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Development of a Novel 3D Human Cartilage Model System to Investigate Changes in Cartilage Associated with Osteoarthritis

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Development of a Novel 3D Human Cartilage Model System to Investigate Changes in Cartilage Associated with Osteoarthritis Development of a novel 3D human cartilage model system to investigate changes in cartilage associated with osteoarthritis Thesis submitted in fulfillment of the requirements of the degree of Doctor of Philosophy April 2015 Andreas Heil, Dipl. Biol. School of Dentistry Cardiff University Acknowledgements I would like to thank my supervisors Professor Daniel Aeschlimann and Professor Rachel Waddington for giving me support, motivation and inspiration during my PhD. I would like to thank Arthritis Research UK and the President’s Research Scholarship for funding my research project. I also wish to acknowledge the Consortium for Functional Glycomics, grant number GM62116, for funding of the microarray analysis. A special thanks goes to my lab members Lea, Magda, Ana, Rhiannon, Katka, and Pascale as well as Dr Vera Knauper and Dr Konrad Beck!! Thank you for the great time we had together. Finally, I would like to thank my family and Lea for their love and support. iii „Wissen und Erkennen sind die Freude und Berechtigung der Menschheit“ „Science and knowledge are the joy and entitlement of humanity” Alexander von Humboldt, german naturalist and explorer (1769-1859) iv Abstract Osteoarthritis (OA) is the most prevalent form of degenerative joint diseases and affects about 13% of the world’s population with patients over 65 years of age reflecting the largest group of patients (Matthews and Hunter 2011). OA is characterized by a progressive degeneration of joint cartilage and results in impaired function of affected joints, pain and negatively affects quality of life of patients. Several biomarkers for the detection of early OA were already described (Mobasheri 2013), but none of them is reliable and quantifiable. Therefore, a need for novel biomarkers for OA exists to improve diagnosis when overt changes in cartilage are not yet detectable. The aim of my thesis was to identify novel biomarkers for OA which can be used for early diagnosis. I focused on changes in the glycosylation of proteoglycans, especially on chondroitin sulphate (CS) glycosaminoglycan (GAG) chains. Using human chondrocyte progenitor cells, a novel 3D cartilage model was developed and characterized. The resulting cartilage constructs showed similar biochemical, histological and mechanical properties like native articular cartilage. To investigate the effects of inflammatory cytokines, which are also present in OA, on changes in expression of glycosylation-related genes, treated and untreated constructs were analyzed using microarrays. Analysis showed that although genes for GAG chain synthesis were down- regulated, the sulphotransferase GalNAc4S-6ST was significantly up- regulated. This enzyme catalyzes the formation of GalNAc4,6diS, which has high biological activity (Mikami and Kitagawa 2013). In addition, the expression of serglycin was strongly increased after inflammatory stimulation. These results show that novel epitopes containing GalNAc4,6diS or serglycin could be potential biomarkers for OA. Besides the experiments with cartilage constructs, analysis of the CS chain of decorin isolated from human skin fibroblasts showed that inflammatory stimulation alters the length and composition of CS chains. v List of abbreviations 2-AB 2-aminobenzamide ACI Autologous chondrocyte transplantation ADAMTS A disintegrin and metalloprotease with trompospondin motif AMAC 2-aminoacridone BMP Bone morphogenic protein BSA Bovine serum albumin BSA Bovine serum albumin C-4-S Chondroitin-4-sulphate chABC Chondroitinase ABC chAC-I Chondroitinase AC-I chB Chondroitinase B CHO Chinese hamster ovary cells CS chondroitin sulphate CT Computed tomography CTX-II collagen II C-telopeptide DAPI 4',6-diamidino-2-phenylindole DMEM Dulbecco’s modified eagle medium DMOADS Disease-modifying osteoarthritis drug DMSO dimethylsulfoxide DS Dermatan sulphate ECM Extracellular matrix EDS Ehlers-Danlos syndrome EDTA ethylenediaminetetraacetic acid EGFR Epidermal growth factor receptor ER Endoplasmic reticulum FBS Fetal bovine serum FGF Fibroblast growth factor FPLC Fast protein liquid chromatography GAG Glycosaminoglycan Gal Galactose GalNAc N-acetylgalactosamine vi GDF Growth differentiation factor Glc Glucose GlcA Glucuronic acid GlcNAc N-acetylglucosamine HA Hyaluronic acid HEK293 Human embryonic kidney 293 cells HexA Hexuronic acid HNK-1 human natural killer cell carbohydrate antigen 1 HPLC High performance liquid chromatography HRP Horseradish peroxidase HS Heparan sulphate IdoA Iduronic acid IGD Interglobular domain IGF Insulin growth factor IGFR Insulin-like growth factor receptor IHH Indian hedgehog IL-1 Interleukin 1 beta KS Keratan sulphate LRR Leucine-rich repeat MAPK Mitogen activated kinase MMPs Matrix metalloprotease MRI Magnetic resonance imaging MW Molecular weight NEAA Non-essential amino acids NSAID Non-steroidal anti-inflammatory drug OA osteoarthritis OSM Oncostatin M PBS Phosphate buffered saline PCR Polymerase chain reaction PD Population doubling PMSF Phenylmethylsulphonyl fluoride PTHrP parathyroid hormone-related peptide RA Rheumatoid arthritis vii SLRP Small leucine rich proteoglycan TAE Tris acetate EDTA buffer TBS Tris buffered saline TBS-T Tris buffered saