5.4 Gene expression based-cells scores comparing controls, MoM and MoP. ................. 91 Figure 5.5 TMA of leucocyte aggregates. .................................................................................... 95 Figure 5.6 Heat-map of cytokines in synovial fluid ..................................................................... 97 Figure 5.7 Dot plots of the concentration of cytokines in synovial fluid. .................................. 103 xv Figure 5.8 Heat map and of the gene-expression of cytokines in the TLOs. .............................. 104 Figure 5.9 Chemotaxis of human monocytes exposed to synovial fluid. ................................... 106 Figure 5.10 Cytokine release by synovial fibroblasts exposed to metal ions ............................. 109 Figure 5.11 Dose-dependent release of cytokines by synovial fibroblasts exposed to metal ions...................................................................................................................................................... 111 Figure 5.12 Nuclear translocation of p-65 NF-kB ...................................................................... 113 Figure 5.13 Secondary effects of Co and Cr exposure of primary human fibroblasts. ............... 116 Figure 6.1 Role of metal ions in the etiology of ALTRs. ........................................................... 123 xvi List of Abbreviations Altrs: Adverse Local Tissue Reactions. Alval: Aseptic Lymphocyte-Dominated Vasculitis-Associated Lesion. Anova Analysis Of Variance. Armd: Adverse Reaction To Metal Debris. C M: Control Metal On Metal C P: Control Polyethylene Cd: Cluster Of Differentiation. Dmem: Dulbecco Modified Eagles Medium. Eds: Energy-Dispersive X-Ray Spectroscopy. Edta: Ethylenediaminetetraacetic Acid. Edx: Energy-Dispersive X-Ray Spectroscopy. Egta: Ethylene Glycol Tetraacetic Acid. Ena-78 Epithelial-Neutrophil Activating Peptide 78, Also Known As Cxcl5. Fbs: Fetal Bovine Serum. Fda: Food And Drug Administration, Us. Fitc: Fluorescein Isothiocyanate. Flt-3l: Fms-Related Tyrosine Kinase 3 Ligand. G-Csf: Granulocyte Colony-Stimulating Factor. Gf: Growth Factor Gm-Csf: Granulocyte-Macrophage Colony-Stimulating Factor. Gro- Melanoma Growth Stimulating Activity Alpha, Also Known As Cxcl1. Hif: Hypoxia-Inducible Factors. Hr: Hip Resurfacing. Huvecs Human Umbilical Vein Endothelial Cells. Icam: Intercellular Adhesion Molecule. Igg: Immunoglobulin G. Ikk: Inhibitor Of Nuclear Factor Kappa-B Kinase. Il: Interleukin Ip-10: Ifn-Gamma-Inducible Protein 10, Also Known As Cxcl10. Kda: Kilo Dalton. Lps: Lipopolysaccharide. Mcp-2: Monocyte-Chemotactic Protein 2, Also Known As Ccl8. Mcp-3: Monocyte-Chemotactic Protein 3, Also Known As Ccl7. Mig: Monokine Induced By Gamma Interferon, Also Known As Cxcl9. xvii Mip-1 Macrophage Inflammatory Protein 1. Also Known As Ccl3. Mom: Metal On Metal Mop: Metal On Polyethylene Nf-B: Nuclear Factor Kappa-Light-Chain-Enhancer Of Activated B Cells. P S: Primary Surgery Pbs: Phosphate Buffered Saline. Rcf: Relative Centrifugal Force. Ripa: Radioimmunoprecipitation Assay Buffer. Rpmi: Roswell Park Memorial Institute (Culture Media). Sem: Scanning Electron Microscopy. Sf: Synovial Fluid Tbet: T-Box Transcription Factor, Also Known As Tbx21. Tbs: Tris Buffered Saline. Tem: Transmission Electron Microscopy Th Cells. T-Helper, Usually Referred To Subsets Of CD4+ T Cells. Tha: Total Hip Arthroplasty Tlos: Tertiary Lymphoid Organs. Tnf-: Tumor Necrosis Factor Alpha. Tritc: Tetramethylrhodamine. Uhmwpe: Ultra-High Molecular Weight Polyethylene. Vcam: Vascular Cell Adhesion Protein. Vegf: Vascular Endothelial Growth Factor. Xas: X-Ray Absorption Spectroscopy. Xrd: X-Ray Diffraction. Xrf: X-Ray Fluorescence. xviii Glossary 510K: FDA Mechanism of approval of medical devices, based on the equivalence to previously approved devices. It is the fastest and more economic procedure for approving new medical devices to be used. Chemokines: subset of cytokines that have in their structure four conserved cysteine amino acids in conserved and specific locations. The name chemokine derives from their primary and firstly described function of being chemoattractant for cells of the immune system. Chemokines are classified into four families according to the location of the cysteine residues that allows their tridimensional folding: CC, CXC, CX3C and XC. Cytokines: Broad category of soluble, low molecular weight proteins (~5–20 kDa) that function as cell signaling molecules. Their effects can reach multiple levels, being of paramount importance in the immune system. Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. Hip capsule: Dense fibrous connective tissue that surrounds the hip joint articulation and attaches the hip and femoral component. It attaches to the margin of the acetabulum in the hip bone, and in the labrum. In the external side, it attaches to the femoral neck and the intertrochanteric line. Hip resurfacing: reconstructive surgical procedure of the hip joint, in which only the surface of the femoral head is removed and replaced, preserving the femoral neck and part of the femoral head. Synovial fluid: viscous fluid that fills the joint cavities of synovial joints. The main role of synovial fluid is the lubrication of the joint surfaces reducing friction. It is composed mainly by water and proteoglycans, such as hyaluronic acid and lubricin. It also contains glucose and nutrients for the avascular articular cartilage that cover the bone surfaces, for which synovial fluid is the main source of nutrients. xix Synovium: or synovial membrane, is a specialized connective tissue that lines the internal surface of the capsule. It is also responsible for the secretion of synovial fluid. Histologically is composed of a dense connective tissue, but not as dense as the hip capsule. The collagen fibers also are randomly oriented in the surfaces plane rather than in a single orientation as is the case of the hip capsule. The sub-superficial layer is composed of a softer tissue, which is highly vascularized and innervated. The internal surface is covered by two types of synoviocytes that form an epithelial sheet. Synoviocytes type A are rounded cells from macrophage-monocyte origin, type B are flat cells that have a mesenchymal origin resembling fibroblasts phenotype. It is accepted that type A cells maintain the synovial fluid by removing debris and tear particles, while type B cells are the ones that secrete the synovial fluid. Th1: Are a subset of CD4+ T-cells characterized by the secretion of interferon gamma (IFN-g), interleukin 2 and tumor necrosis factor beta. Evoke cell mediated immunity and phagocyte dependent inflammation. It dominates in diseases produced by intracellular parasites, virus or cancer. Th2: Are CD4+ lymphocytes that secrete IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13, generating strong antibody response, eosinophil activation, but inhibiting functions of phagocytic cells. Th2 lymphocytes are predominant in infections of extracellular parasites and intestinal nematodes. Total hip arthroplasty: reconstructive surgical procedure of the hip joint, in which the whole femoral head and neck are removed, and replaced by an artificial femoral head, that is kept in position by the presence of a femoral stem inserted in the bone marrow canal. xx Acknowledgements I want to thank to my co-supervisor, Prof. Rizhi Wang, for the opportunity to perform this work, for sharing his knowledge and effort during my years in the program. I am also deeply grateful of Prof. Michael Cox, for his advice during his work, and sharing his enthusiasm and knowledge, as well as his critical analysis of this work. My sincere gratitude to my co-supervisor Dr. Bassam Masri, for his guidance and support. As well I would like to express my sincere gratitude to the orthopaedic surgeons of VGH and UBC hospitals: Dr. Clive Duncan, Dr. Donald Garbuz and Dr. Nelson Greidanus, for their enthusiastic collaboration in the patients recruitment, samples collection and important clinical advice during this years in the department. Special mention to Dr. Tony Ng, who give strong support in the pathology department to perform this work, as well as his wise advice and contributions to this work. Many thanks to Prof. Frank Ko, for accepting to be in my committee and his effort and contribution to this thesis. I would like to give special thanks to the following people that made critical contributions to this work, and obtained no benefit for their help besides my deepest gratitude: Special thanks to Ms. Raman Johal, for her collaboration in the patient recruitment and interaction with external institutions. Thanks to Mr. Simon Cheung and Mrs. Margaret Luk for their support and collaboration in the samples collection. Thanks to Mr. Joe Kudzin, for his collaboration in the sample collection, as well as sharing his invaluable knowledge on implants manufacturing and marketing. You made my OR days much happier Joe. xxi To the personnel of the department of pathology of Vancouver General Hospital, especially to Mr. Michael Chong, and Mrs. Shadan Ibrahim, Angela Furtado, and Maria Bernado. To whom I interrupted their regular work 150 times for specimen collection and I received just a generous smile every time. Thanks to all the personnel in OR 19 and 20 in VGH, and OR 3 and 4 in UBC hospital, for their always helpful collaboration with the collection of surgical specimens. To my friend and colleague Qiong Wang, for his collaboration in the particle analysis in the Canadian Light Source Facility, and many constructive discussions in this project. Thanks to Mr. Jake Noble and Dr. Toni Tang for their collaboration in cell culture work and Second Harmonic Generation imaging. Thanks to Mr. Jacob Kabel, for his helpful assistance during the SEM observation. Especial thanks to Professors, Sergio Sanchez, Eduardo Rodriguez and David Rodriguez, for their friendship, support and advice during my PhD program. xxii Dedication A mi padre, quién ha sido la persona mas importante en mi carrera académica con su constante apoyo y preocupación. 1 Chapter 1: Introduction. About two million people receive a hip replacement every year to resolve the pain and restore the function that has been partially lost mainly due to osteoarthritis, a degenerative disease that affects mostly people in the seventh decade of life (1, 2). Although hip arthroplasty is a generally successful procedure, Adverse Local Tissue Reactions (ALTRs) to metal implants can develop in all types of hip replacements, with a higher rate in Metal on Metal implants (MoM) (3), affecting the prognosis of further clinical solutions, thus decreasing the quality of life of the affected patients (4, 5). ALTRs are benign non-infected lesions that arise in the periprosthetic tissues destroying muscles and ligaments, generating pain and swelling in the patients. The etiology of ALTRs is not clearly understood, but their exclusive development after hip replacement surgeries, suggests a role of the metal components of the hip implants in their development (6). This study is focused on the mechanisms of the development of ALTRs, and the roles of the metal elements derived from the corrosion and wear of the hip replacements in the etiology and pathogenesis of the disease. The following subchapters aim to give a thorough literature review on those elements that will help the understanding of this process from a material prospective and the biological process involved in their etiopathogenesis. 1.1 Epidemiology of hip replacements Hip replacement is a surgical procedure in which the malfunctioning femoral-hip articulation is replaced by an artificial implant. This procedure dramatically improves the quality of life of the patients, while the risk of infection or complications has decreased significantly in the last two decades (7, 8). It is estimated that every year two million hip replacements are implanted worldwide. In Canada, during 2015, there were 49,503 primary hip replacements, which represents a five-year increase of 19% (2). The main reason for hip replacement is osteoarthritis, a degenerative inflammatory disease that affects patients with a mean age of 67 years old and 2 exhibits a higher prevalence in women (9); however, due to fractures and traumatic pathology, there is an increasing number of young active men that requires hip replacement surgery (10). The clinical performance of the hip implants varies depending on factors such as the type of implant, age of implantation, gender, activity, and other clinical variables such as acetabular inclination, head size, etc. It was accepted that the 10 year survivorship of MoM articulations is around 75% with a revision rate of 13% during the first 5 years (11, 12), but later reports showed a failure rate of 48% within the first six years for the Jonhson & Johnson (DePuy) ASR total hip replacements (13), while Johnson and Johnson reported that 37% of their MoM systems are expected to fail in the first 5 years (14, 15). This failure rate is higher than metal on polyethylene (MoP) or ceramic on ceramic implants, and it was proposed in 2012 that Metal on Metal articulations should no longer be used in hip arthroplasty (16). As a result of these poor clinical outcome, international regulatory agencies, including Health Canada, the Medicines and healthcare products regulatory agency (UK) and FDA (US) have issued alerts and safety communications related to MoM hip implants (17–19). To the best of our knowledge, Metal on Metal total hip replacements are not being used today, and limited uses for MoM hip resurfacing systems are still in practice (2, 9). 1.2 Materials and designs in arthroplasty systems Metal alloys, due to their mechanical robustness, are the most used materials in hip replacement systems. Stainless steel, cobalt alloys and titanium alloys could be combined with polymers or ceramics to comply with their functional requirements and biocompatibility. Corrosion and wear in the metal components of the hip replacement systems have been widely reported, and the presence of products of corrosion have been described even in distant organs such as liver and spleen (20). Pathogenic effects of those corrosion products in systems and organs has not been demonstrated, and no carcinogenic or teratogenic risk has been demonstrated in patients with hip implants. Nevertheless, deleterious effects of metals in the tissues around the prosthesis are indicated as responsible for the development of ALTRs. In the following sub-chapters a description of the materials used in hip prosthesis is presented, as well as the mechanisms of material degradation that could lead to ALTRs. 3 1.2.1 Development of hip replacement systems; articulating surfaces and modular components. The modern hip arthroplasty systems were firstly introduced in England in 1951 by G.K. McKee and Watson-Farrar. Their implants were composed of a metal stem, and an also metal acetabular cup, both composed of stainless steel articulating with each other (21). This design was improved in 1962 by Sir John Charnley, who introduced a polyethylene liner between the metal parts in order to minimize friction. Since then, metal on polyethylene has been the most used articulation in hip arthroplasty (22). The introduction of titanium alloys in medicine was carried out by Dr. Per-Ingvar Brånemark, with a series of studies in the 1970’s in Goteborg (23–26); thereafter, the conventional hip replacements were generally composed of a titanium femoral stem, a metal femoral head (mostly Co-Cr-Mo alloy) articulating with an Ultra-High Molecular-Weight Polyethylene (UHMWPE) liner, most frequently supported by a titanium acetabular shell (Fig. 1.1). The lower mechanical behavior of polyethylene produced the generation of wear debris that led to clinical complications involving inflammation and bone resorption (27). To avoid these problems, second generation MoM articulations were developed in the 1990s (Fig. 1.2). The articulating surfaces in the femoral head and acetabular cup in MoM implants are composed 60-65% of cobalt, 27-30% chromium, 5-7% molybdenum and 2-5% other traces elements as manganese, silicon, iron, nickel and carbon (Co-Cr-Mo) (28–30). Through the use of new technologies, it was possible to obtain smoother surfaces (nanometer scale) which theoretically would reduce wear and friction (31). Those theoretical assumptions of lower wear generation were later demonstrated in-vitro (32, 33), at the time that clinical outcomes showed positive results, such as: serum levels of chromium and cobalt in most of the patients were considered acceptable (34), and no higher risk of cancer in patients with MoM compared to the general population were found (35–38). Up to date, there is no evidence of fetal malformations or reproduction impairment associated with hip MoM hip replacements. Only one case report of a pregnant woman with bilateral hip resurfacing systems, who had high levels of Co and Cr in her blood during pregnancy, found that high levels of Co and Cr were also found in the newborns blood. However, After 14 weeks of post-natal development, a 4 totally normal baby was observed with no signs of intoxication, as the levels of Co and Cr in the blood drop down to normality (39). Due to these observations, MoM implants were considered safe to be used in humans. The FDA approved the MoM systems for clinical use through the 510K or “substantial equivalence” to previously cleared devices (40). To avoid dislocation, a common complication, the use of large femoral heads was introduced in order to increase the displacement required to remove the femoral head from the acetabular cup (41, 42). Tribological analyses demonstrated that the increase of friction between larger contact surfaces would not be significant if the clearance between the surfaces is decreased (31, 43). The possibility of having a metallic femoral head, articulating directly over the metal of the acetabular cup, allowed the development of hip resurfacing (Fig 1.3), a surgical procedure that involves minimal bone removal on the femoral head. The design was especially indicated for young active patients, with a good outcome after 5 years of surgery, and a lower hospital stay (2.3 days) compared with total hip replacement (4.1 days) (1, 44, 45) Due to those advantages, 35% of THA performed between 1998 and 2008 in the US and Europe were MoM implants, and it is estimated that more than a million of them were implanted (15, 46). Unfortunately, adverse reactions started to be reported in some patients, clinically characterized by pain, rash, alopecia and the development of a local benign fibroma described as pseudotumor (47, 48). The dramatic increase in the number of reports of adverse reactions led to the recall of ASR implant (manufactured by DePuy, a subsidiary of Johnson and Johnson) from the market in 2010, and subsequently most manufacturers halted the production of metal on metal implants (49). The failure of the MoM implants has recently been described as one of the biggest disasters in orthopedic history, and the consequences of the adverse reactions in the patients is being observed on a daily basis (49). Another technological improvement to hip prostheses was the development of modularity of the femoral components (Figure 1.1). The possibility of selecting independently the femoral stem and head, and (in some implants design) the femoral neck, allowed the surgeons to give a better-fitted solution in all clinical conditions such as angles and length, allows reduction in the inventory in hospitals and manufacturers, and simplifies revisions since not all parts need to be 5 removed (50). However, it has been widely reported that the junctions between the modular parts experience corrosion due to fretting and/or a galvanic process, which can be source of metal particles or ions responsible of the development of ALTRs (51–55). Picture: Qiong Wang – Felipe Eltit. Figure 1.1 Metal on polyethylene total hip replacement. 6 Picture: Qiong Wang – Felipe Eltit. Figure 1.2 Metal on metal total hip replacement. 7 Picture: Qiong Wang – Felipe Eltit. Figure 1.3 Metal on metal hip resurfacing. 1.2.2 Degradation of materials: wear of the articular surfaces. The wear of UHMWPE liner in total hip arthroplasty was widely described in the 1980-1990s, and the reaction to these wear products was described as “the polyethylene disease”. The development of second generation MoM articulations in the 1990s was supported by in-vitro studies, that demonstrated a lower wear rate in these articulations than in the traditional MoP (56). The observation and measurement of wear in the articulating surfaces represents an important technical challenge for researchers and the industry. The first analysis of wear in MoM were based on the weight changes measured in an analytical balance (gravimetric wear). This method is well suited for in-vitro studies but not for the analysis of retrieved implants functioning 8 in-vivo (due to the fact that the exact initial weight of the implant is unknown). Newer technologies allow to analyze the surfaces wear in retrieved implants by different methods: 1) The measurement of roundness: is a highly precise method (detection level 10 nm), based on bi-dimensional observations of selected planes. 2) The coordinate measuring machine: is a method of surface scanning that can give a three-dimensional representation of the surfaces, but with a lower resolution (1 micron) (57). Equations have also been proposed to estimate the total volume wear on the basis of the more precise bi-dimensional measurements (58). Through gravimetric measurement, after pin on disc test and hip simulators, it was demonstrated that high carbon content alloys (0.25 weight %) experience lower wear than low carbon alloys (0.04 weight %) (56, 59). No differences were observed when comparing wrought with cast processing of the alloys (59). Simulation studies also demonstrated that acetabular inclination increase the wear rate (60), and a lower wear rate was observed in larger femoral heads (61, 62). In a detailed study, Catelas et al. (2003) qualitatively analyzed particles generated in a hip simulator, on three different Co-Cr-Mo alloys used in MoM articulations: wrought high-carbon, wrought low-carbon and cast alloy. By means of TEM-EDX, they observed that the average size of the particles was 50 nm. They also observed that particle size of the high-carbon alloy was smaller than the other two alloys. They described two types of particles generated in the simulator: one composed presumably by Cr oxides, which are round shaped, and another type with metallic composition (Co-Cr-Mo) with variable morphology from oval to needle-shaped (63). Despite the promising performance of MoM articulation in-vitro, analysis of failed MoM implants demonstrated the presence of wear in the femoral head and in the acetabular cup (64). Wear is higher in edge-loaded implants, which is a consequence of excessive acetabular inclination (65); moreover, a higher degree of wear of the articulating surfaces has been found in implants that failed due to ALTRs than well-functioning implants (66). The process of metal release from the articulating surfaces has been defined as tribocorrosion, or the combined effect of wear and corrosion. Wear removes the passivated layer of the articulating surface, enhancing the corrosion process in this newly exposed surface (67, 68). It was proposed that metals released through this mechanism are responsible of triggering ALTR 9 in periprosthetic tissues, which was supported by the original description of ALTRs in MoM total hip arthroplasty and MoM resurfacing systems (47, 69). Nevertheless, the later findings of ALTRs in MoP THA, and even in metal unipolar systems, suggests that other mechanisms of metal release are involved in the development of ALTRs (6, 70, 71). 1.2.3 Degradation of materials: corrosion at the tapered modular junctions. The alloys used for orthopedic implants, to prevent corrosion, rely on the formation of a passive film of metal oxides. Materials with more dense and homogeneous passive layer are better in preventing this electrochemical degradation. Titanium and Co-Cr-Mo alloys form a very consistent passive layer, and for that reason, they are the most used metal alloys for orthopedic implants in the present (72). Studies “in-vitro” suggested that the tapered modular junctions composed of Ti-Al-V and Co-Cr-Mo alloys, would not experience corrosion when they contact each other and that they are corrosion-resistant under physiologically simulated conditions (73, 74). In spite of that, the presence of corrosion products in the junction of retrieved modular metal on polyethylene implants was widely reported in the 1990s (75–77). This corrosion process that seems unlikely to happen in-vitro, maybe possible in-vivo due to the effect of mechanical factors such as fretting or micro-motion that may cause the abrasion or fracture of the passive film (78). The effect of fretting is also enhanced by crevice corrosion, which is characteristic of geometrical clearances such as the modular interfaces of hip implants. Immediately after the disruption of the passive layer, oxygen from the body fluid interacts with the exposed metal, forming a new passive layer in the surface of the implant, protecting the metal surface. Within the narrow clearance of the crevice, the oxygen becomes depleted after each re-passivation event, thus retarding the formation of a passive layer, and the metal stay exposed for longer periods of time, increasing the corrosion rate (79). An analysis of corrosion in retrieved modular components of eighty-eight non-cemented metal-on-polyethylene hip implants, was published in 1992 by Collier et al. (76). They divided the implants into three groups: The Co-Cr-Mo alloy head over a Co-Cr-Mo alloy stem, the Ti-Al-V alloy head over Ti-Al-V alloy stems and a group of mixed components of Co-Cr-Mo head and Ti-Al-V stem. They observed that none of the Co-Cr-Mo head over Co-Cr-Mo stems and Ti-Al-V 10 over Ti-Al-V systems presented evidence of corrosion. However, more than 50% of the Co-Cr-Mo alloy head over a Ti-Al-V alloy stem present evidence of corrosion. They proposed then that galvanic corrosion was responsible for the corrosion of modular hip prostheses (76). This hypothesis was promptly disproved after the analysis of one hundred and six mixed-metal head-neck tappers, composed of Ti-Al-V femoral stems and Co-Cr-Mo femoral heads, and forty-two similar alloy (Co-Cr-Mo) systems, that was carried out in the Rush University Medical Center. This study showed that both metal combinations underwent equivalent corrosion process (80). Based on the morphological analysis (SEM) of the articulating surfaces, they proposed that six different manifestation of corrosion were evident in the modular components surfaces: etching, fretting, pitting, selective leaching, interfacial layer formation and intergranular attack. They proposed a combined effect of fluids and fretting as the mechanism of corrosion (mechanically assisted corrosion). The same group in Rush university, led by Dr. Jacobs (1995), studied the morphology of the corrosion products and the chemical composition of them through secondary electron, backscattered-electron imaging and energy dispersive X-ray analysis. The results of the analysis demonstrated that the corrosion compounds had a differential topographical location. Chromium oxides and chlorides were found in the area of contact between head and neck, but chromium orthophosphate was found mainly at the rim of the head bore and on the neck just distal to the head-neck junction (81). Another study of the same group in 1997 described that in the interfacial layer, the products corresponded to mixed crystalline oxides of Cr, Mo and Ti, while in the open crevice, the products were identified mostly as amorphous hydrated chromium orthophosphate. Interestingly, minimal cobalt was found in the corrosion particles on the implant surface or the tissues (82). These observations were later confirmed by other researchers (83, 84). 1.3 Products of material degradation in human tissues Elevated concentration of metal particles and ions in the body released by hip implants could have serious effects in the human body, affecting nervous, renal and cardiovascular functions, with even fatal consequences (85, 86). Although rare episodes, these events illustrate the degree of toxicity that metals used for orthopedic implants can achieve, and thus the long-term effects of these elements are subject of study. 11 More common alterations associated with the metals released from the hip replacements are the local reactions that occur in the joint tissues. The presence of polyethylene particles was indicated as the cause of osteolysis and inflammation in the periprosthetic tissues (87). However, due to the fact that ALTRs are significantly more prevalent in MoM than MoP, polyethylene is not considered as a potential cause of ALTRs, and thus will not be analyzed in the following sub-chapters. 1.3.1 Metal particles: The presence of metal particles in periprosthetic tissues has been widely studied, and many reports have associated the presence of metal particles in the periprosthetic tissues with implant failure (88–90). Early studies in 1970s demonstrated the presence of metal particles in the periprosthetic tissues of MoP and MoM implants (either stainless steel or Co-Cr-Mo alloy) (91, 92). Later, through the observations in SEM-EDX the presence of Co-Cr-Mo alloy particles was described in the tissues of the hip joint (92). The histological examination led the researchers to propose that inflammation produced as a foreign body response to metal particles could be a cause of implant failure (91). After the introduction of the second-generation MoM hip replacements in the 1990s, studies promptly confirmed the presence of metal particles associated to MoM THA and hip resurfacing (84, 88, 93). Those studies also proposed that an immunologic reaction to metal debris would be responsible of the aseptic loosening on the hip replacements. In the study of Huber et al. (2009), the SEM-EDX analysis of the particles in the histological sections showed peaks of Cr, P and O, but no Co (the most abundant element of the implant’s alloy). FTIR analysis suggested that a combination of chromium and iron phosphates is the main composition of these particles (84). Using Synchrotron X-ray fluorescence (XRF) and X-ray absorption spectroscopy (XAS) to analyze tissue sections, Hart et al. (2010), described that Co distributes homogenously along the tissue samples, while Cr generate peaks in specific areas (particles). The peaks of Cr were found in areas that histologically are infiltrated by macrophages. XAS analysis revealed that the particles are compatibles with CrPO4 4H2O, and a second form of particle, composed of a 3:1 mix of metallic Co-Cr-Mo and CrPO4 4H2O (94). In a later study, using TEM-EDX in sections of 12 periprosthetic tissues, the presence of 20-200 nm particles with Cr peaks, but not Co in intracellular compartments of macrophages was found (95). Tridimensional reconstruction after dual beam slice in scanning-TEM (STEM) and scanning transmission x-ray microscopy (STEM)-EELS, demonstrated that the metal particles were fully internalized in vesicles in the tissue macrophages, and they have a broad size distribution ranging from nanometers to micrometers (96). The same study determined the presence of two different types of particles: Cr (III) rich particles with minimum Co, and particles composed by oxidized Cr with metallic Co-Cr core. The evidence of two types of particles with different composition has also been histologically described, and it has been proposed that metal particles are products of wear, while the metal oxides are a-product of corrosion of metal implants (97, 98). The synovial fluid is the anatomical connection between the implant’s surfaces, and the synovial tissues, and it corresponds to a transit zone for the particles and ions. By use of TEM-EDX, and Parallel Electron Energy Loss Spectroscopy (EELS), it has been shown that nano-sized particles in the synovial fluid are chromium rich, with no cobalt and amorphous structure, but larger particles (micron size) are cobalt rich and crystalline (99). The oxidation state of the particles is Cr (III), with the exception of the smallest non-crystalline particles that seems to be formed by a mix of Cr (III) and Cr (IV) (99). In summary, the existent evidence shows that wear particles seem to have the same composition than the original metal, but with some oxidation process undergoing in their surface, they are micron or submicron size and have crystalline structure. Corrosion particles are nano-sized and amorphous, composed of Cr oxides and phosphates, while Cobalt is absent. Both types of particulate material have been observed in synovial fluid and the periprosthetic tissues. 1.3.2 Metal ions: The concentration of metals in blood has been extensively studied with the aim of establishing a simple method to determine potential implant malfunction. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was developed in the 1980’s, and introduced to trace element measurement in body fluids in the 2000, allowing a robust method of analysis that is now considered the gold standard in the measurement of ions in serum of patients with metal implants 13 (100). Due to the scientific evidence, the concentration of Co and Cr in serum is considered a valid tool to screen the performance of MoM hip replacements. Furthermore, Medicines & Healthcare Products Regulatory Agency (MHRA, UK) issued an alert in 2015 regarding the Birmingham MoM hip implants. They recommend monitoring the metal concentration in blood, establishing the maximum threshold for patients with MoM implants to be 7 ppb of Co or Cr, as an indicator of potential development of soft tissue reaction (17). Similarly, in a consensus statement, the American Association of Hip and Knee Surgeons, the American Academy of Orthopedic Surgeons, and The Hip Society, recommended the systematic evaluation of patients with dual modular neck systems, including the analysis of serum ion levels together with clinical and radiological evaluation to optimize the management of these patients (101). The report acknowledged that the increases in Co and Cr in patients with MoP dual modular neck implants is lower than the observed in MoM articulations, and noted a shift in the Co/Cr ratio. To the best of our knowledge, there are no alerts regarding metal or serum ion levels in MoP systems without a modular neck. Elevated metal ion concentrations in the blood of patients with MoM articulations has been widely reported in the past 20 years. High concentrations of Co and Cr in patients with MoM articulation than those with MoP articulation were described just a few years after the introduction of MoM systems (34, 102, 103). It has also been demonstrated that there is a higher concentration of metals in serum of patients with a higher inclination of the acetabular cup (104). Studies have been contradictory when comparing total hip arthroplasty (THA) with hip resurfacing systems. Moroni et al. (2007) found no differences between a group of 48 mm head resurfacing and a 28 mm head total hip arthroplasty (105), and similar results were described later by Venditolli et al (2010) (106). However, Garbuz et al (2010) found significantly higher values in large head THA than HR (100). They explained that the presence of adaptor sleeve in large head THA introduce a second tapper junction, combined with a larger wear surface increase the release of ions from the implants surfaces. These observations were later confirmed by the analysis of 144 patients with different implant models. The group with the lower values of Co and Cr was the group that used Biomet system, which uses a Ti sleeve, thus offering only one surface for Co-Cr-Mo fretting (107). Another interesting observation is that there is a different Co/Cr proportion in serum of patients with THA and HR, being Co relatively lower in patients with HR compared with THA. This can 14 be explained by the different mechanisms of ion release from the different systems, since HR experiences wear in the articulating surfaces, but minimum corrosion since it does not have modular junctions, THA experience both processes, wear of the articulating surfaces and corrosion of the head-neck junction. The concentration of metals in the synovial fluid should be critical in the development of ALTRs; however, there are limited studies that have reported concentration of metals in synovial fluid, and only a few of them have tried to associate the metal ions levels to implant failure. A study published by De Smet et al. (2008) showed that the levels in synovial fluid are at least one order of magnitude higher than the observed in serum, and the concentration of Cr and Co in patients with metallosis are 20 and 50 times higher than those with well-functioning implants respectively (108). In their report, they also proposed a correlation between the ion levels in synovial fluid and the levels in serum, however due to the limited sample number in the study (n = 26), strong conclusions are lacking. A more extensive analysis of metal presence in synovial fluid was published in 2010 by a group led by Allister Hart and John Skinner in UK. They analyzed synovial fluid of 92 consecutive patients that underwent revision of MoM hip replacement systems (83 resurfacing, 9 THA), due to unexplained pain. They observed high levels of cobalt and chromium in the patients with failed implants, and found no association between the metal concentration in synovial fluid and clinical variables as implant size, manufacturer, acetabular inclination and concentration of metals in serum (109). In summary, the concentration of Co and Cr in blood is accepted to be a useful parameter to monitor implant performance; however, it has not been possible to establish a relationship between the concentration of metal ions in serum and synovial fluid, which would represent the local environment where ALTRs develop. No studies have associated the presence of ALTRs with the ion concentrations in the synovial fluid. 1.4 Adverse local tissue reactions to metal implants ALTRs have been histologically described as the growth of cystic or solid fibrotic masses originated in the synovial membrane of patients with hip implants (110–114). ALTRs generate 15 pain and discomfort, as well as pressure effects on vein and nerves, but some can also be asymptomatic (115, 116). Different terms have been used to describe lesions associated to hip replacements systems, the most common being: “Pseudotumor”, “Adverse Local Tissue Reactions (ALTR)”, and “Aseptic Lymphocyte Vasculitis Associated Lesions (ALVAL)”. These denominations were primarily used for the pathologies associated with MoM implants, but similar reactions occur in MoP implants(6). 1.4.1 Nomenclature: The clinical name “Pseudotumor" was used in the 80s to describe soft tissue growth in a MoP implants (117), it was thought to be a reaction to the acrylic cement of the femoral components, but were later observed in non-cemented systems (118). The same term was reincorporated for the lesions found in second generation MoM implants in the study of Pandit et al. (2008) (47). Pseudotumor refers to the Greek pseudo = false + tumor = increase in volume. The actual pathology observed surrounding the implants correspond to a true volume increase, but since the term “tumor” has been associated to neoplasms, the prefix pseudo was added to reduce concerns on the malignancy of these inflammatory lesions. Pseudotumor then, correspond to a clinical and imaging description of a volume growth associated to a hip replacement system. The term ALVAL (Aseptic Lymphocyte-dominated Vasculitis-Associated Lesion), is considered to be introduced by Campbell et al. (2010) (119), although the lesion of the blood vessels (Vasculitis) was previously described by Willert et al. (2005) (93). They introduced a method of quantifying the severity of the lesions based on the histological observations (ALVAL score), which is now being used in most of the studies to describe the severity of the lesions around hip implants. The term ALVAL corresponds to the histological description of the lesions that are found in the clinically described “pseudotumors”. Adverse Reaction to Metal Debris (ARMD) is a term used to define the pathology associated with MoM implants; however, this term will not be used in this thesis, because it suggests that metal debris are the main cause of the adverse reactions, which is still being researched. Adverse Local Tissue Reactions is a term that includes a broad spectrum of pathological changes due to the presence of hip replacements. It was used in the 70s and 80s to describe the pathology in the tissues surrounding hip arthroplasty (120). It was reintroduced to the failure of second generation metal on metal hip prosthesis in a literature review by Carli et al. 16 (2011) (121), and is currently the most used term to define aseptic periprostehtic lesions of the hip (6, 97). 1.4.2 Histological description of ALTRs: Histologically, ALTRs are described as solid masses or cystic cavities, in which the ulceration of the synovial surface with or without fibrin deposition, is accopmapied by subsuperficial necrosis, mononuclear cell infiltration and variable number of eosinophils and giant multinucleated cells, in a thickened synovial membrane composed of dense connective tissue (48, 97, 111). It is accepted that, as other reactions to foreign bodies, the immune system plays a critical role in the development of ALTRs. It has been described the presence of a difuse infiltrate of T cells with an equivalent number of CD4+ and CD8+, and few plasma cells or eosinophils (48). The presence of perivascular mononuclear aggregates, composed of T (CD3+) and B (CD20+) cells has been descrived as a characteristic of high-grade ALTRs (48, 111). By the use of immunohistochemistry, a high level of organization in which the B cells are located in the central zones surrounded by the T cells has been described (122). Because of the cell organization, and molecular interactions between cells it has been proposed that they correspond to tertiary lymphoid organs, similar to those observed in peripheral organs with chronic inflammation (122, 123). The presence of macrophages in the tissues has also been described, and due to their phagocytic function, the assocaition between them and metal particles has been studied. The presence of metal particles inside the macrophages has been determined either by OM or by TEM (84, 95), and a direct relationship between the amount of wear and the number of macrophages in the tissue has been established (119). TEM observations of cells within the tissues have shown that in living macrophages the particles are inside of phagosomes, but when the cells die, the membranes disintegrate and the particles are released. No particles have been observed in fibroblasts, and their morphological changes suggests that an apoptotic process is the cause of the disappearance of these cells in the necrotic zones (95). 17 1.4.3 Etiology and pathogenesis of ALTRs: The role of the degradation products (both metal ions and solid particles) released from the implants as a consequence of corrosion or wear have been widely accepted as etiological factors (13, 16). However, studies have failed in correlating the amount of wear or corrosion in the implants with the histological and clinical observations (124). The most accepted hypothesis is that ALTRs correspond to a type IV or delayed hypersensitivity reaction triggered by metals (48, 84, 125). Type IV hypersensitivity corresponds to the harmful effects of the cell-mediated immune response, in which the generated damage by the immune system is exaggerated in comparison to the damage that the etiological agent may produce. In some cases, type IV reactions are related to chronic infections (such as tuberculosis). Cytokines secreted by T cells induce macrophages to fuse and form multinucleated cells around the damaged area (granuloma), leading to tissue fibrosis and necrosis (126). All these features (granuloma, fibrosis, necrosis, multinucleated cells and T-cell infiltration) are found in ALTRs (97, 98, 111, 119), giving strong support to this theory. However, how this reaction is triggered in the ALTR, and the role of the metal components in the process has not been clarified so far. It is not clear how metals can induce hypersensitivity, but a molecular mechanism by which metals ions induce T-cell mediated hypersensitivity (type IV) has been recently described for beryllium. The ion conjugates with part of the HLA-peptide complex (HLA-DP2), generating structural changes in the molecule, with this modified-HLA molecule being recognized by the T-cell receptor, triggering the immune reaction. In beryllium hypersensitivity, this process triggers a disease that generates severe lung destruction due to chronic inflammation in 1-15 % of people exposed to beryllium (127–129). This pathway may also be part of the mechanisms of immunogenicity of the metals from the implants; however, studies have failed to demonstrate association between metal hypersensitivity (allergy) and implant loosening (130). The study of lymphocyte activation in the presence of cobalt and chromium showed no difference between patients with failed MoM implants and the control group, which suggests that metal hypersensitivity should not play a role in the development of ALTRs (131). These observations together with the high prevalence of ALTR in MoM but lower in MoP systems, suggests that a complex process involving different mechanisms such as cellular damage induced by metal ions, 18 cell death, and immunogenicity of metal ions could be involved in this reaction (132). 1.4.4 Hypersensitivity as an event in ALTRs: The term “hypersensitivity” defines a subset of pathologies in which the damage generated by the immune response is exaggerated in relation to the potential harm generated by a pathogen. This term can be misleading since some of the pathology associated to hypersensitivity could be developed by a completely normal immunological response (126). Classically, it is accepted that there are four mechanisms of hypersensitivity: Type I or allergies, are reactions mediated by immunoglobulin E. Type II are antibody (Immunoglobulin G or M) – mediated reactions to tissue-fixed antigens. Type III are responses to inadequate circulating antibody – antigen complexes. Type IV or cell – mediated are due to sensitized T Cells (126, 133) (Table 1.1). Type IV hypersensitivity is thought to be involved in the development of ALTRs. It is composed by two simultaneous types: 1) Delayed hypersensitivity, initiated by CD4+ T cells. 2) T-cell mediated cytotoxicity, generated by CD8+ T cells. Delayed hypersensitivities (CD4+ mediated) begin with the activation of CD4+ T cells, that recognizes antigens with their receptors (TCR) during the antigen-presentation by antigen presenting cells. This process generates a subtype of memory CD4+ cells that remain in circulation for years, mostly with a Th1 phenotype. After subsequent encounters with the antigen, the “sensitized” CD4+ cells respond by secreting cytokines, with the most relevant in this reaction being: IL-12, IFN-, IL-2 and TNF- (126). Granuloma is a special form of delayed hypersensitivity that develops in the presence of non-degradable antigens. The CD4+ T cell infiltrate is progressively replaced by macrophages that became rapidly activated. The active macrophages adopt a flat shaped morphology (epithelioid cells) and are surrounded by lymphocytes (134). The macrophages also could fuse to form giant multinucleated cells in an attempt to phagocytose large antigens (135). Most of the models for 19 granuloma formation include the activation of a subset of CD4+ cells called Th17. These Th17 cells secrete large amounts of IL-17 and IL-22 in addition to IFN- (). Table 1.1. Classification of hypersensitivity reactions. Type Immune mechanism Prototype disorder I Anaphylactic (immediate) Allergen cross – link IG-E antibody. This activates mast cells to secrete heparin and histamine, which generates vasodilation, vascular leakage leading to edema, mucus secretion and muscle contraction. Pollen allergy, asthma, anaphylactic reaction to drugs. II Antibody to tissue antigen Antibody (Immunoglobulin G or M) binds antigen on cell surface. Antibodies on cell surface elicit phagocytosis by macrophages, and complement activation, producing tissue destruction. Hemolitic anemia, Pemphigus vulgaris. III High level of circulating antigen-antibody complex Antigen – antibody complexes in circulation activate complement, neutrophils release of oxygen radicals producing vascular damage. Systemic lupus, Glomerulonephritis. IV Cell mediated (Delayed) T cells recognizes antigens. The activation of T cells induces the secretion of cytokines mediating the immune response. Activated CD8+ T cells destroy cells and tissues. Tuberculosis, contact dermatitis, transplant rejection. T-cell mediated cytotoxicity is a form of type IV hypersensitivity in which sensitized CD8+ T cells destroy antigen-bearing cells in the tissues. CD8+ cells play a critical role in the response to virus or other intracellular pathogens. Two principal mechanisms of cell elimination have been described in CD8+ lymphocytes: 1) the expression of FAS-L, which is recognized by the cell receptor FAS as a death signal, triggering apoptosis in the affected cell. 2) the secretion of perforins, proteins that polymerize in the cells membrane, generating pores in the cell surfaces, generating a water flux into the cell, which produces osmotic lysis (126, 136). 20 Delayed hypersensitivity has been proposed as the mechanisms of ALTRs (84, 119, 137). This hypothesis has been supported mostly by the histological descriptions of ALTRs, in which the described elements of type IV hypersensitivity reaction are present, such as CD4+ and CD8+ T cell infiltration, granuloma, giant multinucleated cells. However, none of the previously described molecular mechanisms have been demonstrated to occur in ALTRs. Moreover, Kwon et al (2010) did not find an increased sensitivity to Co and Cr in lymphocytes from patients with ALTRs compared to a control group of patients with implants but not ALTRs (131). Metadata analysis demonstrated that the frequency of positive metal sensitivity test increases in patients that receive a hip replacement, and the increase is even higher in patients with failed implants. However, hypersensitivity testing was not able to discriminate between stable and failed joints since it has no predictive value for ALTRs (130). In conclusion, despite being mostly accepted that delayed hypersensitivity plays a role in the pathogenesis of ALTRs, the mechanisms are not clear, and it has not been proven how this immunological reaction can be triggered or activated by the presence of metal hip replacements. 1.4.5 Tertiary lymphoid structures and lymphocyte activation in ALTRs: One of the histological characteristics of ALTRs is the presence of perivascular mononuclear infiltrates that can organize in tertiary lymphoid structures (TLOs) (122). These structures are observed in 10 to 30 % of ALTRs (97), and have been classically associated with a higher degree of inflammation (114, 119). Tertiary lymphoid structures are accumulations of lymphocytes that arise in non-lymphoid organs during chronic inflammation through a process called “lymphoid neogenesis”. TLOs are composed of densely-grouped T and B cells with a high degree of organization, and are considered to be equivalent to secondary lymphoid organs. They can be observed in different types of chronic inflammation diseases, originated by viral or bacterial infections, autoimmune diseases, smoking, environmental conditions, idiopathic fibrosis, and cancer (138–143). TLOs differentiate from secondary lymphoid organs in the fact that they develop in response to a pathologic event and are not constitutive of the human organism, whose function, organization and signaling are considered 21 to be equivalent (144–147). TLOs are considered “an adaptation of the body to an increased demand for a localized immune response” (123). The role of TLOs in disease progression could be beneficial or detrimental, for instance they have been shown to play an important role in the antitumor response in cancer, but enhance the auto-immune response in rheumatoid arthritis (148, 149). Despite their critical role in disease, the function of TLOs in ALTRs remain unknown, as well as their characterization in terms of the type of immune reaction that they may drive in the periporsthetic tissues of patients with hip replacements. 1.4.6 Etiology of ALTRs, the effect of Co and Cr in cell populations: The role of cobalt and chromium products in triggering the cellular phenomena observed in ALTRs such as inflammation, cell death and fibrosis, has been partially demonstrated. Studies carried out at the Rizzolli Orthopaedic Institute (Bologna, Italy) have shown that high concentrations of Co ions generate necrosis in human peripheral mononuclear cells, while lower concentrations induce apoptosis in the same cell population. On the other hand, Cr ions at high concentrations induce apoptosis in the cells and no effects are observed in lower concentrations (150). The same group also reported that peripheral monocytes respond to the exposure of ion extracts of metal particles by secreting cytokines such as TNF-, IL-1, IL-6, sIL-2R, IFN-, and GM-CSF (150). Another study in macrophage-type cells, but this time a cell line (J774), showed that after 24 hours of exposure to elevated cobalt and chromium concentrations the cell death is due to apoptosis, but after 48 hour of exposure, the cells experience necrosis (151). The same group, in a later report by Catelas et al. (2005) showed that chromium ions need higher concentrations than cobalt (250 ppm vs 8 ppm) to generate similar effects in terms of necrosis or apoptosis of the J774 cells, suggesting that a high concentration of these ions "in-vivo” could be responsible of the cell death in the tissues. Co and Cr particles have been demonstrated to have toxic effects for human cells. Studies in keratinocytes (HaCaT cells), cultured with different concentration of nanoparticles of CrO and 22 Cr-Cl, showed an increase in cell death due to apoptosis when the amount of particles is increased (152). Another study showed that nanoparticles of chromium oxide can induce apoptosis in a macrophage cell line (J774), but the secretion of cytokines after the exposure is low. The authors proposed that this should not be the triggering mechanism of ALTRs (153). Low cytokine secretion was also observed when nanoparticles of Co-Cr-Mo alloy were used in a macrophage cell line (U937), but an increase in cytokine secretion was observed if the cells were previously exposed to Co ions (154). A study using dermal fibroblast showed that nanoparticles of Co-Cr-Mo alloy produce a higher rate of cell death than a similar volume of micron-size particles, perhaps because the higher surface of the nano-sized particles generate a higher ion release. They also showed a lack of cytokine response (IL-6) to the metal particles (155). This lack of secretion of Il-6 was previously reported for fibroblasts obtained from the knee ligaments (156). In summary, the fact that the metals can cause deleterious effects in different cell populations is well accepted, but the mechanisms of cell toxicity of metals are not totally understood. It has been demonstrated that metals can increase the oxidative stress of cells, by inhibiting enzymes of mitochondrial respiration. Recent studies have demonstrated that those ions can bind proteins and enzymes that are essential for cell function (152, 157). 23 1.5 Research questions The numerous studies that in the past 10 years have been published regarding the problems related to aseptic hip implant failure, had epidemiologically and clinically described the problem. Studies had also give some answers to the metallurgical, biological and clinical problems that are related to ALTRs. However, there are many questions still unsolved about the etiology and pathogenesis of ALTRs: What is the etiological factor of ALTRs? Why do ALTRs happen? Why do some patients develop ALTRs and others do not? Is the concentration of metal ions or particles relevant in triggering the inflammatory process? What are the pathogenic processes that participate in the development of the lesions? Are the processes of ALTRs similar in different types of implants? Is the necrosis of the tissue originated by the metal ions or by hypoxia? What triggers the fibrosis that is described in the hip capsule? What is the role of the immune system in the pathogenic events of ALTRs? Some of these questions motivated this work, and will be answered in this thesis that will focus on the etiology and pathogenesis of ALTRs. The studies previously analyzed in the literature review presented in the precedent sub-chapters, suggests that fibroblasts are not capable of triggering an inflammatory reaction through the secretion of cytokines after exposure to metals, and that macrophages do not significantly increase the secretion of cytokines after being exposed to metal particles. However, the above-mentioned studies have not used synovial fibroblasts from patients with previous episodes of osteoarthritis, which is the actual condition of the patients with hip replacements (that may enhance the secretion of cytokines). Fibroblasts are the major cell population in the hip capsule and are the most exposed to metals in the hip joint. Fibroblasts also seems to be a key participant in the ALTRs pathogenesis, since all the histological descriptions include an extensive fibrosis in the hip joint. Thus, is valid to ask: what is the role of fibroblasts in the etiology of ALTRs? How are fibroblasts affected by metals released from the hip implants? The following chapters will describe the studies driven to answer these questions. 24 Chapter 2: Hypothesis and aims. ALTRs are one of the main causes of the failure of hip implants. The association of metals originating from the implants and the presence of ALTRs is widely accepted, but the mechanisms behind the development of ALTRs are poorly understood. And questions such as: What is the role of metal elements in the development of ALTRs? What type of immunological response develops in ALTRs? Is the immunological reaction triggered by metal ions or particles? Is it a hypersensitivity reaction? What are the biological mechanisms in the adverse reaction to metal implants? have not been successfully answered thus far, and it is therefore the aim of this thesis to provide evidence for answering these questions. The aim of the work presented in this thesis was to test the hypothesis that metal elements released from hip implants are responsible for the development of ALTRs. This process is started by the cellular damage that is generated in the cells of the hip joint, which through the secretion of specific cytokines and chemokines, triggers the migration and activation of cells of the immune system, leading to chronic inflammation and tissue necrosis. To test this hypothesis, three studies were conducted: 1) Histological analysis of adverse local tissue reaction in MoM and MoP hip replacements (chapter 3): The aim of this study was to analyze the morphological characteristics of MoM and MoP ALTRs tissues. This study provided critical information on the cell populations in ALTR lesions, and this knowledge drove the mechanistic study presented in this thesis (chapter 5). 2) Determination of the presence of corrosion products in tissues and body fluids of patients with hip replacements (chapter 4): The qualitative and quantitative analysis of ions and metal particles in the synovial fluid, serum and ALTR tissues, aimed to identify the elements (ions 25 or particles) that were associated with ALTRs. The analysis conducted in this study presented critical information that was used in the cell culture study (chapter 5), in which we aimed to determine the pathologic process in the development of ALTRs. 3) Pathologic mechanisms of adverse local tissue reactions (Chapter 5): Gene-expression analysis of cells of the immune system in ALTRs (described in chapter 3), as well as proteomic analysis of the synovial fluid of patients with ALTRs, were used to determine the type of immunological reaction in ALTRs. Finally, a cell culture study was performed, where cells from the hip joint were exposed to metal ions (as described in chapter 4). The subsequent cellular and immunological reaction was analyzed to prove the hypothesis that the presence of these metals is able to trigger the development of ALTRs. Three studies corresponding to the above-mentioned aims are described in the following chapters (chapters 3-5). They are presented with the description of the methodology, results and discussion. A final discussion and conclusions for the whole project are presented as an independent chapter (chapter 6). 26 Chapter 3: Histological description of ALTRs in Metal on Metal and Metal on polyethylene hip replacement systems. 3.1 Rationale: The traditional histological description of ALTRs includes the ulceration of the synovial surface with or without fibrin deposition, various degrees of necrosis, and mononuclear cell infiltration in a thickened synovial membrane (97, 111). However most descriptive studies correspond to ALTRs associated with MoM implants, and only a few case reports on ALTRs in MoP systems have been published so far (6, 158–160). A recent comparative histologic analysis between large head MoM THA, MoM hip resurfacing and dual modular non-MoM systems, showed that the ALTRs have a common spectrum of events. The same study demonstrated that some differences were observed associated to different implant types, which may be indicative of differences in the biological response to the different implant designs (97). Previous studies have revealed that similar types of reactions are observed between metal on metal total hip arthroplasty and hip resurfacing (10, 119), and no systematic studies have been described in non-modular neck MoP. The aim of this study was to describe the histological characteristics of ALTRs in MoM and non-modular neck MoP and compare them with control cases (patients with implants but no ALTRs), through morphological analysis and immunohistochemistry. Additionally, a pilot study on the presence of inflammatory cytokines in the tissues was performed in order to confirm the similarities or differences in the pathogenesis of ALTRs associated to MoM and MoP total hip arthroplasty. 3.2 Patients and Methods With approval of the ethics board of the University of British Columbia, 34 cases of ALTR associated with total hip arthroplasty (THA) were submitted for analysis. These included 17 patients following revision of MoM THA (8 females, mean age 69.1 years old, mean implantation 27 time 59 months) and 17 patients who had undergone revision of non-modular MoP THA (10 females, mean age 69.9 years old, mean implantation time 48 months). During the period 2012-2015, sixty-two revision operations dues to ALTRs were performed at Vancouver General Hospital (37 MoM, 25 MoP). We only included patients in whom a diagnosis of ALTR was made preoperatively and an informed consent for participating in the study was obtained preoperatively. The implant types are shown in detail in Table 3.1. The reasons for revision and the diagnostic imaging used in each patient are shown in Table 3.2. Ultrasound was used as a routine screening examination in symptomatic patients, since it has similar sensitivity and specificity for the diagnosis of ALTRs as MARS MRI, with advantages in cost and availability (Fig. 3.1) (161, 162). In all the cases, an experienced pathologist confirmed the presence of ALTR. Patients with evidence of bone resorption were excluded, since it has been demonstrated that osteolysis is associated with different gene expression than ALTRs, and thus may correspond to a different pathology (163). Patients with infection or previous revision operations were also excluded. A metal allergy was not noted in any of the patients, as is a contraindication for a metal femoral head. 28 Table 3.1Hip replacement systems analyzed in the study. All MoM THA systems included the use of a Co-28Cr-6Mo adaptor between the femoral stem and head none of the MoP systems had an adaptor sleeve. All femoral heads are composed of forged high carbon Co-28Cr-6Mo alloy, all femoral stems are composed of forged titanium alloy. System Manufacturer Stem Cup Head Head Diameter (mm) No MoM (17) Zimmer ML Durom Metazul 42 5 44 2 46 2 48 2 50 4 54 2 MoP (17) Zimmer (10) ML Trilogy Versys 28 1 32 2 Versys Trilogy Versys 32 2 36 5 Stryker (7) Accolade Trident Trident 28 2 32 3 Accolade Tritanium Tritanium 32 2 29 Table 3.2 Chief complaint, diagnostic images and volume of ALTR in MoM and MoP total hip replacements. Case Symptomatology Implantation time (months) Diagnostic images Appearance Size of lesions in ultra sound (cm) Tissue volume (mL) MoM 1 Pain 52.3 US + MRI Cystic 7.6 x 4.8 x 3.3 83 MoM 2 Pain 63.2 US + MRI Cystic 6.5 x 4.3 x 5 105 MoM 3 Discomfort and alopecia 75.2 US + MRI Cystic 2.6 x 2.2 x 6.5 101 MoM 4 Pain 63.8 US + MRI Cystic 4 x 1 x 1 1 MoM 5 Pain 71.0 US + MRI Cystic 11.7 x 5.5 x 4 121 MoM 6 Pain 63.0 US Solid 7 x 3 x 3.1 32 MoM 7 Asymptomatic 61.6 US Mixed 10.9 x 4.8 x 3.4 100 MoM 8 Pain 67.7 US + CT Cystic 9 x 4.3 x 2.1 69 MoM 9 Pain 45.2 US + MRI Cystic 14.2 x 3.7 x 6.5 143 MoM 10 Pain 60.1 US + MRI Mixed 6.8 x 3.6 x 6.2 35 MoM 11 Pain 89.3 US Cystic 3.5 x 1.3 x 6.1 50 MoM 12 Pain 95.4 US No evidence 17 MoM 13 Asymptomatic 94.0 US Cystic 4.9 x 3.7 x 1.6 + 7.7 x 3.6 x 2.4 9 MoM 14 Pain 85.9 US Mixed 8.8 x 7.8 x 5.1 280 MoM 15 Pain 84.0 US + MRI Cystic 3 x 5.5 x 12 245 MoM 16 Discomfort 70.0 US Mixed 8.8 x 4.2 x 3 143 MoM 17 Pain 74.3 US Cystic 5.6 x 5.2 x 2.8 10 Mean MoM 71.5 91 Case Symptomatology Implantation time (months) Diagnostic images Appearance Size of lesions in ultra sound (cm) Tissue volume (mL) MoP 1 Pain and stiffness 33.6 CT Mixed 225 MoP 2 Pain 42.0 US Solid 11.2 x 4.1 x 6.2 563 MoP 3 Dislocation 39.4 X- Ray No evidence 91 MoP 4 Dislocation 77.6 X- Ray No evidence 52 MoP 5 Dislocation (Rec) 57.0 X- Ray No evidence 40 MoP 6 Pain 9.6 US Solid 9 x 10 x 6 462 MoP 7 Pain 34.2 MRI Solid 4.9 x 8 x 4.3 498 MoP 8 Pain 27.5 MRI Solid 5.5 x 4.8 x 3 60 MoP 9 Pain 38.0 MRI Solid 11.5 x 7.3 x 4.2 344 MoP 10 Pain 38.3 US Solid 12.4 x 8.5 x 3.1 22 30 MoP 11 Pain 54.8 US Mixed 13 x 5 x 3 35 MoP 12 Dislocation (Rec) 65.4 X- Ray No evidence 266 MoP 13 Pain 39.9 US Solid 7.4 x 7.4 x 5.9 552 MoP 14 Pain 37.5 US + MRI Solid 3 x 4 x14 405 MoP 15 Pain 47.3 US Solid 7 x 6 x 6 81 MoP 16 Pain 61.3 US + MRI Solid 8.5 x 3.9 x 4.6 + 5.7 x 1.3 x 8.3 172 MoP 17 Pain 44.7 MRI Mixed 17.5 x 9.5 x 8.1 1204 Mean MoP 44.0 242 Figure 3.1 Characteristic ultrasound images of ALTRs. A) Cystic lesion in MoM total hip arthroplasty. B) Solid mass in MoP hip implant. With the aim of comparing the ALTR patients, with a baseline of MoM patients without ALTR, we selected a control group of 9 patients (3 female, mean age 57.9 years old, mean implantation time 75.7 months) who underwent revision surgery of MoM implants for reason other than ALTR (2 cup loosening, 2 extra capsular fractures, 3 recurrent dislocation, and 2 concerned about the presence of MoM THA). The clinical evaluation, ultrasound and histopathology of those patients did not show the presence of ALTR. 31 3.2.1 Histology and scoring. Five tissue specimens were obtained from each lesion. The specimens were selected from the areas were the surgeon indicated to represent the most abnormal appearance. All the specimens were collected from the operating room by the same researcher and a suture was used to indicate the orientation of the specimens. The specimens were sectioned perpendicular to the synovial surface in order to obtain slides containing the whole thickness of the lesions; they were fixed, dehydrated and paraffin embedded. Five-micron thick sections were stained with hematoxilin-eosin, Masson’s trichrome, Picrosirius red and Prussian blue. The slides were examined and photographed under bright field, dark field and polarized light. Necrosis was scored according to the classification described by Mirra et al. and modified by Doorn et al. (114, 164). The samples were graded as 0) when no necrotic areas were observed, 1) when 1-2 mm of the slide was necrotic, 2) for 3-9 mm necrosis per slide, and 3) when more than 9 mm of necrosis was observed in the slide. The extension of the necrotic areas (observed as areas without cells, presence of ghost lines and absence of blood vessels) was measured by use of the Zen software (Zeiss). The ALVAL score was assigned using the criteria of Campbell et al. (110) Table 3.3, by an experienced pathologist who was blinded to patients’ information. 32 Table 3.3 ALVAL score for periprosthetic tissue. Feature Observation Value Synovial Lining Intact synovial surface. 0 Focal loss of the epithelium, fibrin deposition may be found. 1 Moderate loss of synovial epithelium + Fibrin deposition. 2 Marked loss of Epithelium + necrosis + fibrin deposition. 3 Inflammatory infiltrate Minimum cell infiltration. 0 Predominantly macrophages. 1 Macrophages and lymphocytes, perivascular aggregates < 200 µm. 2 Macrophages and lymphocytes, perivascular aggregates of 200-500 µm. 3 Predominantly lymphocytes, with perivascular aggregates > 500 µm. 4 Tissue organization Normal tissue arrangement. 0 Small areas of synovial hyperplasia, focal necrosis may be observed. 1 Appearance of cellular and acellular zones, thick fibrous layers. 2 Thick fibrous layers with large acellular zones. Lymphocyte aggregates may be observed. 3 3.2.2 Cellularity: Cell count was used as a confirmation of the evaluation of necrosis in ALTRs. For this purpose, 7 MoM and 7 MoP cases were randomly selected. In each sample, three tissue slides were stained with Hoechst. In each of the slides two representative regions were selected and three 33 consecutive collinear areas of 515 x 385 µm were photographed under 20x lens, analyzing a total length of 1.5 mm from the surface to the tissue depth. The fluorescent nuclei were counted by use of ImageJ software (NIH). The mean values for each sample were compared with two-tailed Wilcoxon Mann-Whitney (WMW) test. The WMW is a test for non-parametric samples, and is based on ranking the samples giving them an ordinal value instead of the absolute value of the individual measurements (165), avoiding the bias of not normal distributed samples. P-values < 0.05 were considered significant. 3.2.3 Immunohistochemistry. To determine the T and B cell populations present in the ALTRs, immunohistochemistry was performed on the tissue slides. The following primary antibodies were used: anti-CD20 (MABF250, Millipore), anti-CD3 (C7930, Sigma-Aldrich), and CD45 (4KB5, invitrogen). Immunodetection was observed by means of the peroxidase reaction using the Histostain kit (Invitrogen, Burlingame, CA). 3.2.4 Cytokine microarrays. Tissue sections from 16 specimens (6 MoM, 5 MoP, and 5 controls) were flash frozen and stored at -80 C until processed. Sections of 100 mg including the whole thickness of the tissue were used since a high variability in the distribution of cytokines among the tissues has been reported (166). They were mechanically pulverized under liquid nitrogen, suspended in RIPA buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1%Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, and 140 mM NaCl) for 48 hours at 4C and clarified by centrifugation at 10,000 g x 5 min. The supernatants were diluted in RIPA buffer to normalize the total protein concentration to 1 mg/ml. Aliquots of 100 µl (100 µg of total protein) were used to semi-quantitatively assess the presence of 120 human proteins, including cytokines and other soluble signaling molecules, using a sandwich-ELISA antibody array (G-1000 Human Cytokine Antibody Array: AAH-CYT-G1000, Raybiotech) according to manufacturer’s instructions. The arrays were read in laser scanner GenePix 4000B (Molecular Devices) with a wavelength of 532 nm, a pixel size of 10 µm and a digital resolution of 16 bits. The subsequent quantification was performed 34 using Bio Discovery ImaGene® 9.0 software (Bio Discovery). Signal intensity medians were background corrected, and the mean of the replicates were calculated. WMW test was used to compare the concentration of cytokines between groups, p values < 0.05 were considered significant. Unsupervised hierarchical clustering was applied to observe how the cytokines concentration allows grouping the samples, and if those clusters correspond with the clinical and morphological characteristic. Hierarchical clustering is based on the complete linkage method using a Euclidian distance metric, “consist of nested sets of objects such that any pair of sets are either disjoint or one includes the other” (167). The results of these calculations are shown in a 2D heat map where samples and cytokines are ordered by their similarities. A dendrogram is added to show the arrangement of clusters of samples and cytokines according to their closeness. These calculations were performed by use of the R statistical environment (R Foundation for Statistical Computing). A dendrogram is the graphical representation of the closeness of the samples. Dendrogram is a diagram, in which the differences are shown graphically in the shape of a family tree, with the more similar individuals clustered together, and the differences between individuals and clusters represented with the length of the connecting branches (167). Figure 3.2 is an example of dendrogram. The analyzed group is composed of 5 members, with the respective values: A = 3, B = 4, C = 7, D = 11, E = 15. The horizontal distance between the individuals is fixed. The vertical bars that connect corresponds to the Euclidian differences between the individuals. Once clustered, the length vertical bars are determined by the largest differences between the individuals in the clusters. In figure 3.2 the distance between A (3) and B (4) = 1, and they group as the most similar individuals. The individual C (7), is more similar to the A – B cluster (value = 3 since is the bigger distance to C) than to D (12) or E (15). The distance between C and the A – B cluster is 4, but the length of the branch between A and B (1) is subtracted, thus the branch emerging from the A B cluster has a length of 3. The difference between D and E is 3, and they cluster together, since their difference (3) is lower than the difference with the cluster A B C. The difference between the clusters A B C and D E is 12, which is the largest differences between individuals that form each cluster (A and E). then a vertical bar with a length of 12 should connect the clusters, but the length of the already present vertical bars is rested. This example is a 35 simplification of what is done in the unsupervised hierarchical clustering, in which the closeness between the clusters and individual is determined by the average differences found in each of the 120 cytokines analyzed. Figure 3.2 Example of dendrogram, as used in unsupervised hierarchical clustering. 36 3.3 Results 3.3.1 MoP ALTRs are fibro-necrotic lesions, while MoM ALTRs are cystic-inflammatory. Macroscopic evaluation of the specimens showed that MoM ALTRs were typically cystic cavities with a thickened synovial membrane, from which none of the 17 cases exceeded 1 cm of thickness (Fig. 3.3A), while MoP ALTRs were typically solid or mixed solid-cystic in structure with a synovial wall between 1 and 6 cm in thickness (Fig. 3.3B, Table 3.4). Based on those differences, we carried out a qualitative and quantitative histological assessment. The most evident difference between the groups is the extension of necrosis that was significantly higher in the MoP group (Table 3.4). This result was confirmed by the cellularity analysis, in which significantly lower cell counts were observed in MoP ALTR when compared to MoM (p < 0.001; Fig. 3.3, Fig. 3.4 and Table 3.5). ALVAL score was significantly higher in the MoP group (Table 3.6). No association between the histological assessment and clinical information was found, although the time of implantation was significantly lower (p < 0.05) in the MoP group. The younger age of MoM patients (p < 0.05) is due to the fact that MoM implants were offered to younger patients. 37 Figure 3.3 Macroscopic representative sections of ALTRs. A) Thin cystic wall of in MoM. B) Solid mass in MoP. Table 3.4. Extension of necrosis in MoM and MoP ALTRs. Evaluation following method of Mirra: Grade 0 = no necrosis, grade 1= 1-2 mm of necrosis, grade 2= 3-9 mm of necrosis, grade 3=more than 9 millimeters of necrosis. MoM n=17 MoM % MoP n=17 MoP % Necrosis Grade 0 0 0% 0 0% Grade 1 6 35% 0 0% Grade 2 7 42% 1 6% Grade 3 4 23% 16 94% 38 Figure 3.4 Measurement of cellularity. Images of typical HOETSCH-stained samples of MoM (A) and MoP (B) ALTRs. they correspond to images from the synovial surface (left of the picture) to a 500 µm depth. Nuclei are observed as bright blue dots. 39 Figure 3.5 Measurement of cellularity. Images of typical HOETSCH-stained samples of MoM (A) and MoP (B) ALTRs. they correspond to images from 500 µm (left) to 1 mm depth (right). Nulei are observed as bright bue dots. 40 Table 3.5. Cellularity values for MoM and MoP ALTRs in the tissues depth. Expressed in cells/ mm2 Mean St. Dev. Median MoM Zone 1 (0-0.5mm) 1617.7 863.4 1472.7 Zone 2 (0.5-1mm) 1749.0 936.1 1555.9 Zone 3 (1-1.5mm) 1886.0 1237.7 1626.5 MoP Zone 1 (0-0.5mm) 73.1 90.1 30.3 Zone 2 (0.5-1mm) 54.8 65.6 22.7 Zone 3 (1-1.5mm) 68.6 74.7 47.9 Table 3.6. ALVAL assessment of the pseudotumors in MoM and MoP hip replacement. Value MoM n=17 Mean MoM MoP n=17 Mean MoP Synovial lining 1 4 1.94 0 2.88 2 10 2 3 3 15 Inflammatory infiltration Not observed 0 3.00 10 3.30 1 2 0 2 3 1 3 5 3 4 7 3 Tissue Organization 1 5 2.06 0 3.00 2 6 0 3 6 17 SUM 7.00 9.18 41 3.3.2 MoM and MoP ALTRs exhibit common morphological elements. Given the described differences between the MoM and MoP lesions, we next analyzed the microscopic structure of the lesions. In MoM ALTR the thickened synovial tissue generally corresponds to a 5-8 mm thick membrane in which we observed 3 histological layers as described by Davies et al. (168). Layer 1 corresponds to the ulcerated synovial surface with or without fibrin deposition. Layer 2 is characterized by the infiltration of macrophages, delimiting the areas of necrotic tissue. Layer 3 has an intense mononuclear infiltration that can vary from diffuse cellular infiltration to large perivascular aggregates (Fig. 3.6A). In contrast, MoP ALTRs are solid masses of more than 1 cm thickness, characterized by extensive necrotic areas covering most of the slides (Fig. 3.6B), but in some cases cellular areas can be observed in the external part of the samples (Fig. 3.6C). Aiming to analyze the cell distribution in the MoP ALTRs, we analyzed areas of the resected tissue at the boundary between pathologic and normal appearance. Those areas were found to be organized in a three-layer structure similar to the described for MoM (Fig. 3.6D), with perivascular lymphoid aggregates, in which T cells (CD3+) and B cells (CD20+) have similar distribution to that described for MoM systems by other authors (122) (Fig. 3.7A-B), many of which are in a proliferative phase (Ki67+) (Fig. 3.7C). Interestingly, fibroblasts of all the analyzed samples were positive for alpha-smooth muscle actin (SMA), suggesting their reparative role and a myofibroblasts phenotype (Fig. 3.7D) 42 Figure 3.6 Histological structure of ALTRs in MoM (A) and MoP (B-D) implants. A) Histological layers described in MoM lesions. B) Typical image of ALTR in MoP systems, extensive necrosis from the synovial surface extending for a few centimeters into the tissue depth. C) Higher magnification of an area in B, showing the cellular zone of the slide. D). Lateral zones of the lesion in B, at the border with tissue with normal appearance, the typical layered structure described in MoM ALTRs can be observed. Stain: Hematoxilin-eosin. 43 Figure 3.7 Immunohistochemistry of perivascular lymphocyte aggregates in MoP ALTRs. A) T cells in ALTRs (CD3+). B) B cells in ALTRs (CD20+). C) proliferative cells (Ki67+). D) Myofibroblasts (-SMA+) 3.3.3 Degenerative changes in blood vessel’s walls associated to MoP ALTRs. Thickening of the walls of blood vessels leading to complete occlusion of the lumen were observed in the seven MoP ALTRs in which a cellular zone could be analyzed (Fig. 3.8). The vascular wall appears as thickened by the presence of mononuclear infiltrate and foam cells and deforms the bood vessel from round to a banana-shape in its lumen (Fig 3.8A), in smeome cases fibrilar material could be observed in the blood vessels accompanig a prismatic shaped endothelium instead of the regular flat shaped cells (Fig. 3.8B). Finally the disapearence of blood vessels is characteristic of the necrotic zones, in which evidence of hemorrhage could be evident, and the presence of “ghost-like” cells in the characteristic coagulative necrosis areas (48). 44 Figure 3.8 Structural changes in the vascular wall of blood vessels in non-modular MoP ALTRs. A, thickening of the vascular wall (asterisks), with foam cells. B, vessel occlusion with endothelial cells adopting a cubic shape (arrows). Necrotic tissue with lack of blood vessels, ghost-like cells in which the preexisting macrophages fade gradually reducing its normal nuclear and cytoplasmic staining (arrowheads) and hemorrhagic areas (asterisk). 45 3.3.4 ALTRs showed a distinctive cytokine pattern in the periprosthetic tissues. Out of the 120 soluble signaling molecules analyzed, 18 were detected at significantly higher concentrations in MoM ALTRs than in controls and are listed in Table 3.7; Only IL-7 showed lower concentration in MoM ALTR than control samples. Cytokines in MoP cases in general showed low levels and only 4 of them had significantly higher concentration than in the controls (FGF-7, I-309, Il-8 and TIMP-1). Two of the 120 analyzed molecules showed higher concentration in MoP than MoM ALTRs, but 27 showed higher levels in MoM than in MoP. In order to determine the morphological features that could be related to the concentration of cytokines, the samples were ranked for necrosis and ALVAL, then the unsupervised hierarchical clustering was run by an expert in statistics who was blind to the type of implant or patient’s information. The clustering was then compared with the type of implant and histological observations of the lesions. The analysis generated for each of the two sets of samples (eight in each microarray slide) created two basic families: one of MoM ALTRs, and the other with MoP ALTRs and control samples, with the highest affinity within the control samples (Fig. 3.9). Nevertheless, this analysis failed to differentiate between the MoP sample with a low degree of necrosis and ALVAL score (MoP 5) and regular MoM ALTR, and between the MoM with a low degree of necrosis and low ALVAL score (MoM 4) and Control samples. 46 Table 3.7. Cytokines and biomarkers that exhibit significant higher values in the MoM group than the Control group. The values represent the average of arbitrary measurement of the fluorescence laser scanning of the antibody microarrays. * significant differences between MoP and MoM, ** significant differences between MoP and Control group, *** significant differences between MoM and Controls. Significance = p-value > 0.05. Marker Significance MoP ALTRsMoM ALTRs ControlAgRP * 173.5 230.4 232Angiopoietin-2 * *** 632.5 3637.8 956.6Axl * ** 413.9 1887.7 1840.8bFGF * 143.3 5221.4 486.5BLC * *** 63.1 472.9 15.9CNTF * ** 169 224 227.2ENA-78 * 86.6 458.3 129.6Eotaxin *** 85.9 182.7 110Fas/TNFRSF6 *** 324.9 805.3 411.8FGF-6 *** 131.9 209.6 80.3FGF-7 ** *** 152.1 243.4 53.3Flt-3 Ligand * 231.9 581.4 657.7GITR *** 647.6 517.7 162.8GM-CSF * ** 285.7 413.1 435.3GRO * 247.9 626.8 349.5HCC-4 *** 146.5 201.1 178.3I-309 ** *** 39.2 24.5 3.7IFN-gamma * ** 249.3 327.2 398.1IL-10 * ** 283.7 394.1 418.2IL-13 * 418 535.2 578.5IL-15 * ** 269.1 357.3 396.2IL-1alpha * ** 415.6 510.5 521.5IL-1ra * ** 160.1 222.2 211IL-2 * ** 289.5 415.5 457.9IL-3 * 189 235.2 246.9 47 Marker Significance MoP ALTRsMoM ALTRs ControlIL-7 * ** *** 293.1 384.6 477.2IL-8 ** 2207.1 1904.4 661.6I-TAC * *** 73.4 134.7 94MCP-1 *** 2840.8 6685.7 1221MCP-2 *** 170.1 225.3 96.9MCP-3 *** 145.1 96 31.3M-CSF * ** 79.9 180.1 257.9MIG * ** 229.4 337.4 374.7MIP-3beta *** 6109.3 4784.6 6598.2MSP-alpha * *** 110.4 486.3 126.3Osteoprotegerin *** 870.8 1450.9 364.4RANTES * 77.6 888.1 382.3SDF-1 *** 104.1 140.3 50.9TGF-beta 1 * ** 457.5 575.8 588.5TIMP-1 ** 4924 4480.1 3124.9TNF-alpha * ** 308.4 397.5 432.4TNF-beta * ** 343.6 461.9 492.6TRAIL R3 *** 2039.4 1699.6 745.2 48 Figure 3.9 Unsupervised hierarchical clustering of the cytokines in MoM and MoP ALTRs and control samples. The clustering was performed in two directions, in the horizontal direction the cytokines were clustered according to their similarities in concentration, in the vertical direction the patients were clustered according to similarities in the cytokine concentrations, the clustering of cytokines was omitted for facilitating the observation of the chart. In the heat map the white-light yellow boxes represent high concentration of cytokines and intense red color represent low concentration. The dendrogram on right shows the clustering among patients with similar concentration of cytokines. The length of the horizontal lines of the dendrogram to a common node between two samples is inversely proportional to the similarity between the samples. 49 3.4 Discussion 3.4.1 MoP ALTRs are highly destructive lesions. ALTR in non-modular MoP THA are highly destructive lesions that affects the integrity of the synovial tissues of the hip: The histological findings in this study revealed significant damage induced by ALTRs in the joints with MoP systems. These changes were observed to be even more dramatic than the observed in MoM systems in terms of volume of the destructed tissue and extension of necrosis. A recent histological comparison between ALTRs in 18 MoM and 55 dual modular neck MoP systems, reported similar degree of tissue necrosis between the groups (111). These disparate results may be explained by the fact that in the mentioned study, slides with more than 75% of necrosis were excluded. In the present study 16 out of 17 of the MoP and only one of 17 MoM samples showed more than 75% necrosis in the slides. The presence of similar histological features in the ALTRs in MoM and non-modular MoP hip replacements, suggests that the reactions should have similar origin and development. The observed differences in the extension of necrosis and thickness of ALTRs in MoM and MoP, could be due to specific susceptibility of the patients, considering that a much lower percentage of patients develop ALTRs in non-modular MoP than in MoM systems (169). Another possible reason for this is that a milder symptomatology in MoP implants combined with less awareness of clinicians, allowed the damage in MoP become more persistent, as opposed to cases of MoM, where the clinical index of suspicion was much higher, and the threshold for revision would have been lower than in MoP. The degenerative changes in the vessels wall may be an important finding in the mechanisms of cell death in MoP ALTRs. Although the number of samples analyzed in this study is small, the hypothesis of vascular changes that lead to the infarction of the tissue is coherent with the type of necrosis described, with minimum degradation of the extracellular matrix (coagulative necrosis) was observed in all MoP cases. These alterations have been previously described in MoM ALTRs and pointed as potential mechanism of tissue damage (111). 50 3.4.2 Cytokines could play a key role in ALTR’s etiology and pathogenesis. Our work includes an analysis of signaling molecules extracted from tissue homogenates in MoM and MoP ALTRs as well as controls. We observed higher levels of cytokines in the MoM group than the MoP, which could be explained by a lower cell count in the MoP group. Our results showed that 20 molecules have significantly higher values in the MoM ALTR group than in controls. Those molecules belong to four families: C-C and CXC chemokines, TNF receptors, and fibroblast growth factors. Eotaxin, I-309, HCC4, MIP3-alpha, MIP3-beta, MCP1, MCP2 and MCP3 belong to the C-C chemokine family (CCL11, CCL1, CCL16, CCL20, CCL19, CCL2, CCL8 and CCL3). They have similarities in structure and function; moreover, their genes localize in the same zone of the chromosome 17q12, and are chemotactic for macrophages and eosinophils (170, 171). BLC, I-TAC and SDF are chemokines of the CXC family (CXCL13, CXCL11 and CXCL12); in this group the genes of BLC, and I-TAC map in 4q21, and SDF in chromosome 10q11. These chemokines are related to the chemotaxis and activation of B cells, monocytes and T cells (171). The TNF-receptor superfamily is composed by death receptors such as FAS and Trail, activating receptors as GITR or decoy receptors as OPG (172). While Fas and Trail trigger apoptosis or programmed necrosis through activating the NF-kB pathway (173), GITR has a role in apoptosis but also in the activation and survival of regulatory T cells (174). OPG has an important role in bone metabolism by inhibiting osteoclastogenesis (172). A recent study, through analysis of RNA from tissue homogenates, also found high expression of CC and CXC chemokines in ALTR (CCL19, CCL13 and CCL8, and CXCL9, CXCL10 and CXCL13) (163). The same study also found high expression of MIG and IP-10; proteins that are up regulated by IFN- which is responsible of the Th1 switch in the immune response. This is somehow contradictory with the results that the same group described previously, in which by immunohistochemistry they described the T-cell population of the lesions as “mixed pattern with predominant GATA3 positivity (Th2 lymphocytes) but also substantial T-bet (Th1) and FOXP3 (Treg) expressing lymphocytes” (97). In our study, we didn’t find any clear pattern of cytokines that allows determining a specific type of immune response, and thus further studies are needed to determine the type of immune reaction in ALTRs. More importantly, it will be important to identify the role of metal components in triggering the synthesis and secretion of these molecules 51 in order to understand the ultimate etiologic factor of ALTR, and how the cellular interactions through soluble molecules determine the pathogenesis of ALTRs, in order to develop effective diagnostic and therapeutic tools. 52 Chapter 4: Degradation of materials: ions and metal particles in periprosthetic tissues. 4.1 Rationale The concentration of Co and Cr in serum has been associated with implant malfunction; however, there are no conclusive studies that associate the levels of metals in synovial fluid with the presence of ALTRs. Moreover, only a few studies have reported ion concentration in synovial fluid of patients with hip implants. This is missing in the study of ALTRs, since the synovial fluid represents the medium of direct contact with the implants surfaces, and is the anatomic connection between the implants and the soft tissues of the joint. Particles in tissues have been studied in MoM implants, but the presence of metal particles in MoP systems has only been reported in a couple of articles as “corrosion products”. This is mainly due to the assumption that wear should not exist on metal femoral heads articulating with a polyethylene liner. In the present study, we analyzed the presence of metal products of implant degradation (particles and metal ions) in synovial fluid, periprosthetic tissues and serum. The aim was to associate the presence of these products with the presence of ALTRs. 4.2 Methods A group of 90 patients undergoing revision surgeries due to ALTR were included in this study (49 MoM and 41 MoP). Twenty-two patients with MoM implants and 12 with MoP implants, undergoing revision for reasons other than ALTR, were included as a control group. A total of 25 patients undergoing primary surgery were also recruited to define baseline conditions for the study. All patients were contacted and gave consent between one month and two weeks prior to surgery. From all of the patients, we aimed to obtain serum, synovial fluid and periprosthetic tissue for the analysis; however, it was not always possible to obtain all of these elements due to: 1. Patients did not always get the blood test, 2. Insufficient synovial fluid in the joint to be analyzed, 3. The amount of resected tissue was not enough for diagnosis and research. Thus, the specific number of samples analyzed will be specified in each study. 53 4.2.1 Analysis of chromium and cobalt ions concentrations in serum and synovial fluid. Samples of peripheral blood were obtained from the patients 24-72 hours prior to the surgery. The synovial fluid was obtained by aspiration during the surgical procedure, before the joint capsule was opened. Blood and synovial fluid were collected using a plastic 7mL non-additive tube (Trace Element, Serum, REF 368380, Becton Dickinson). The fluids were centrifuged at 1,400 RCF for 15 minutes. The supernatant of serum and synovial fluid were transferred using a plastic pipette into a polypropylene tube, and stored at -20 C prior to analysis. The protein concentration was measured with a protein assay following the manufacturer instructions (Pierce, ThermoFisher Scientific). Aliquots of serum and synovial fluid were shipped to the Trace Elements Laboratory (London, Ontario). This laboratory uses the Thermo Fisher Element 2 high-resolution sector field inductively coupled plasma mass spectrometer (HR-SF-ICP-MS; Thermo Scientific) for measurement of metal ions. Inductively coupled plasma mass spectrometry (ICP-MS) is an element analysis technology capable of detecting elements at concentrations in the nanograms / L range. ICP-MS is an ionization source that fully decomposes a sample into its constituent elements by high energy field in argon gas solution. The samples are first treated and taken into a liquid form, generally by the use of strong acids or base for digestion. In a second step the high energy is coupled to the argon gas solution to form a plasma. Because of the acceleration of the atoms produced by the electrons in the argon gas, the plasma reaches temperatures in the order of 10,000 K. ICP-MS has the advantage of analyzing multiple elements simultaneously, reducing time and sample volume for the analysis. The measured values of the concentration of Co and Cr were Log transformed, and compared between groups by use of one-way ANOVA. In order to reduce type-I error due to multiple comparison analysis, the obtained p-values were corrected by use of Benjamini-Hochber. ANOVA is a statistical method used to analyze the differences between the means of different groups. ANOVA provides a statistical test of whether the means of several groups are equal, and therefore generalizes the t-test to more than two groups. ANOVA is useful for 54 comparing three or more groups for statistical significance. It is conceptually similar to multiple two-sample t-tests, but is more conservative (results in less type I error, and is therefore suited to a wide range of practical problems). 4.2.2 Analysis of particles in the tissues: The resected tissues were macroscopically observed by a pathologist of bone and soft tissue at Vancouver General Hospital. After selecting the most representative sections for diagnosis, tissue specimens were cleared. The obtained samples of each patient were divided into two groups: one group was frozen at -80 for posterior cryosectioning, and the remaining was fixed in buffered formalin (10%) for 24 hours. The fixed samples were observed and photographed under a stereomicroscope to observe their macroscopic structure and the presence of metal particles. Then the samples were processed for histologic observation as described in chapter 2. Briefly, they were dehydrated in ethanol, cleared in xylene substitute and embedded in paraffin. Sections of 6 m thickness, were stained with Hematoxylin-eosin (H-E), Masson’s trichrome and Sirius red. When the presence of particles was confirmed, 10 micron-thick sections were mounted on plastic microscope slides. Paraffin was carefully removed by use of xylene substitute (Thermo Fischer Scientific), which prevents the toxic effect of xylene with similar effectiveness in paraffin removal and optical microscopy results (175, 176). After SEM-EDX observation, the slides were stained with hematoxylin for histological observation. SEM-EDX: Tissue slides with metal particles were analyzed by use of energy-dispersive x-ray spectroscopy in a scanning electron microscope (Hitachi S3000N VP-SEM-EDX), under variable pressure mode (20 Pa) and 20 kV, at a working distance of 12 mm. Synchrotron XRF and XRD: Synchrotron technology is an advanced method to obtain a high amounts of radiation, obtained from the release of energy by ultra-high speed sub-atomic particles that are accelerated by magnetic fields which are synchronized to increase the kinetic energy of the particles. The large amount of energy released by the particles is then used in the different beamlines. The main advantage for particle analysis in this project resides in that the synchrotron radiation allows particle analysis without changing environmental conditions of the 55 analytes (e.g. pressure, vacuum, humidity), and thus our samples were analyzed in humid conditions as they are found in the patient’s body, avoiding the changes produced by dehydration and pressure changes. Frozen specimens were observed under stereo microscope. In the specimens in which the presence of particles was confirmed, sections were prepared for observation in synchrotron XRF and XRD as follows: tissue specimens were embedded in optimal cutting temperature compound (OCT), which is composed of 10.24 % polyvinyl alcohol, 4.26 % polyethylene glycol, in a base of non-reactive ingredients. The samples were freeze-sectioned in a cryostat (Leica Microsystems GMBH, Germany) by use of metal avoidance technique. For this purpose, low profile polytetrafluorethylene-coated microtome blades were used to cut 10 micron-thick sections. The samples were manipulated avoiding contact with metal instruments and surfaces, and mounted in plastic microscope slides. The OCT was removed by immersion in deionized water (Milli-Q), and subsequently the slides were fixed in 100 % alcohol. VESPERS beamline (Canadian Light Source) was employed to conduct the site-specific analysis on corrosion particles at different locations. The X-ray beam at VESPERS is tunable from 6 to 20 KeV and the flux could reach 2 ×10-9 at 15 KeV with Si monochromator. The sample is positioned at 45 degrees with respect to the incident 4×4 m beam. The one micron-sized beam and the high flux of photons permitted studying the area of interest at a micrometer scale with high sensitivity. Monochromatic X-ray at 20KeV was used for synchrotron X-ray fluorescence (XRF) analysis to study the chemical composition of the samples. Synchrotron X-ray diffraction (XRD) with incident X-ray energy at 14 KeV was used to study the crystal structure of the observed particles. 56 4.3 Results 4.3.1 Significantly higher concentrations of chromium and cobalt in synovial fluid of patients with ALTRs. Five to ten mL of synovial fluid were obtained from 35 MoM patients, 18 of the 30 MoP patients (with the other 12 of them having dry joints), 10 control MoM, 6 control MoP, and 4 primary surgery patients (out of 25). Out of the 18 MoP samples with ALTR, six corresponded to the regular amber-translucent color (Fig. 4.1 A), nine of them had an opaque cafe-au-lait color with a creamy-sandy consistency (Fig. 4.1 B), and three were dark grey color with fluid consistency (Fig 4.1 C). The samples obtained from MoM patients were mostly amber-translucent; one was café-au-lait type and one dark grey, all control samples and primaries were a regular amber translucent color. Protein concentrations in the synovial fluid samples varied in accordance with their optical appearance, the cafe-au-lait type had higher protein concentration (p < 0.05) (mean 81 g/L) compared to amber ones (42 g/L) and dark ones (40 g/L). The Cr and Co concentration was also significantly higher in the café-au-lait type (p < 0.05) with a mean ten times higher than amber or dark ones. The comparison by one-way ANOVA of the concentration of Co and Cr between groups revealed statistical differences between groups (p < 0.000001 for Cr and Co). When comparing specific groups, the concentration of chromium in the synovial fluid of patients with ALTRs was significantly higher than controls in both MoM (p - value < 0.05) and MoP (p - value < 0.01) and in both cases the values were significantly higher than in patients undergoing primary surgeries (p - value < 0.0001). There were no differences in the concentration of chromium in synovial fluid of patients with well-functioning implants (Control MoM and MoP) and the primary surgeries group (Table 4.1, Fig. 4.2, Fig. 4.3). No differences were found between MoM ALTRs and MoP ALTRs, and between Control MoM vs Control MoP (Table 4.1, Fig. 4.2 and Fig. 4.3). Combined, these results show that patients with ALTRs have higher concentrations of Co and Cr in the synovial fluid than patients with well-functioning implants. 57 Figure 4.1 Synovial fluid of ALTR patients. Table 4.1. Chromium and cobalt concentration in synovial fluid. Expressed in g / L. MoM ALTR MoP ALTR Control MoM Control MoP Primary Surgeries Chromium Mean 249.5 2476.1 52.7 4.7 3.21 Median 131.4 474.0 11.8 3.8 3.21 St. Dev. 303.0 4786.8 73.0 2.3 1.5 Cobalt Mean 521.4 4059.7 44.3 6.1 4.78 Median 304.9 1547.0 33.5 4.8 4.78 St. Dev. 570.1 5924.0 49.3 4.3 3.6 58 Figure 4.2 Dot plot of the concentration of Cr in synovial fluid. Mean and standard error shown. Figure 4.3 Dot plot of the concentration of Co in synovial fluid. Mean and standard error shown. 59 4.3.2 Elevated concentration of metal ions in serum of patients with ALTRs in MoP. The concentration of Co and Cr in the serum of patients with ALTRs in MoM (n = 23), MoP (n = 22), Control MoM (n = 6), Control MoP (n = 6), and primary surgeries (n = 11) was analyzed through ICP-MS. The concentration of chromium in patients with ALTR was significantly higher than in control patients (p - value < 0.05 for MoM, and p - value < 0.0001 for MoP), and primary surgeries (p – value < 0.00001 for MoM and MoP). The control MoM group exhibited significantly higher values than the primary group (p – value < 0.001), but this difference was not observed in the MoP controls (Fig. 4.4). The concentration of chromium was significantly higher in patients with metal on metal articulation in both ALTRs and control cases (p – value < 0.0001 for ALTR groups, and p – value < 0.01 for controls). Cobalt was also significantly higher in patients with ALTR compared with controls (p – value < 0.01 for MoM and MoP), and compared to primary patients (p – value < 0.0001 for MoM and MoP). The control MoM group displayed significantly higher values than primaries (p – value < 0.05), but that difference was not observed in control MoP (Fig. 4.5). Different from what was observed for chromium, there were no differences in the concentration of cobalt in synovial fluid of patients between MoM and MoP in either the ALTR groups or the non-affected groups (control MoM and control MoP). Linear regression showed no association between the concentration of metal ions in serum and in synovial fluid. No association between the metal ion concentration and clinical variables such as age, time of implantation, or severity of the lesions was observed. 60 Figure 4.4 Dot plot of the log-2 concentration of Cr in synovial fluid. Mean and standard error are shown. Figure 4.5 Dot plot of the log-2 concentration of Cr in synovial fluid. Mean and standard error are shown. 61 4.3.3 Similar amount of metal particles in ALTR and non-ALTR joints. Different types of particulate material can be described according to their optical properties. We defined three different types of particles: 1) Wear particles: dark small spots (< 1 micron) observed in the extracellular matrix or in the cytoplasm of phagocytes and fibroblasts, occasionally forming clusters that are usually surrounded by T cells or macrophages (Fig. 4.6). We define them as wear particle due to the fact that their chemical composition is concordant with the composition of the original alloy, and it has also described by others (98). 2) Corrosion particles: Prismatic-shaped translucent structures, bigger in size (10-200 microns) found mostly in the extracellular space or associated to giant multinucleated cells (Fig. 4.7 and Fig. 4.8). We defined as product of corrosion due to their differences with the original alloy (98). 3) Brown spotted vesicles: observed in the cytoplasm of macrophages. These structures corresponded to hemosiderin as it was confirmed through SEM-EDX due to their high iron content (Fig. 4.10). The wear particles were observed in 57 % (19 of 33) of the MoM ALTR cases, 56.2 % of the control MoM cases (9 of 16) and 43.3 % (13 of 30) of the analyzed cases of MoP ALTR (Table 4.2). Corrosion particles were observed in 42 % of the MoM ALTR cases (14 of 33), 37.5% of the control MoM cases (6 of 16) and 30 % of MoP ALTR cases (9 of 30). 62 Figure 4.6 Microphotographs of wear particles in periprosthetic tissues. A) isolated particles in macrophages cytoplasm. B) Clustered particles in leucocyte aggregates (arrows) C) BSE imaging of cluster in B. 63 Figure 4.7 Corrosion particles in periprosthetic tissues. A and B) Light microscope imaging of corrosion particles associated to giant multinucleated cells. C) BSE imaging of a corrosion particle mechanically extracted from tissue. 64 Figure 4.8 Corrosion particles in periprosthetic tissues. A) Light microscope imaging of corrosion particles. B) SEM images of the section in A. C) higher magnification of area in B, shows lamellar structure of particles. 65 Figure 4.9 SEM-EDX spectra of wear particles (A), or corrosion products (B). both spectra correspond to particles analyzed in tissue sections. 66 Figure 4.10 Iron rich vesicles in macrophages, compatible with hemosiderin. A) histological image. B) SEM-BSE image of section in A. C) SEM-EDX spectra. 67 Table 4.2. Metal particles observed in ALTR tissue sections. Wear particles Corrosion particles No of cases % No of cases % MoP (n=30) 13 43.3 9 30 MoM (n=33) 19 57.5 14 42.4 Control MoM (n=16) 9 56.2 6 37.5 4.3.3.1 Wear particles and corrosion products have different chemical composition and both are found in MoM and MoP ALTRs. After the histological observation, the particles derived from wear or corrosion of hip replacements in the tissue sections were analyzed through SEM-EDX. Wear particles of 18 cases were analyzed (10 MoM, 4 MoP, 4 Cont. MoM). They showed similar composition in all cases, with a 61 % of cobalt (St. dev +- 6.2%), 31 % of chromium (St. Dev. +- 2.44), 2 % of molybdenum (St. Dev. +- 0.8%) and traces of other elements (Fig. 4.11). Under BSE looked like bright spots, which was consistent with their elevated metal content (figure 4.6C). Fifty-two corrosion particles were analyzed: 26 MoM (12 patients), 21 in MoP (8 patients), and 5 controls (3 patients). All the particles showed a major composition of chromium (61.6 % MoM, 55.2 % MoP, St. dev +- 7.1 % and +- 6.82 % respectively), with minimal or no cobalt and molybdenum. The particles had a high content of calcium and phosphorous (Fig. 4.12). Although the general composition of these particles is similar among the cases, the particles in MoM cases revealed a higher amount of Cr than MoP (p < 0.05). 68 Figure 4.11 Average composition (percentage) of wear particles in periprosthetic tissues. 69 Figure 4.12 Average composition (percentage) of corrosion products in periprosthetic tissues. These results suggest that wear particles correspond to relatively un-corroded particles generated by wear of the articulating surfaces or by fretting in the modular joints. The wear particles maintain the proportion of Co and Cr of the original alloy; however, molybdenum seem to be leaching out since its proportion is decreased to half of the original alloy. We hypothesized that the corrosion particles are formed due to precipitation of metal in solution. The more soluble cobalt (177), does not form salts and tends to remain in the ionic state, while the less soluble chromium precipitates in conjunction with other abundant ions in body fluids such as P and Ca, which are especially highly concentrated around bone tissue. Although it is generally accepted that 70 the metallic state particles correspond to wear in the articulating surfaces, and thus they should be only present in MoM articulations, we observed metallic particles in 13 MoP patients, with the corresponding results in SEM-EDX. 4.3.3.2 Corrosion products observed as amorphous chromium-rich particles. In order to analyze the crystal structure of the corrosion particles found in the tissues, we performed an x-ray diffraction analysis (XRD), and an element distribution analysis by use of x-ray fluorescence (XRF) in the VEESPERS beamline of synchrotron facility at the Canadian light source (Saskatoon). Since the wear particles previously described preserved the chemical composition of the original alloy, and thus should correspond to particles in the original metallic state, we focused on the analysis of the corrosion particles that showed a Cr-rich composition. For that purpose, four tissue slices of 10 mm thickness, mounted on plastic tissue slides, were analyzed. The distribution of Co, Cr and Mo within the particles showed a relative co-localization of these elements in the particles as shown in figure 4.13. The image shows relative values of concentration, and does not represent the total metal content of the particles, thus comparison between different metals content is not possible. 71 Image by Qiong Wang. Figure 4.13 Synchrotron-XRF mapping of corrosion particles in periprosthetic tissue. 72 In the analysis of XRD, the particles displayed a weak signal at 1.43 Å (Fig. 4.14), which prevents determining a specific crystal structure, and suggests that these particles are mostly amorphous but some crystalline elements could be contained in the particles. This observation is similar to the one that has been described at the interface between the metal surfaces in the modular joints (52). Images by Qiong Wang. Figure 4.14 Synchrotron-XRD analysis of corrosion products in periprosthetic tissue. 4.4 Discussion: The products of degradation of materials of hip replacements are thought to be the main etiologic cause of adverse local tissue reactions to hip replacements. The presence of both, metal ions and particulate material has been proposed to be cytotoxic and trigger complex reactions of the immune system that can be responsible for tissue destruction and necrosis (93, 137, 178). The obtained results show that metal ions and particles can be found in MoM and MoP ALTR patients, at the time that a specific composition of particles was obtained from the patient’s tissues. The significantly higher levels of Co and Cr in MoP and MoM ALTRs than in controls, suggests that they may have a direct role in the development of ALTRs, and thus, their potential role in triggering the reaction will be explored in the next chapter of this thesis (Chapter 5). 73 4.4.1 Metal ions in blood as predictors of function of MoP implants. The presence of elevated concentrations of Co and Cr in serum of patients with MoP compared to controls, is a major finding of this study. Although these high levels of Co and Cr in serum of patients with MoM implants is well known, there was only one report recently published that associated Co and Cr in serum of patients with MoP implants and the risk of ALTRs. In that study, published by the group of Rush University, they retrospectively investigated patients that underwent revision of MoP systems, and contrasted the pre-surgical analysis of metal ions with the biopsy description. They found that in the 44 revised patients with a diagnosis of ALTRs, Co and Cr were significantly higher than in the 20 patients with no ALTR in the pathology description. They determined that cobalt was the best for diagnosing the presence with a cutoff value of 1 g / L that showed a sensitivity of 100% and a specificity of 90%, and these values were very similar when analyzing the non-modular neck implants only (179). Our results confirm this recent description in terms of the difference observed in the serum ion levels in MoP patients with ALTRs vs MoP patients without ALTRs, but the cutoff value of 1 g / L in our cohort shows a 91% sensitivity, and 80% specificity. Our numbers are still small to drive strong conclusions (22 MoP ALTRs and 5 controls MoP), and thus, it represents a good opportunity for future research. Moreover, considering that MoP implants are the most used design world-wide, its malfunction could affect millions of patients. 4.4.2 Local concentration of metals as a pathogenic element in ALTRs. The analysis of synovial fluid presented in this study demonstrates that the synovial fluid of patients with failed MoM and MoP hip replacements due to ALTR have significantly higher values of Co and Cr than the control groups. This suggests that the presence of elevated metal ions in the local environment of the hip joint could be an etiologic factor in the development of ALTRs. Our results of Co concentration in synovial fluid revealed a mean value of 249.5 g / L in MoM ALTRs, and 2476 g / L in MoP ALTRs. This is the first report of metal ion concentration in synovial fluid in patients with MoP implants, and only a few studies have reported previously the values in MoM cases. The study of Davda et al. (2011) reported values of 12,744 g / L for 74 Cr, and 1,249 g / L for Co, both in 93 ALTR MoM patients. They described a weak correlation between the concentration of metals in synovial fluid and serum, and no association with the head diameter or implant manufacturer (109). A previous study of De Smet et al (2008) in patients with MoM implants with evidence of metallosis showed median value of Cr of 201.0 g / L (range, 19.0 to 29,080.0 g / L), and median value of Co of 235.0 g / L (range, 13.0 to 5120.0 g / L) (108). The first study to show association between metal ion levels and the presence of adverse reactions to metal implants was the report of Langton et al. (2010). This study, similarly to all the analyses of joint fluid, showed a wide range of values from tens to tens of thousands of g / L (180). Our results are within the range of the values described in the literature, as is the wide variance observed in our study. Experimental studies have demonstrated that Co induces apoptosis in different cell populations (150, 181), and a concentration of 6 mg/L (6 mg / L) of Co generates necrosis in cultured J774 macrophages after 48 hours of exposure (151). In our study, four of the 19 MoP ALTR samples analyzed in this study showed a concentration higher than 6 mg / L in the synovial fluid. This high concentration of ions combined with a long-term exposure may lead to important cellular effects, and thus, the study of the effects of ion concentrations in cells of the hip joint will be described in the next chapter. 4.4.3 Particles in periprosthetic tissues: no association with pathologic events in the hip. The presence of two basic types of particles in tissues surrounding hip replacements has been previously described. A recent study of the analysis of 285 biopsies, described the presence of wear metallic particles and corrosion products in tissues of the hip joint. They observed wear particles in 8% of dual modular non-MoM total hip replacements, and in 30% of MoM systems, while corrosion products were found in 95% of the non-MoM group and in 100% of the MoM (98). Their results are different from the observations described in the present study, where metal particles were observed in 43% of MoP and 57% of MoM ALTRs, while the corrosion products could be observed in 30% of MoP tissues and 42% of MoM. These differences could be due to a slight difference in the type of implant analyzed, since their non-MoM was a dual modular neck, while we analyzed only single modular MoP and single modular MoM. Another possibility could 75 be due to the absence of a standard observation protocol for these particles (mainly magnification in the light microscope), which could be important considering that the size of the particles can vary from a few nanometers to millimeters, thus being the resolution for the light microscopy the main limitation for this type of quantification (96). The results of the chemical speciation of metallic particles and corrosion products in the synovial tissues confirm previous descriptions, in which the presence of corrosion products with Cr, P, O and Ca, but lacking Co and low levels of molybdenum have been described (84, 94, 95, 182). The crystalline pattern found in the products of corrosion in tissues through synchrotron-energized XRD, has not been described previously and is commonly accepted that these particles correspond to amorphous compounds. Due to the weakness of the observed signal (Fig. 3.10), no further conclusions could be made, but that some of the material in the corrosion particles may have a crystalline structure. In summary, the presence of particles in periprosthetic tissues is a matter of further study, and their presence could be relevant since they might also be source of soluble ions that may affect the physiology of the synovial tissues. The finding of a significantly higher concentration of metals in the synovial fluid of ALTR joints, led us to the assumption that soluble metal ions could be more directly associated to the development of ALTRs. This assumption is supported by experimental studies, in which the culture of murine macrophage cell line in presence of nanoparticles and soluble ions, have demonstrated that Co-Cr-Mo nanoparticles and cobalt ions have similar deleterious effects. That finding was associated to a linear correlation between the amount of Co-Cr-Mo particles and the release of ions to the culture media, suggesting to the authors that the toxicity of the particles may be due to the release of ions and not by the particles themselves (183). Another study showed that metal extracts of Co-Cr-Mo particles are cytotoxic to human lymphocytes, leading to apoptosis when cobalt is in low concentrations and to necrosis in high concentrations (180, 183). However, recent studies have demonstrated that metal particles can induce apoptosis in human lymphocytes and reduce viability in human monocyte cell line while cobalt ions do not have these effects (154, 184). This discrepancy could be explained by the concentration of metals used in different studies. In the studies that demonstrated the toxicity of cobalt ions the cells were exposed to 1 mM of Co ions, but the studies that showed that cobalt ions 76 do not generate a response used 0.1 M. This difference of 10,000 times makes the studies not comparable. With the data obtained in this study of metal ion concentration in synovial fluid, accurate experiments to evaluate the effect of these ions were conducted and described in the following chapter. 77 Chapter 5: Biological effects of Cobalt and Chromium on the periprosthetic tissues of the hip. 5.1 Rationale: The corrosion products of the metal components of the hip implants have been proposed as a major etiological factor of ALTRs, but their role in triggering the reaction, as well as their participation in cell and tissue destruction, are yet to be understood (71). As discussed in the previous chapter, studies on cell culture have not established a clear role of metal ions and/or particles, mainly due to the difficulties in finding the appropriate concentration of metal ions, and chemical composition of the particles found in the tissues. The results of chapter 4 demonstrate that there are no statistical differences in the presence of particles and their composition between controls and the ALTR patients; however, the concentration of ions of Co and Cr in synovial fluid is significantly higher in ALTRs patients. We therefore hypothesized that the presence of metal ions could develop a cellular response that may be the etiologic factor in triggering the ALTRs. The histological characteristics of ALTRs depends on the type of implant: ALTRs in MoM articulations are predominantly cystic with more joint fluid and lower ALVAL score than cases in MoP implants, which are more fibrotic and with a higher degree of necrosis (Chapter 2). It is not clear if those differences are due to a different immunological response or if they correspond to different severities of the same reaction. All ALTRs are characterized by a similar spectrum of inflammatory-degenerative changes, such as: synovial ulceration, variable degree of necrosis, and mononuclear infiltration. The infiltration of mononuclear cells can vary from a diffuse distribution of macrophages and/or lymphocytes to the presence of highly organized perivascular lymphocyte aggregates that resemble tertiary lymphoid organs (TLOs) (119, 122). The TLOs are structures composed of densely grouped T and B cells with a high degree of organization. They are considered to represent “an adaptation of the body to increased demand for a localized immune response” (123). TLOs have been described in different types of chronic inflammation diseases, which could originate from viral or bacterial infections, autoimmune diseases, smoking, environmental conditions, idiopathic fibrosis, and cancer (139–143). The role of TLOS in the progression of the disease could be beneficial or detrimental. For instance, TLOs have shown to 78 play an important role in the antitumor response in cancer, but they enhance the auto-immune response in rheumatoid arthritis (148, 149). Despite the critical role of TLOs in inflammatory diseases, their function in ALTRs has not been studied. Cytokines are intercellular communication molecules of the immune system. Cytokines can trigger tissue events that drive inflammation and necrosis, and thus, they are a key element to study when characterizing immune reactions. By using immunohistochemistry, a semi quantitative analysis of cytokine secretion showed that IL-6 and IL-1 are highly secreted in patients with failed implants (166). Analysis of synovial fluid demonstrated a higher concentration of MCP-1 in patients with MoM than patients with ceramic on ceramic hip replacements (185). This research suggest that cytokines are related in some way to implant failure, but their characterization and role in ALTRs have not been determined thus far. The results of the pilot study of cytokines in periprosthetic tissues (Chapter 2) revealed differences in the concentration of cytokines between ALTRs and tissues of patients with implants but without ALTRs. These studies, although limited, suggest that cytokines are associated with the presence of ALTRs and warrants further research in the etiology and diagnosis of ALTRs. This chapter presents a comprehensive analysis of the gene expression in TLOs in MoM and MoP ALTRs. The objective was to determine the similarities/differences in the immune-response related to the different implant designs, and to characterize the type of immune reaction in those lesions. To determine if the previously defined immunological characterization could be associated with the presence of cytokines in synovial fluid, a quantitative analysis of cytokines in the synovial fluid of patients was carried out. Finally, the effect of metal ions in the initiation of the immune response was studied. Primary synovial fibroblasts were exposed to Cr and Co ions, and their cytokine secretion was quantified, as was their ability to trigger an immune response through the activation of human endothelial cells and chemotaxis of human monocytes. 5.2 Study design and patients 79 Patients were recruited for this study after clinical suspicion of ALTR and ultrasound confirmation. Serum samples were obtained prior to surgery and frozen at -20 °C until analyzed. A total of 60 patients that underwent arthroplasty between May 2012 and December 2016 were included in the study: Twenty-six with MoM ALTRs, 15 with MoP ALTRs, 11 with MoM implants but without ALTRs (control MoM), five with MoP implants without ALTR (control MoP), and six undergoing primary hip replacement with no previous prosthesis. During the surgical procedure, synovial fluid samples were collected by aspiration, and centrifuged at 1400 rcf. The supernatants were stored at -20 °C until analyzed. Three tissue specimens were analyzed to determine the presence of Gramm + and Gramm – species. Patients with positive microbiological results were excluded from this study. 5.2.1 Gene expression analyses by use of Nanostring n-counter system: Up to ten microscope sections of five-micron thickness were stained with hematoxylin-eosin and Masson’s trichrome. Samples with lymphocyte aggregates larger than 1 mm, were selected for gene expression analysis by Nanostring n-counter system, and to perform immunohistochemistry in tissue microarrays (Fig. 5.1). 80 Figure 5.1 TLOs in ALTRs. Stain = H-E (A and C) and Prussian Blue (B and D). Prussian blue stain demonstrates the presence of macrophages (blue dye) in the TLOs. Bar = 1 mm. After the histological observation and confirmation of the presence of TLOs (by size, and presence of macrophages), the location of perivascular aggregates was determined in the paraffin embedded tissues of 10 MoM ALTRs and 10 MoP ALTRs. As a control group, 4 lymph nodes extracted during plastic surgery from healthy donors were used. The TLOs were punched out of the cassettes, and a formalin-fixed paraffin-embedded RNA-extraction kit was used according to manufacturer instructions (Roche). The RNA concentration and purity was determined by 81 spectrophotometry on Nanodrop ND-100 spectrophotometer (NanoDrop Technologies, Wilmington), the quality of the extracted RNA was confirmed by the RNA integrity number (OD 260/280). The RNA samples were diluted to a final concentration of 100 ng/l. Samples of 100 ng of RNA from each patient were assayed on a nCounter Digital Analyzer (NanoString), following the manufacturer’s instructions, at the Vancouver General Hospital. The RNA samples were subjected to analysis by nCounter GX Human Immunology V2 kit assay (NanoString, Seattle, WA), which includes 579 genes related to Immune system function and activation, as well as 15 housekeeping control genes. The Nanostring system consists of single target color-coded barcodes probes that are hybridized for 16 hours at 65 °C. These barcoded probes are detected by the 400x microscope included in the nCounter Digital Analyzer. The collected data was analyzed using the nSolver Analysis Software. After normalization and validation of the data, two outliers were excluded from the analysis: one patient in the ALTR MoM group and one in the ALTR MoP group. 5.2.2 Determination of cytokines in synovial fluid and serum. Samples of synovial fluid of 29 MoM ALTRs, 15 MoP ALTRs, 11 control MoM (With MoM implants but without ALTR), 5 control MoP (with MoP implants but without ALTR), and 5 patients undergoing their primary hip surgery (no implants), were used to analyze the cytokine profile of patients with ALTRs. The Bio-Plex Suspension Array System was used to quantify sixty-five different cytokines, chemokines and growth factors (65 plex-Eve technologies, Calgary): 6Ckine (CCL21), BCA-1(CXCL13), CTACK (CCL27), EGF, ENA-78 (CXCL5), Eotaxin (CCL11), Eotaxin-2 (CCL24), Eotaxin-3 (CCL26), FGF-2, Flt-3L, Fractalkine (CX3CL1), G-CSF, GM-CSF, GRO (CXCL1), I-309 (CCL1), IFNα2, IFNγ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, 82 IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-16, IL-17A, IL-18, IL-20, IL-21, IL-23, IL-28a, IL-33, IP-10 (CXCL10), LIF , MCP-1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), MCP-4 (CCL13), MDC (CCL22), MIP-1alpha (CCL3), MIP-1 (CCL4), MIP-1 (CCL15), PDGF-AA, PDGF-AB/BB, RANTES (CCL5), SDF-1 a+b (CXCL12), sCD40L, SCF, TARC (CCL17), TGF-α, TNF-α, TNF-β, TPO, TRAIL, TSLP, and VEGF. The same technology was used to analyze 11 other markers, corresponding to different panels (Human Cytokine P3 and Human Cytokine P4): M-CSF, I-TAC (CXCL11), MIG (CXCL9), MIP-3a (CCL20), HCC-4 (CCL16), HMGB1, BRAK (CXCL14), MIP-4 (CXCL18), IFNB, IL-34, YKL40/CHI3L1. The obtained results were compared to internal calibration curves, and thus the results were obtained as pg/mL. One-way ANOVA with Tukey post-hoc and Benjamini-Hochber procedure to decrease the false discovery rate, were used to determine the cytokines differences among the groups. Unsupervised hierarchical clustering was performed with the cytokines that showed significant differences between groups. A heat map that illustrates the relative concentrations of cytokines was rendered. A dendrogram showing the closeness of samples and cytokines in their distribution was generated to graphically illustrate the differences among the groups. These calculations were performed by use of the R statistical environment (R Foundation for Statistical Computing, Vienna, Austria). 5.2.3 Biomarkers in patient’s serum. The previously described multiplex (65-plex) analysis used for synovial fluid was performed in serum. Samples of 15 MoM ALTRs, 15 MoP ALTRs, 10 Control MoM, 5 Control MoP and 5 primaries were analyzed. This analysis was complemented by ELISA of other 7 molecules: Fibronectin 1, PLG2A2, C1s, TNFAIP6 and CCL18, M-CSF, IL-1RA. The ELISA kits (Raybiotech) for the above-mentioned molecules were used following manufacturer’s instructions. 83 5.2.4 Cell culture of human primary synovial fibroblasts. Synovial tissues from seven patients undergoing primary total hip arthroplasty due to osteoarthritis (4 women with a mean age of 67 years old) were obtained during the surgeries and preserved in Dulbecco's Modified Eagle Medium (DMEM) with 1% Penicillin / Streptomycin on ice for one hour. The tissues were sectioned and mechanically disrupted into small pieces (1 x 1 x 1 mm), then digested with 0.4 % Collagenase I (Gibco) in DMEM with 1% Penicillin / Streptomycin for 90 minutes at 37 °C under agitation. The solution was then filtered with 40 m grids (Falcon), and the obtained suspension was washed with PBS and pelleted, resuspended in DMEM and seeded in 10 cm petri dishes. Adherent cells were washed after 12 hours with PBS, and cultured with DMEM 10% fetal bovine serum, 5% glucose and 1% Penicillin / Streptomycin. In passage 3 the cells were transferred to 6-well plates to perform the metal exposure. 5.2.5 Analysis of cell cycle and cytokine release by metal exposed fibroblasts. Wells with 80% confluence were then exposed to 0.1 and 0.01 mM of Co or similar concentrations of Cr, in protein-free medium (DMEM with 1% Penicillin / Streptomycin) for 24 and 48 hours. The experiments were done in triplicate. Survival rate and cell cycle were analyzed by propidium iodine staining and flow cytometry. Aliquots of the culture media were obtained from all the samples and were analyzed for cytokine concentration through the Bio-Plex Suspension Array System as described previously (65-plex, Eve-technologies). As a positive control, fibroblasts were exposed to bacterial lipopolysaccharide (LPS), which triggers the secretion of inflammatory cytokines by different types of mesenchymal cells (186). A one-way ANOVA was performed to analyze the differences in the concentration of cytokines between the groups. 84 5.2.6 NF-kb activation In order to understand the mechanisms of cytokine release in metal exposed fibroblasts, a phosphorylation of NF-b assay was performed in the cell culture plates. The described primary fibroblasts were cultured in the presence of Co and Cr at concentrations of 0.1 mM and 0.01 mM. LPS at a concentration of 1 µg / mL was used as positive control. After 24 hours of exposure, the cells were incubated with 1:100 anti-phospho NF-B p65 (Ser536) antibody (93H1, Cell Signaling Technologies), followed by FIT-C conjugated secondary antibody incubation. 5.2.7 Preparation of synovial fluid and conditioned media concentrates. Synovial fluid samples extracted from patients were thawed, and centrifuged at 4,000 RCF for 15 minutes to discard possible metal particles. 1 ml of supernatant was then obtained and filtered in 200 nm syringe filters. The filtered solution was then concentrated by centrifugation using 3 kDa centrifugal filter tubes (Amicon Ultra Centrifugal Filters-4), at 4,000 g x 25 minutes at 4 °C concentrating the molecules with a molecular mass of 3 kDa or higher, and discarding water and metal ions. The final volume of synovial fluid was reduced to 1/5th, and thus, in the following experiments a 20% of the concentrate was used for cell stimulation. One mL of cell culture media obtained from the fibroblasts exposed to Co and Cr was filtered in 200 nm syringe filters and concentrated by centrifugation using 3 kDa centrifugal filter tubes (Amicon). The volume was reduced to 1/10 of the original volume, and therefore a concentration of 10% was used for cell stimulation. This conditioned media was frozen at -20 °C until used. 5.2.8 Expression of V-CAM and P-selectin in human endothelial cells. 85 HUVEC-derived endothelial cell line, EA hy926 (187) (EC), were cultured in DMEM-Low with 10 % FBS, 4 mM L-glutamine, 1 g/L D-glucose, 100 µg/mL penicillin–streptomycin and 2.5 µg/mL amphotericin. Cells were grown in an incubator at 37 ºC in a humidified atmosphere (95 % air and 5 % CO2). To analyze the effect of the conditioned media from fibroblasts exposed to metal ions, the described conditioned media of primary fibroblasts exposed to 0.1 mM of Co, 0.1 mM of Cr, and a negative control was used. The media corresponding to fibroblasts of three different patients was used in a concentration of 10 % in DMEM-Low with 1 % fetal bovine serum 4 mM L-glutamine, 1 g/L D-glucose, 100 g/mL penicillin–streptomycin and 2.5 g/mL amphotericin B for 24 hours. After 24 hours of incubation in triplicate, ECs were lysed in cold lysis buffer (4 ºC) with protease and phosphatase cocktail inhibitor. Then, the proteins were extracted and centrifuged at 15,000 RPM for 30 minutes at 4 ºC. Supernatants were collected and stored in the same lysis buffer. Protein extracts (with denaturing loading buffer) were subjected to 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis for 3 hours at 60 Volts and resolved proteins were transferred to polyvinylidene fluoride membrane in Trans-Blot® Turbo™ Transfer System (BIO-RAD). The membrane was blocked with 5 % non-fat milk in TBS-0.1% Tween-20 (TTBS) for 1 hour and incubated over night at 4 °C in agitation with the following primary antibodies: anti-VCAM-1 (PA5-29179, Thermofischer Scientific), anti P-Selectin (710281, ThermoFisher Scientific), and anti-−actin (ab8226, Abcam). Then, the membrane was washed twice with TTBS for 10 minutes and incubated with a peroxidase-conjugated IgG secondary antibody (anti-mouse AP307P, and anti-rabbit AP307P, Merk-Millipore) for 1 hour at room temperature. Bands were revealed using the Fotodyne™ FOTO/Analyst System™ (FOTODYNE Inc.). -actin was used as a loading control. The densitometric analysis was performed using image J program, and statistically compared by a one-way ANOVA, followed by a Tukey post-hoc. 5.2.9 Chemotaxis of THP-1 cells. 86 Human monocytes-type cell line THP-1 was used to test the effects of the cytokines secreted by synovial fibroblasts exposed to Co and Cr, and those present in the synovial fluid of patients. They were cultured in RPMI with 10 % heat-inactivated FBS. The chemotaxis assay of synovial fluid was performed in duplicates using synovial fluid obtained from 12 ALTR MoM and 10 ALTR MoP, 10 control MoM, 5 control MoP, 5 Primary surgery patients. Cobalt and chromium in concentration of 0.1 and 0.01 mM as well as LPS as positive control were also used. In the lower compartment of Boyden chambers (Cellbiolabs), 150 L of concentrated synovial fluid were placed in a 20 % concentration in DMEM high with 1 % Penicillin / streptomycin. In the upper chamber 100,000 THP-1 cells suspended in 100 L of DMEM were placed. After 24 hours incubation at 37 °C, the migration of cells was detected with the kit provided by the manufacturer and read at 490 nm. The chemotaxis assay of conditioned media was performed in triplicate. Conditioned media of fibroblasts from six patients that were exposed to 0.1 mM of Co or 0.1 mM of Cr for 24 hours were used. In the lower compartment, the concentrated conditioned media were plated on a 10% concentration in fresh medium. In the upper compartment a total of 100,000 THP-1 cells suspended in 100 L were incubated. After 24 hours of incubation, the number of cells that migrate to the lower chamber through the porous membrane was measured by use of the detection kit provided by the manufacturer, and read in a fluorescent plate reader at 490 nm. The migration in different conditions was compared by one-way ANOVA followed by Tukey post-hoc. 87 5.3 Results 5.3.1 Th1/Th2 immune reaction is characteristic of ALTRs. To characterize the immune reaction present in the ALTR lesions, we performed a gene expression analysis by use of Nanostring platform. After normalization and validation of the data, two outliers were excluded of the analysis one patient in the ALTR MoM group and one in the ALTR MoP group. Clinical variables such as patient’s age and gender, time of implantation, size of the lesions and concentration of cobalt and chromium in synovial fluid and serum had no effect on the gene expression in the lymphocyte aggregates (Table 5.1). Table 5.1 comparison of gene expression in TLOs according to clinical variables. P < 0.01 P < 0.05 Adj P < 0.01 Adj P < 0.05 total targets Age 43 127 1 2 480 Gender 6 37 1 1 Metal vs Poly 9 61 2 2 Size 4 14 2 2 Time 16 60 2 3 Metal vs Control 191 254 95 149 434 Poly vs Control 200 257 89 153 Diff. Exp. Metal vs Ctrl unique probes 16 Diff. Exp. Poly vs Ctrl 20 Diff. Exp. Metal vs Ctrl and Poly vs ctrl common probes 133 The comparison of the gene expression of MoM ALTRs and MoP ALTRs was performed for 480 of the 579 genes. The genes that showed less than the minimum threshold in 50 % of the samples were not considered in the study. The analysis showed that only two genes (out of 480) are differentially expressed between MoM and MoP, confirming that the immune reaction to both 88 types of articulations is biologically the same. When comparing ALTR MoM vs Control and ALTR MoP vs control, 149 and 153 genes showed significant differences, respectively, of which 133 were the same for MoM vs control and MoP vs control (Fig 5.2). The comparison of gene expression in MoM ALTRs vs control and MoP ALTRs vs control is shown in volcano plots (Figs 5.3 and 5.4). A list with the 50 genes with the higher increase in expression in MoM and MoP compared to controls, is presented in Appendix F. Genes were also ranked according to their expression in each of the groups. The genes that showed bigger changes in the ranking of expression in ALTRs compared to controls are shown in Appendix G. Figure 5.2 Venn diagram of genes differentially expressed between MoM ALTRs V/S Controls, and MoP ALTRs V/S controls. 89 Figure 5.3 Volcano plot of the comparison of gene expression in TLOs of MoM v/s Control. 90 Figure 5.4 Volcano plot of the comparison of gene expression in TLOs of MoP v/s Control. 91 The expression of cell – specific genes was used to generate relative cell scores for the analyzed samples. The lymphocyte aggregates of ALTRs in MoM and MoP hip replacements showed lower B-cell, NK cell, and Th17 cell scores than the controls (healthy lymph nodes), and a higher score for exhausted CD8 cells, Th1 and Th2 cells than in the control lymphoid tissue (Figs. 5.5 – 5.10). There were no differences in the cell scores between MoM and MoP ALTRs. The results of the gene expression analysis indicated that the immunological response in ALTRs is independent of the type of implant, and is a predominantly T-cell response, with no Th17 component. The presence of Exhausted CD8 confirms that ALTRs correspond to persistent chronic inflammation. The distribution of Th1 (T-Bet +), Th2 (GATA3+) and Th17 (IL-17+) cells, the T-cell regulators PD-1 (CD279), and B7-H3 (CD276) in controls MoM and MoP lymphocyte aggregates are observed in Figure 5.11. Figure 5.5 Gene expression based-cells scores for B-cells versus total lymphoid cells, comparing controls, MoM (Metal) and MoP (poly). 92 Figure 5.6 Gene expression based-cells scores for exhausted CD8+ cells versus total lymphoid cells, comparing controls, MoM (Metal) and MoP (poly) Figure 5.7 Gene expression based-cells scores for exhausted CD8+ cells versus total lymphoid cells, comparing controls, MoM (Metal) and MoP (poly). 93 Figure 5.8 Gene expression based-cells scores for exhausted Th1 cells versus total lymphoid cells, comparing controls, MoM (Metal) and MoP (poly). Figure 5.9 Gene expression based-cells scores for exhausted Th2 cells versus total lymphoid cells, comparing controls, MoM (Metal) and MoP (poly). 94 Figure 5.10 Gene expression based-cells scores for exhausted Th17 cells versus total lymphoid cells, comparing controls, MoM (Metal) and MoP (poly). 95 Figure 5.11 immunohistochemistry of TLOs in MoM ALTRs, MoP ALTRs and Controls. Shows distribution of Th1, Th2, Th17 cells as well as the check points inhibitors PD-1 and B7-H3. 96 5.3.2 Elevated concentration of inflammatory cytokines in synovial fluid of ALTRs affected joints. Next, we analyzed the concentration of 65 cytokines in the synovial fluid of patients with ALTRs in MoM implants, ALTRs in MoP implants and patients with MoM or MoP implants but not ALTRs (Control MoM and Control MoP groups). As a baseline, the synovial fluid of patients with no history of orthopedic implants, but undergoing their first hip replacement surgery, (Primary surgery) was used. The one-way ANOVA analysis of the concentration of 65 cytokines in synovial fluid showed significant differences in 28 of them between the groups. In order to visualize the differences in the distribution of the cytokine concentration between groups, we selected the 28 cytokines that showed significant differences in the one-way ANOVA analysis, and performed an unsupervised hierarchical clustering and heat-map based on the groups means (Fig. 5.12). The dendrogram showed that the highest similarity is observed between the control MoM and control MoP groups, which once clustered are more similar to primary patients than the affected MoM and MoP groups. Affected MoM and affected MoP groups were closer to each other than to any control group. These results confirm that there are important differences in the cytokine concentrations in the synovial fluid of patients with ALTRs, when compared with patients without ALTRs. 97 Figure 5.12 Heat-map and dendrogram generated by unsupervised hierarchical clustering of the concentration of cytokines in synovial fluid between the groups. C M = Control MoM, C P= Control MoP, P S = primary surgery, M A = MoM ALTRs, P A = MoP ALTRs. 98 Eleven of the analyzed cytokines had the highest concentration in patients with ALTRs (IL-1a, IL-1RA, IL-5, IL-6, IL-8, IL-13, IL-16, MCP-2, MCP-3, MIP-1a, ENA78). The plots for the cytokines that revealed a higher concentration in ALTRs compared to controls are shown in figures 5.13 to 5.23. Generally, the value of these cytokines is low in synovial fluid of primary surgery patients, increases in control patients, and reach their highest values in the presence of ALTRs, suggesting that the sole presence of implants is important to trigger an increase in concentration of these cytokines and affected patients have a higher concentration. Figure 5.13 Dot plots with mean and standard error of the concentration of ENA78 in synovial fluid of primary surgeries (PS) controls MoM and MoP and ALTRs. 99 Figure 5.14 Dot plots with mean and standard error of the concentration of IL-1 in synovial fluid of primary surgeries (PS) controls MoM and MoP and ALTRs. Figure 5.15 Dot plots with mean and standard error of the concentration of IL-1RA in synovial fluid of primary surgeries (PS) controls MoM and MoP and ALTRs. 100 Figure 5.16 Dot plots with mean and standard error of the concentration of IL-5 in synovial fluid of primary surgeries (PS) controls MoM and MoP and ALTRs. Figure 5.17 Dot plots with mean and standard error of the concentration of IL-6 in synovial fluid of primary surgeries (PS) controls MoM and MoP and ALTRs. 101 Figure 5.18 Dot plots with mean and standard error of the concentration of IL-8 in synovial fluid of primary surgeries (PS) controls MoM and MoP and ALTRs. Figure 5.19 Dot plots with mean and standard error of the concentration of IL-13 in synovial fluid of primary surgeries (PS) controls MoM and MoP and ALTRs. 102 Figure 5.20 Dot plots with mean and standard error of the concentration of IL-16 in synovial fluid of primary surgeries (PS) controls MoM and MoP and ALTRs. Figure 5.21 Dot plots with mean and standard error of the concentration of MCP-2 in synovial fluid of primary surgeries (PS) controls MoM and MoP and ALTRs. 103 Figure 5.22 Dot plots with mean and standard error of the concentration of MCP-3 in synovial fluid of primary surgeries (PS) controls MoM and MoP and ALTRs. Figure 5.23 Dot plots with mean and standard error of the concentration of MIP-1 in synovial fluid of primary surgeries (PS) controls MoM and MoP and ALTRs. 104 5.3.3 Cytokines that are highly concentrated in synovial fluid are not expressed in TLOs To determine whether the highly concentrated cytokines in synovial fluid are a product of the secretion of T cells in the lymphocyte aggregates, we then analyzed the gene expression of those cytokines in the Nanostring platform previously described through an unsupervised hierarchical clustering. Out of the 28 cytokines that showed significant differences in synovial fluid, 24 were analyzed in their gene expression, of which 13 did not reach the minimum threshold for analysis. Of the remaining 11, only IL-8 and RANTES have significantly higher expression in TLOs of MoM and MoP patients (Fig. 5.24), but four of them have significantly decreased expression (TNF-, Eotaxin-2, IL-16 and sCD40L), and no difference was observed in five of them. These results demonstrate that despite their critical role in ALTR, the TLO-like structures are not accountable for the differences of cytokines concentration in the synovial fluid. Figure 5.24 Heat map and dendrogram of the gene-expression of cytokines in the TLOs. 105 5.3.4 Cytokines highly concentrated in synovial fluid showed no differences in serum. The 65 cytokines, chemokines and growth factors were analyzed in serum obtained from 10 patients with MoM ALTRs, 10 patients with MoP ALTRs, 7 Control MoM, 4 Control MoP, and 3 primary surgeries. None of the analyzed cytokines, chemokines or growth factors revealed significant differences in serum of patients with ALTRs compared to controls. ELISAs of the molecules that had significant differences in the gene expression and/or their concentration in SF were also performed in serum from the described patients. The analysis of the concentration of PLA2G2A, fibronectin-1 (FN-1), CCL-18 (MIP-4), CCL-13 (MCP-4) and complement subunit 1s (C1S) did not show differences between the groups. 5.3.5 Chemotaxis of THP-1 cells is induced by ALTRs synovial fluid, but not by metal ions. In order to observe if the cytokines and other biomolecules and/or metals in the synovial fluid affects the inflammatory condition of the periprosthetic tissues of ALTRs, we analyzed the chemotactic potential of the synovial fluid of patients on human monocytes (THP-1 cells). Samples of synovial fluid from patients with MoM and MoP ALTRs, as well as control MoM and MoP, and primary surgeries were centrifuged to avoid the presence of metal particles, and the supernatant was concentrated by centrifugation in filtration tubes with a 3 kDa molecular weight cut-off, thus cytokines, chemokines and growth factors remain in the 20 % volume that was conserved, while water, ions and metabolites were discarded. The effect of synovial fluid of patients with ALTRs over human monocytes was analyzed by a chemotaxis assay. There was a significantly increased migration rate to synovial fluid of patients with ALTRs compared to controls (p-value < 0.05 for MoM and MoP), and the synovial fluid of controls MoM 106 and MoP have a higher chemoattractant potential than the synovial fluid of patients without implants (p-value < 0.05) (Fig. 5.25). Cobalt and chromium ions in concentrations of 0.01 mM and 0.1 mM had no effect in cell migration after 24 hours (not shown). This result demonstrates that cytokines, and perhaps other biomolecules present in the synovial fluid of patients with ALTRs, induce migration of leucocytes, and this capability is not produced by the metal ions. Figure 5.25 Chemotaxis of human monocytes exposed to synovial fluid. P S = synovial fluid from primary surgeries, C M = Control MoM, C P = Control MoP, M A = MoM ALTRs, P A = MoP ALTRs. 107 5.3.6 Synovial fibroblasts release inflammatory cytokines when exposed to concentrations of Co and Cr observed in ALTRs. To determine the role of mesenchymal cells of the hip joint in the secretion of cytokines during ALTR, as well as to determine the primary stimuli in the development of ALTRs, we exposed primary synovial fibroblasts to cobalt and chromium. To have a clinically relevant approach, we analyzed the concentration of Co and Cr in the synovial fluid of the patients with ALTRs in MoM and MoP. As we reported in chapter 4, cobalt in synovial fluid shows mean values of 536 g/L (0.009 mM) in MoM patients and 4225 g/L (0.072 mM) in MoP cases, while Chromium shows mean values of 243 g/L (0.004 mM) in MoM and 1665 g/L (0.032 mM) in MoP. Then concentrations of cobalt and chromium of 0.1 mM and 0.01 mM were selected to perform the subsequent studies. The secretion of 65 cytokines was measured in the culture media after 24 hours of exposure to metals. Synovial fibroblasts exposed to 0.1 mM of cobalt secrete significantly higher concentrations of Il-8, VEGF-, IP-10, IL-6 and FLT-3L (Fig. 5.26). A similar concentration of chromium induced the secretion of FLT-3L and MCP-1. 108 Figure 5.26 Release of IL-6 (A), IL-8 (B) and VEGF (C) by synovial fibroblasts exposed to metal ions in concentrations observed in synovial fluid of patients with ALTRs. 109 Figure 5.27 Release of FLT-3L and MCP-1 by synovial fibroblasts exposed to metal ions in concentrations observed in synovial fluid of patients with ALTRs. 110 To evaluate if the secretion of cytokines was dose-dependent, and also to observe if other cytokines could be secreted when exposed to higher concentrations of chromium and cobalt, we exposed the fibroblasts to increasing concentrations of chromium and cobalt (0.1, 0.25, 0.5, 0.75, 1.0 mM). Higher concentrations of chromium increase the release of FLT-3L and VEGF, with no increase in the concentration of IL-6 and IL-8 (fig 5.28). High doses of cobalt significantly increased the secretion of 12 cytokines in a dose dependent manner (IL-6, IL-8, VEGF, G-CSF, IP-10, TNF-alpha, GRO-alpha, IL-4, GM-CSF, Flt-3L, and IFN-y) (Fig. 5.29). The release of IL-6 and IL-8 increases up to 100 fold compared to the baseline (fig 5.29). Combined, these results demonstrate that synovial fibroblasts of patients, when exposed to the concentrations of Co and Cr that are observed in patients with hip replacements, secrete pro-inflammatory cytokines. Figure 5.28 Dose-dependent release of cytokines by synovial fibroblasts exposed to Cr. The chart show the mean values and standard deviation from cells obtained from 7 patients. 111 Figure 5.29 Dose-dependent release of cytokines by synovial fibroblasts exposed to Co. The chart shows the mean values and standard deviation from cells obtained from 7 patients. 112 5.3.7 Cobalt and Chromium ions induce activation of NF-B in synovial fibroblasts: To understand the molecular mechanisms of the above-mentioned secretion of cytokines, we then analyzed the activation of the nuclear factor NF-b (nuclear factor kappa-light-chain-enhancer of activated B cells) in primary synovial fibroblasts exposed to Co and Cr. NF-b is a nuclear transcription factor that is present in almost all animal cells, and its activation enhances the secretion of cytokines and chemokines. NF-b in its inactive form is located in the cytoplasm combined with its inhibitor IKK. When activated, the phosphorylated NF-b translocates to the nucleus and binds DNA to promote the gene expression of cytokines. In our experiment, phosphorylated NF-b was detected in the nuclei of fibroblasts exposed to Co and Cr at both concentrations (0.1 and 0.01 mM), and also in the positive control (LPS), but not in the negative control (Fig. 5.30), demonstrating that the secretion of cytokines after exposure to metal ions is associated to NF-b activation. 113 Figure 5.30 Nuclear translocation of phospho-NF-B in cells exposed to 0.01 mM of Cr and Co. Antibody conjugated with FITC, cytoskeleton stained with phalloidin. 114 5.3.8 Conditioned media of metal-exposed fibroblast induce chemotaxis in human monocytes and activates human endothelial cells. To prove that the secretion of cytokines by fibroblasts exposed to Co and Cr could have a direct effect in triggering inflammation, we evaluated the endothelial activation and the chemotaxis of monocytes after stimulation with the fibroblast’s secreted molecules. The activation of Human endothelial cells was observed by quantifying the expression of P-Selectin and ICAM by HUVECs cells. Both (ICAM and P-Selectin) are cell adhesion molecules that allow the rolling and firm adhesion of leucocytes to the endothelium during inflammation. The adhesion of leucocytes to the blood vessel is a key process during inflammation, allowing the presence of inflammatory cells in the injured tissue. Monocytes are the circulating precursors of macrophages. They are recruited by chemoattractant molecules such as cytokines and chemokines during inflammation. We used a porous membrane that separated monocytes from the conditioned media from fibroblasts, and the migration of cells through the membrane was evaluated by fluorescence. ICAM and P-Selectin significantly increased their expression after the exposure to conditioned media of fibroblasts exposed to Co and Cr ions (Fig. 5.31). Metal ions do not have a direct effect on the expression of P-Selectin and ICAM after 24 hours of exposure. The migration of THP-1 cells exposed to conditioned media from fibroblasts incubated with cobalt was significantly higher than the control group (conditioned media from fibroblasts with no metal added), but no difference was found in the conditioned media of fibroblasts exposed to chromium. Interestingly, cobalt and chromium themselves do not have any effect on cell migration (Fig 5.32). 115 These results demonstrate that the sole exposure of fibroblast to a concentration of metals similar to the one observed in synovial fluid of patients with ALTRs, can activate the endothelium and recruit cells of the immune system, which is the starting point of inflammation. Figure 5.31 ICAM (A) and P-selectin (B) expression in HUVECS cells incubated in conditioned media obtained from Co and Cr exposed primary synovial fibroblasts. 116 Figure 5.32 Monocyte chemotaxis to conditioned media of fibroblasts exposed to Cr and Co. Chemotaxis of THP-1 cells was measured by use of a trans-well insert. 117 5.4 Discussion: 5.4.1 ALTRs in MoM and MoP hip replacements correspond to a similar immunological entity. The results of the analysis of gene expression in the perivascular aggregates of ALTRs, showed no differences in the reaction to MoM and MoP hip replacements. A few studies previously described differences in the histological appearance of the lesions (97); however, all histological descriptions of ALTRs in MoP implants described similar morphological elements as necrosis, mononuclear infiltration and the presence of perivascular monocyte infiltrates (6, 71, 97). The differences in the extension of necrosis reported previously in this work (chapter 3), and by other authors, could be related to the clinician’s suspicion rate and awareness, that may extend the pathologic process for a longer time in MoP implants in comparison with MoM, in which there are established protocols and follow-ups. However, the higher extension of necrosis that has been previously described in MoP ALTRs does not correlate with the implantation time, which was similar for both types of implants. 5.4.2 Hypersensitivity may not play a role in ALTRs etiology. Considering the histological features of ALTRs, as the presence of granuloma and giant multinucleated cells, some authors have proposed that a type IV hypersensitivity response is present in the pathogenesis of ALTRs (84, 93). The results obtained in the gene expression analysis of perivascular lymphocyte aggregates exhibited a low score for TH17 cells, which has been indicated as a driver of type IV hypersensitivity diseases, such as atopic dermatitis, granuloma and sarcoidosis, multiple sclerosis and tuberculosis (134, 188–191). These results of the gene expression are consistent with the concentration of IL-17 in synovial fluid, which is lower in patients with ALTRs than in controls and in patients undergoing primary surgery. This cytokine is mainly secreted by Th17 cells and it has been highly related to allergic and autoimmune diseases 118 such as lupus, rheumatoid arthritis, asthma, allograft rejection, psoriasis and multiple sclerosis (192, 193). Thus, if involved in ALTRs pathogenesis, hypersentivity mechanisms do not up-regulate the Th17 pathway and do not enhance the secretion of IL-17. In our cell culture study, we observed that synovial fibroblasts exposed to relatively low concentrations of cobalt ions, can significantly increase the secretion of pro-inflammatory cytokines, which are able to induce chemotaxis in human monocytes. These experiments demonstrated that the cells of the hip joint can react and trigger an immune reaction when exposed to Co and Cr ions in serum free media. This suggests that inflammatory events may happen without the presence of a hapten-carrier reaction. A T-cell activation could happen as consequence of the inflammation and perhaps an hapten-carrier reaction occurs in the periprosthetic tissues, but the inflammatory changes could be triggered in absence of such a reaction. 5.4.3 Th1-Th2 reaction in ALTRs. The gene expression analysis also revealed that the type of reaction corresponds to a Th1 and Th2 reaction, which is not common since these two types of responses are known to be mutually inhibitory (194). Our observations confirm a previous description based on immunohistochemistry by Perino et al (2014). In their study, the perivascular aggregates showed cells positive for T-Bet and cells positive for GATA3, which are transcription factors of Th1 and Th2 respectively (97). However, the meaning of these findings are yet to be understood, since the plasticity of the commitment of CD4 cells to a specific lineage (Th1, Th2, Th17) has been recently demonstrated, and the classic canonic view is matter of current review (195, 196). Indeed, it has been demonstrated that Th2 committed Cd4 cells, can adopt a Th2+1 phenotype when exposed to a Th1 environment, keep this phenotype for months after the exposure (197). As such, our results may be explained by the presence of a chronic inflammation, that persist by experiencing permanent cellular damage due to the presence of metal ions, and enhanced phagocytosis induced by the particulate material that could be observed in the tissues. 119 5.4.4 Presence of cytokines in synovial fluid. This work includes a thorough analysis of cytokines in synovial fluid of patients with ALTRs and compared them with control cases and patients with osteoarthritis, but no previous metal implants in their bodies. Cytokines in synovial fluid have been used in the research of the pathogenesis and as possible diagnosis tools for osteoarthritis (198, 199), and infections of hip prosthesis (200, 201), but limited studies have analyzed the presence of cytokines in synovial fluids associated to ALTRs. A previous study determined the increase of MCP-1 in synovial fluid in patients with MoM articulations and ceramic on ceramic, but the increase in the first group was significantly higher, and both groups of implants was higher than the control group (185). This observation is confirmed with the results of our study, in which MCP-1 was increased in MoM and MoP ALTRs. In a different study, through antibody microarrays, Kolatat et al. reported that IP-10 and MIG are significantly higher in synovial fluid of patients with ALTRs when compared to patients with osteolytic lesions associated to hip replacements (202). Previously the presence of the pro-inflammatory cytokines IL-1, IL-6 and TNF- in the periprosthetic tissues of failed MoM THA and HR was reported by Catelas et al. (2003). They analyzed the presence of the mentioned cytokines through immunohistochemistry in histological sections, finding no differences according to the implant type and time of implantation, and showing a weak correlation with the particle load of the tissues. These aforementioned studies aimed to establish a comparison within implant types or lesions, and do not present a control group, which prevent conclusions regarding the presence of implants in the secretion of the cytokines (166, 202). In our study, we included a comparison of ALTRs with control patients and the baseline conditions in patients without implants. Then the results of elevated concentration of cytokines in synovial fluid of patients with ALTRs has no precedent in the scientific literature, and may set the basis for accurate diagnosis of the implants conditions with minimally invasive procedures. 120 5.4.5 Effects of metals in cytokine secretion of synovial fibroblasts. The effects of cobalt and chromium ions in different cell populations have been matter of study in the recent years, their capability to induce cell death and cytokine secretion in peripheral mononuclear cells or monocyte cell lines has been demonstrated (Granchi, Cenni, et al. 1998; Granchi, Verri, et al. 1998; Huk et al. n.d.), but there are limited studies on their effect on fibroblasts. A study in dermal fibroblasts showed that nanoparticles of Co-Cr-Mo alloy produce a higher rate of cell death than similar volume of micron-size particles, suggesting that the higher surface of the nano-sized material allowed a higher ion release, which would increase the cell damage. They also showed a lack of cytokine response (IL-6) of this type of cells to the metal particles (155). This lack of secretion of Il-6 was previously reported for fibroblasts obtained from the knee ligaments exposed to cobalt alloy particles (156). Our results differ from those previous reports. The primary synovial fibroblasts from the hip joint used in this study were able to secrete elevated levels of several pro-inflammatory cytokines and chemokines. Moreover, under higher concentrations of metals, the secretion of IL-6 and IL-8 was increased up to 100 times higher than the basal state, and an increase of 10 to 20 times was observed for VEGF, IP-10, G-CSF and TNF-. These differences may be due to the use of metal ions in our case compared with the use of particles in the precedent studies (155), and thus, the secretion of cytokines seems to be caused by the metals in solution and not by particulate material. More importantly, in our study we used fibroblasts from patient with osteoarthritis, which is a chronic inflammatory disease and the initianl condition of most patietns that ereceive hip replacements. This inflammatory environment could affect the secretion of inflammatory cytokines by the fibroblasts during cell culture experiments. The activation of HIF nuclear factor in monocytes exposed to cobalt nanoparticles was recently demonstrated by Nyga et al. (2014) in cell culture experiments using human macrophage cell lines and alveolar macrophages. They demonstrated that some of the genes downstream of 121 HIF could get upregulated, such as VEGF and TNF- and this effect could be caused by nanoparticles and by cobalt ions (157). They demonstrated in their study that the activation of HIF pathway induced by nanoparticles is independent of ROS, but the mechanism is unclear. In our study, VEGF was highly secreted by synovial fibroblasts, which suggests that a similar mechanism affects synovial fibroblasts, and that they are able to respond through the secretion of cytokines. Furthermore, in our experiments, we demonstrated that during the exposure of synovial fibroblasts to Co and Cr, there is an activation and nuclear translocation of NF-b, which has been shown to be an upstream regulator of HIF (205). 122 Chapter 6: Conclusion The main conclusion of this work is that the presence of elevated levels of metal ions in the hip joint triggers the release of cytokines, which are able to initiate the inflammatory response observed in ALTRs. This conclusion was possible to achieve through the development of the three research chapters of this thesis. The first chapter defined histologically the ALTRs in MoM and MoP implants, identifying cellular components and providing a pilot study on cytokines in the tissue that gave evidence of the involvement of the immune system in the pathogenesis of ALTRs. The second chapter demonstrated that the concentration of Co and Cr ions in the hip joint fluid is significantly higher in patients with ALTRs, and thus a possible factor in the etiology of ALTRs. In the third chapter, the knowledge of the histology that was gathered in the first chapter was used to perform an in-depth gene expression analysis in the perivascular lymphocyte aggregates. This analysis provided novel information about the activity of the immune system in ALTRs, as well as helping to define the characteristics of the immune reaction in ALTRs. Following the evidence of cytokines in periprosthetic tissues that was observed in the first research chapter, a full analysis of cytokines in synovial fluid was carried out. Significant differences in the cytokine concentration between patients with ALTRs compared to patients without ALTRs were demonstrated. Finally, the data obtained from the fourth chapter, regarding the concentration of Co and Cr in the synovial fluid was used to establish "in-vitro” conditions, in which synovial fibroblasts obtained from the hip capsule were exposed to Co and Cr. The results of this study showed that synovial fibroblasts respond to the presence of metals through the secretion of cytokines. These cytokines are capable of activating human endothelium and inducing chemotaxis of human monocytes, which are starting points of inflammation, and may be the triggering event for the development of ALTRs (Fig. 6.1). 123 Render by estudiokbza.cl Figure 6.1 Role of metal ions in the etiology of ALTRs. The histological differences observed between MoM and MoP ALTRs described in chapter 3, did not find a relationship with the gene-expression in the lymphocyte aggregates as described in chapter 5; while histologically the MoM lesions are described as cystic – inflammatory lesions, the MoP ones are observed as fibro – necrotic, the gene expression analysis showed that they correspond to identical immunological reactions. The reason for this could not be explained in this thesis, and the analysis of the necrosis, and the process of cell death in the tissues and cell culture systems is part of the future work in the project. 124 6.1 Limitations. Number of patients with ALTRs: The number of MoM and MoP ALTRs, although a relevant complication is low, especially the MoP ones that have a much lower prevalence. The numbers presented in this work (40 MoM and 30 MoP) are comparable to most of the prospective studies in the field, but smaller than studies that are based on analysis of clinical files, histopathology reports, or analysis of retrieved implants, that can include hundreds of cases (80, 98, 206). Low number of Primary surgeries and control MoP patients: These are the most critical numbers in our study (5 primary surgeries, 6 Control MoP); The main reason for the low numbers in the analysis of synovial fluid of these patients is due to the fact that these patients present minimum amounts of synovial fluid. In our case, we attended 30 surgeries for primary hip replacements, and obtained synovial fluid enough to be analyzed (500 microliters) in only 5 cases. Similar case, but not as dramatic is the MoP ALTRs, in which out of 30 surgeries, synovial fluid could be obtained only in 15 cases. Fortunately, the primary surgery group was very homogeneous in the concentration of cytokines and metals, which gives us a valid baseline to compare the affected patients. The MoP controls are not as homogeneous as the primary surgeries, but their results are backed up by their similarity with the results of the control MoM group (no differences in ANOVA and grouped together in unsupervised hierarchical clustering). The analysis of particles in the periprosthetic tissues: The main one is related to the detection of particles is limited to the resolution of the optical microscope, thus particles smaller than 0.5 micron were not observed. Thus, the quantification presented is based on large particles or clusters of them. It is know that the particles generated by the corrosion of the metal implants usually size in the submicron level (95, 181). Similar limitation has the analysis performed with SEM and SEM-EDX. The EDX can provide accurate information of chemical composition on elements with a depth of 5-10 um, but limited information can be obtained from smaller samples. 125 The synchrotron-energized XRD analysis presented in this study correspond to the analysis of two particles in the periprosthetic tissues. This is due to the limited access to this technology. However, the results agree with previous descriptions of a mostly amorphous structure of the corrosion particles. The study of Co and Cr concentration in synovial fluid performed in this work, by use of ICP-MS, did not include analysis of particulate material in this fluid. The presence of small particles was avoided by sample centrifugation, but this mechanism may not be sufficient to remove nanometer-sized particles, and thus a more thorough mechanism of quantification of soluble ions needs to be developed. To support the main conclusion of this project, that cytokines secreted by synovial fibroblasts exposed to metal ions can trigger pathological events such as cell migration (Chapter 4), an experiment that block cytokines need to be performed in order to confirm that are cytokines and no other elements are responsible for triggering the chemotactic effect. This experiment of blocking was not possible to perform due the fact that there is a myriad of cytokines that could be highly expressed in the metal exposure conditions. Damage associated molecular pattern (DAMPs): It is well know that there are molecules released by cells under stress of undergoing a process of cell death, that could be parts of their structure, or functional proteins (i.e. myosin, DNA, ATP), that can act as chemoattractant or activating blood vessels, thus triggering inflammation (207). In this study, we did not analyze DAMPs (neither tissues, synovial fluid and cell culture). This is part of the future work that should be performed in this project. A major limitation in measuring the effects of metals in synovial fibroblasts, and their consequent secretion of cytokines was performed in 24 hours, and no longer periods to observe chronic effects of the metals were included in this work. 126 6.2 Future work This work made progress in understanding the mechanisms involved in the development of ALTRs by tissue analysis of metal elements, histology and also a mechanistic study. The cell culture study allowed us to propose mechanisms that are dependent on the presence of metal ions and release of cytokines by the synovial fibroblasts. However, this model ignores the participation of cells of the immune system, at the time to avoid the complexity of the cellular relations that exists in the tissues. To improve our understanding of the pathogenic mechanisms of ALTRs involving those variables, an animal model for the study of the disease is needed. This will provide more elements for the analysis of this complexity. The obtained results showed that fibroblasts are a key player in the initiation of the adverse reaction to metal implants, and also seem to be involve in the final outcome of the disease by generating the extensive fibrosis that can be observed in ALTR cases. Nevertheless, fibroblasts were not characterized in the lesions. Are they myofibroblasts? How is their proliferation state? Do they respond by proliferating after being exposed to the cytokines? These questions should be answered in a future work to explain the whole pathogenic process and fibrosis in ALTRs. Our study described the presence of wear particles in tissues surrounding MoP implants. A further analysis of these particles, how and where they are originated may be key to understand the development of ALTRs in MoP articulations, which is the most common type of implant used currently. It is known that damage associated molecular patterns (DAMPs) are released in situations of cell stress and cell death. In our study, we focused on the release of cytokines, but the effects of DAMPs (if present) in triggering inflammation could be critical in the development of ALTRs, and thus their presence in synovial fluid as well as their release in cells exposed to metals should be analyzed to fully understand the etiology of ALTRs. The mechanisms of cell damage and death associated to the presence of Co and Cr were not studied in this project. It is well documented that cell death is one of ALTRs main 127 characteristics. The presence of Co is known to generate hypoxic stress in different cell populations, but the mechanisms of cell death are not known. This is a fundamental question to understand the tissue damage generated by orthopedic implants. 128 6.3 Contributions. The main contribution of this thesis is the demonstration that inflammatory changes that initiate the development of ALTRs, can be triggered by the presence of metal ions in the joint fluid. Those ions trigger the secretion of pro inflammatory cytokines by the fibroblasts, which is the structural and functional cell in the hip capsule. Previous studies defined fibroblasts as a non-reactive cell, and most of the studies of the effects of metals in cell populations used monocytes or lymphocytes. This demonstration of the reactivity of fibroblasts is essential to explain the etiological mechanisms of ALTRs. A second major contribution is the description of the immune reaction in ALTRs as a Th1/Th2 reaction, and no Th17 component, as observed in the gene expression analysis and confirmed by the low levels of IL-17 in the synovial fluid. This combined with the effect in cytokine secretion of fibroblasts after exposure to metal ions, gives evidence to prove that ALTRs are non-specific chronic inflammation reactions, and not associated with metal hypersensitivity. A third contribution is the demonstration of significant differences in the concentrations of metals in synovial fluid of patients with ALTRs compared to control patients for MoM and MoP implants. Limited studies have analyzed synovial fluid in MoM ALTRs, and no studies in MoP ALTRs were published before our work. This could lead to development of diagnostic mechanisms of ALTRs in MoP. The fourth contribution is the description of differential cytokine concentration in synovial fluid associated to ALTRs when compared to control patients in both MoM and MoP implants. 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Stain H-E. 152 Appendix D Microscopic imaes of collagen structure in ALTRs: A picrosirius red under light microscopy. B Picrosirius red under polarized light. C picrosirius red under second harmonic generation. 153 Appendix E Bonferroni correction of ANOVA analyzed concentration of cytokines in tissue homogenates. The data show the cytokiens that showed significant differenes between the groups, specifying which groups have significant differences in their concentration. Bonferroni Group 1=MoP, group 2=MoM, Group 3 =Control Molecule Group V/s Group Mean Difference (I-J) Std. Error Sig. Angiogenin 1 2 -11009.5833* 3072.5113 .011 3 -11156.1500* 3193.0474 .013 2 3 -146.5667 2882.2711 1.000 Angiopoietin-2 1 2 -3005.3333* 1014.6563 .036 3 -324.1000 1054.4618 1.000 2 3 2681.2333* 951.8320 .047 Axl 1 2 -1473.7917 549.5156 .060 3 -1426.9250 571.0733 .084 2 3 46.8667 515.4913 1.000 CNTF 1 2 -55.0833* 18.0542 .030 3 -58.2000* 18.7625 .027 2 3 -3.1167 16.9364 1.000 Fas/TNFRSF6 1 2 -480.4583* 162.2431 .036 3 -86.9250 168.6080 1.000 2 3 393.5333 152.1975 .072 FGF-6 1 2 -77.7917 40.3818 .234 3 51.5750 41.9660 .728 2 3 129.3667* 37.8815 .015 HGF 1 2 -3252.0833* 1036.5797 .026 3 -1393.8500 1077.2452 .660 2 3 1858.2333 972.3979 .241 I-309 1 2 25.2500 11.1446 .128 3 46.0500* 11.5819 .006 2 3 20.8000 10.4546 .210 IFN-gamma 1 2 -77.7917 48.2207 .398 3 -148.7250* 50.1124 .035 2 3 -70.9333 45.2350 .429 IL-10 1 2 -110.4167* 26.8098 .004 3 -134.4500* 27.8615 .001 2 3 -24.0333 25.1498 1.000 154 Bonferroni Group 1=MoP, group 2=MoM, Group 3 =Control Molecule Group V/s Group Mean Difference (I-J) Std. Error Sig. IL-12 p40 1 2 41.1667 16.4810 .084 3 46.9500 17.1276 .054 2 3 5.7833 15.4606 1.000 IL-13 1 2 -117.1667 54.6662 .160 3 -160.5000* 56.8108 .046 2 3 -43.3333 51.2815 1.000 IL-15 1 2 -88.2083* 24.0463 .010 3 -127.0750* 24.9896 .001 2 3 -38.8667 22.5574 .332 IL-1alpha 1 2 -94.8750* 21.2493 .002 3 -105.8750* 22.0830 .001 2 3 -11.0000 19.9336 1.000 IL-1ra 1 2 -62.0417* 20.6904 .033 3 -50.8750 21.5021 .107 2 3 11.1667 19.4093 1.000 IL-2 1 2 -126.0000* 24.6163 .001 3 -168.4000* 25.5820 .000 2 3 -42.4000 23.0921 .274 IL-7 1 2 -91.4583* 29.2565 .026 3 -184.0750* 30.4043 .000 2 3 -92.6167* 27.4451 .017 I-TAC 1 2 -61.2917* 19.7871 .028 3 -20.6250 20.5633 1.000 2 3 40.6667 18.5619 .147 M-CSF 1 2 -100.2083 37.7511 .063 3 -178.0250* 39.2320 .002 2 3 -77.8167 35.4136 .145 MIG 1 2 -108.0417* 38.4769 .047 3 -145.3250* 39.9864 .010 2 3 -37.2833 36.0946 .966 MSP-alpha 1 2 -375.8750 138.5798 .057 3 -15.9250 144.0164 1.000 2 3 359.9500 129.9994 .051 sTNF RII 1 2 -1583.3750* 568.4854 .049 3 -627.3250 590.7874 .928 2 3 956.0500 533.2866 .295 TGF-beta 1 1 2 -118.2500* 28.5542 .004 3 -131.0000* 29.6744 .003 155 2 3 -12.7500 26.7862 1.000 Bonferroni Group 1=MoP, group 2=MoM, Group 3 =Control Molecule Group V/s Group Mean Difference (I-J) Std. Error Sig. TIMP-1 1 2 443.9167 571.3582 1.000 3 1799.1000* 593.7729 .031 2 3 1355.1833 535.9815 .079 TNF-alpha 1 2 -89.1250 33.1332 .059 3 -124.0250* 34.4330 .011 2 3 -34.9000 31.0817 .850 TNF-beta 1 2 -118.2917* 39.9497 .036 3 -148.9750* 41.5169 .011 2 3 -30.6833 37.4761 1.000 VEGF 1 2 495.3750 183.2569 .058 3 462.5250 190.4462 .095 2 3 -32.8500 171.9102 1.000 156 Appendix F Fold change and p-value of genes in TLOs from MoM and MoP ALTRs. Only the 50 genes with higher fold change differences are shown GENE Fold change MoM Fold change MoP P-value MoM vs Cont P-value MoP vs cont PLA2G2A 4205.1 2460.51 0.001500842 0.000485504 CLEC5A 175.92 94 0.015918388 0.012924357 FN1 125.41 80.09 0.000246716 0.000238391 TNFAIP6 121.58 64.67 0.004232587 0.000621344 CCL18 75.93 40.91 0.001715493 0.035011751 CCL13 46.1 48.18 0.005867489 0.027668468 IL1RN 44.41 38 0.006311689 0.019909214 SELE 38.83 30.73 0.001642792 0.032339337 FCGR1A/B 45.3 17.39 0.000241625 0.002390591 MUC1 27.83 25.12 7.01882E-05 9.65951E-06 SPP1 37.87 13.73 0.020336887 0.015972994 MSR1 26.15 19.48 0.00116558 0.000868405 LILRA6 23.59 13.64 0.000215522 0.003413333 CCL11 18.09 17.19 0.021663761 0.087652155 FCGR3A/B 19.19 14.92 0.000163064 0.00153347 IL8 15.41 13.68 0.046149845 0.037129734 LILRB3 12.82 11.74 8.66931E-06 0.000763896 THY1 13.95 10.54 0.044434358 0.000165519 PLAU 11.05 11.15 5.80466E-07 6.24625E-05 CXCL10 8.76 12.47 0.010256864 0.003447306 CCL7 12.46 8.49 0.029370883 0.100347572 CXCL9 9.42 9.85 0.006608383 0.000122952 C1R 10.24 7.98 2.38567E-05 3.0888E-05 FCGR2A 8.92 8.63 4.51401E-05 0.000252851 S100A9 9.92 5.77 0.00185861 0.001589825 CD276 7.78 7.11 0.011773371 0.000473566 157 GENE Fold change MoM Fold change MoP P-value MoM vs Cont P-value MoP vs cont CCR8 6.61 8.09 0.008677376 0.005918778 CLEC4E 7.04 7.4 0.007887231 0.009179924 CFB 7.64 6.77 0.003162575 0.001142242 CD9 7.48 6.33 0.000437589 0.002127746 CCR1 7.87 5.29 0.000170088 0.00297983 CD70 8.99 4.17 0.000486551 0.00442514 CCL8 6.23 6.9 0.002326453 0.014939493 CLEC6A 5.65 7.21 0.051197778 0.068363343 TLR3 7.78 4.96 0.000129222 0.004130815 ARG2 7.18 4.79 0.093647925 0.003610206 DEFB1 10.25 1.56 0.017061264 0.205022273 S100A8 7.65 4.03 0.048614665 0.004604671 LILRB4 5.97 5.37 0.001083751 0.010839182 TLR8 5.61 5.2 0.001267892 0.000363509 CFD 6.07 4.63 0.023911424 0.045446226 C1S 5.98 4.56 2.83284E-06 6.45217E-05 CFH 5.87 4.63 0.0001085 0.001745687 CD163 5.95 4.25 5.21613E-05 0.001171531 LILRA3 5.96 4.05 0.060659945 0.090920541 IL12B 6.83 2.13 0.064901235 0.131930423 CLU 6.37 2.38 0.017863605 0.094896832 TNFSF15 6.02 2.14 0.000709199 0.03135275 HAMP 6.2 1.44 0.047431115 0.380446534 158 Appendix G Rank changes of genes in TLS in Control MoM and MoP. Genes are organized according to their rank differences between ALTRs and Controls. Only genes with rank changes bigger than 50 are shown. Ranking Rank difference Gene Control MoM MoP MoM - Control MoP - Control PLA2G2A 491 9 13 -482 -478 CCL13 459 90 67 -369 -392 SPP1 371 21 19 -350 -352 CCL18 330 8 8 -322 -322 MSR1 455 103 142 -352 -313 IL8 464 281 154 -183 -310 CXCL10 426 140 126 -286 -300 FCGR3A/B 341 64 57 -277 -284 CLEC5A 537 323 255 -214 -282 FN1 283 4 7 -279 -276 PLAU 367 110 93 -257 -274 THY1 327 56 64 -271 -263 FCGR2A 334 82 81 -252 -253 CFB 358 105 124 -253 -234 CD276 340 109 107 -231 -233 LILRB4 304 76 77 -228 -227 CD9 300 72 83 -228 -217 PLAUR 352 215 140 -137 -212 CXCL9 244 31 35 -213 -209 CCL8 461 311 253 -150 -208 S100A9 277 45 71 -232 -206 S100A8 384 120 188 -264 -196 CCR1 386 177 190 -209 -196 LAG3 451 309 268 -142 -183 PTAFR 361 196 186 -165 -175 EGR2 437 372 263 -65 -174 TLR2 332 188 168 -144 -164 CLEC4E 475 331 312 -144 -163 LILRA6 481 260 319 -221 -162 ITGAM 261 134 99 -127 -162 159 ITGA5 243 101 82 -142 -161 CFH 208 59 54 -149 -154 DPP4 440 272 287 -168 -153 TNFAIP6 516 316 369 -200 -147 NT5E 317 153 171 -164 -146 IL1RN 535 443 395 -92 -140 FCGR2A/C 190 53 51 -137 -139 SELE 497 365 358 -132 -139 GBP1 253 143 115 -110 -138 TFRC 254 164 118 -90 -136 CFD 282 112 148 -170 -134 CXCL11 458 344 324 -114 -134 C3 177 60 45 -117 -132 GBP5 398 310 267 -88 -131 TLR8 460 318 331 -142 -129 FCGR2B 223 94 98 -129 -125 CLEC7A 432 320 307 -112 -125 CCL4 234 187 117 -47 -117 LILRB3 486 376 371 -110 -115 RUNX1 299 200 184 -99 -115 CEBPB 183 96 68 -87 -115 CCL2 236 98 122 -138 -114 C2 200 67 87 -133 -113 LGALS3 166 40 53 -126 -113 PDGFRB 156 55 50 -101 -106 CDKN1A 247 154 141 -93 -106 CCL26 533 509 427 -24 -106 C1R 123 17 18 -106 -105 HAVCR2 375 257 271 -118 -104 CCR5 414 327 310 -87 -104 CD163 140 36 43 -104 -97 LTBR 281 156 185 -125 -96 TNFSF13B 296 197 200 -99 -96 LILRB2 348 271 254 -77 -94 IL13RA1 195 92 102 -103 -93 CCL11 539 480 446 -59 -93 CFI 312 157 221 -155 -91 CD58 314 206 224 -108 -90 C1S 120 26 30 -94 -90 160 GZMA 264 190 174 -74 -90 PDCD1LG2 315 246 226 -69 -89 NOD2 431 366 342 -65 -89 CMKLR1 197 83 110 -114 -87 IFNG 502 436 417 -66 -85 DUSP4 467 406 383 -61 -84 CD86 380 287 297 -93 -83 MUC1 510 422 429 -88 -81 CCL5 165 119 86 -46 -79 CCL7 540 489 462 -51 -78 NFIL3 407 374 333 -33 -74 NLRP3 434 362 362 -72 -72 STAT1 124 63 52 -61 -72 SRC 342 280 272 -62 -70 MME 396 425 327 29 -69 GPI 159 93 91 -66 -68 GZMK 203 158 135 -45 -68 IL1R1 362 270 295 -92 -67 FCER1A 435 349 368 -86 -67 CD274 370 348 303 -22 -67 CAMP 509 456 443 -53 -66 RARRES3 230 178 166 -52 -64 CLEC4A 349 325 285 -24 -64 TGFBR1 221 168 159 -53 -62 IL6 484 474 422 -10 -62 CCRL2 447 359 387 -88 -60 BST1 390 333 330 -57 -60 LILRA5 446 368 388 -78 -58 TGFBI 81 24 23 -57 -58 CD14 82 30 24 -52 -58 LIF 469 465 411 -4 -58 SLC2A1 415 420 357 5 -58 ITGAX 131 89 75 -42 -56 CCL3 229 261 173 32 -56 FCGR1A/B 538 464 483 -74 -55 AHR 116 54 61 -62 -55 CLEC6A 542 504 488 -38 -54 SLAMF7 269 234 215 -35 -54 LY96 251 201 198 -50 -53 161 TLR3 476 389 425 -87 -51 CCBP2 487 449 436 -38 -51 FAS 454 423 404 -31 -50 CSF2 560 560 510 0 -50 ICAM1 149 128 100 -21 -49 FCER1G 95 46 47 -49 -48 CD45R0 113 68 65 -45 -48 MIF 111 73 63 -38 -48 PRDM1 279 253 231 -26 -48 SERPING1 69 23 22 -46 -47 PSMB5 224 182 180 -42 -44 LILRA3 501 470 457 -31 -44 IL27 546 530 503 -16 -43 TNFSF15 472 338 430 -134 -42 ENTPD1 284 179 242 -105 -42 CSF1R 357 304 315 -53 -42 KIT 372 438 413 66 41 IL23R 492 547 534 55 42 ATG16L1 268 303 311 35 43 CD1A 474 528 517 54 43 IL6ST 32 65 76 33 44 ILF3 44 86 88 42 44 CCL19 14 57 58 43 44 IL4R 92 139 136 47 44 JAK1 56 100 101 44 45 CIITA 210 238 256 28 46 EGR1 87 173 133 86 46 IKBKG 249 254 296 5 47 KLRC2 420 440 468 20 48 CD83 119 183 167 64 48 IL2RB 155 193 204 38 49 TIRAP 393 433 442 40 49 GFI1 379 435 428 56 49 CD82 226 290 275 64 49 GZMB 220 305 270 85 50 IKZF2 346 390 397 44 51 TNFSF10 74 155 125 81 51 KLRC3 427 455 479 28 52 162 CXCL13 54 87 106 33 52 RAG1 490 534 542 44 52 C6 391 392 444 1 53 SLAMF1 365 416 418 51 53 IL6R 215 220 269 5 54 LTF 308 314 363 6 55 RAG2 506 553 561 47 55 IL22RA2 507 555 562 48 55 ATM 429 484 485 55 56 IL18RAP 428 490 484 62 56 JAK3 112 161 169 49 57 MX1 107 160 164 53 57 IL2RG 55 121 112 66 57 CASP8 93 144 151 51 58 IL7R 61 133 119 72 58 TNFRSF9 350 412 409 62 59 ABCB1 356 424 415 68 59 CUL9 286 343 346 57 60 SLAMF6 373 437 433 64 60 S1PR1 259 298 320 39 61 NFATC3 173 222 234 49 61 LAMP3 293 352 354 59 61 TAGAP 135 191 197 56 62 TRAF2 204 266 266 62 62 CD5 245 302 309 57 64 TRAF3 152 211 216 59 64 PPARG 412 481 476 69 64 IKBKE 181 269 245 88 64 XCR1 382 419 448 37 66 NFKB2 97 141 163 44 66 STAT4 295 357 361 62 66 CCRL1 404 477 470 73 66 CSF2RB 47 124 113 77 66 IL18R1 318 396 385 78 67 PTPN6 78 136 146 58 68 CCND3 141 209 211 68 70 NCR1 424 503 494 79 70 CASP2 205 249 277 44 72 GP1BB 368 397 441 29 73 163 IRAK4 201 256 274 55 73 IL7 409 482 482 73 73 IRF4 157 231 230 74 73 ITGAL 76 176 149 100 73 MAF 316 340 390 24 74 IKBKB 188 223 262 35 74 CD45RB 217 293 291 76 74 MALT1 144 218 219 74 75 VCAM1 85 130 161 45 76 TNFAIP3 35 135 111 100 76 TRAF1 160 263 236 103 76 ITGA4 184 248 261 64 77 LTB4R 274 354 351 80 77 IL11RA 292 380 372 88 80 LCK 162 247 243 85 81 IRF7 222 286 305 64 83 KLRC1 392 460 475 68 83 LILRA4 423 498 508 75 85 CD160 410 501 495 91 85 KLRB1 207 301 292 94 85 TNFRSF17 285 382 370 97 85 ICOSLG 214 283 301 69 87 TRAF4 322 411 410 89 88 CCL16 418 515 506 97 88 KLRF1 408 497 497 89 89 TAL1 377 441 467 64 90 AICDA 436 521 526 85 90 LTA 313 405 403 92 90 CD3E 108 216 199 108 91 CD3D 103 213 195 110 92 BTK 196 295 289 99 93 NOS2 413 496 507 83 94 CD27 83 175 177 92 94 CCL20 421 525 516 104 95 IKZF1 260 364 355 104 95 CD96 164 255 260 91 96 ICAM3 115 208 212 93 97 CD6 187 285 284 98 97 IKZF3 126 229 223 103 97 164 TNFRSF11A 355 439 453 84 98 SIGIRR 175 268 273 93 98 LTB4R2 265 363 364 98 99 CD36 46 150 145 104 99 TNF 266 351 366 85 100 EBI3 374 479 477 105 103 IL16 40 145 143 105 103 IRF8 50 148 155 98 105 GNLY 275 377 380 102 105 MAP4K1 129 232 235 103 106 CD28 252 342 360 90 108 TRAF5 150 252 258 102 108 CEACAM1 403 518 515 115 112 CD247 174 273 288 99 114 CD48 58 181 172 123 114 MS4A1 17 137 132 120 115 BCL2 88 186 206 98 118 ZAP70 106 224 225 118 119 TLR9 338 429 459 91 121 BLNK 258 355 381 97 123 CD7 232 383 356 151 124 CR1 48 189 176 141 128 CD79A 23 170 153 147 130 TNFSF11 369 491 500 122 131 ZBTB16 240 373 374 133 134 CD40LG 248 369 384 121 136 CXCL2 57 259 196 202 139 EDNRB 276 428 416 152 140 TCF7 53 194 194 141 141 PTGS2 278 476 419 198 141 LEF1 189 330 332 141 143 ICAM2 178 315 326 137 148 BTLA 169 299 318 130 149 CD1D 301 453 450 152 149 POU2F2 171 262 321 91 150 CCR6 287 446 439 159 152 SELL 59 203 214 144 155 TNFRSF13B 256 399 412 143 156 CD79B 37 163 203 126 166 165 AIRE 345 516 512 171 167 MAP4K2 163 334 337 171 174 TNFRSF13C 63 278 238 215 175 HLA-DOB 139 288 317 149 178 CFP 238 371 420 133 182 CD209 62 294 250 232 188 CD45RA 79 250 276 171 197 CCL24 280 493 478 213 198 CD19 77 239 281 162 204 CD22 41 225 247 184 206 CCR7 128 337 334 209 206 CD24 91 230 302 139 211 PAX5 176 393 399 217 223 C7 29 408 316 379 287 CR2 49 462 455 413 406 Total 278 166 Appendix H Cells scores obtained from the gene expression analysis in ALTR TLOs. Box plot of relative cell score for exhausted CD8 vs total CD8 cells. Significant higher number of exhausted CD8 are present in ALTRs than controls. Box plot of relative cell score for CD8 cells vs total leukocytes. No differences are observed between the groups. 167 Box plot of relative cell score for neutrophils vs total leucocytes. Significant higher number of neutrophils are present in ALTRs than controls. Box plot of relative cell score for cytotoxic cells vs total leucocytes. No differences are observed between groups. 168 Box plot of relative cell score for T-cells vs total leucocytes. Significant lower number of T-cells are present in ALTRs than controls. Box plot of relative cell score for macrophages vs total leucocytes. Significant higher number of macrophages are present in ALTRs than controls.
Thesis/Dissertation
eng
Biomedical Engineering
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Roles of corrosion products of metal hip replacements in the etiology of adverse local tissue reactions
Text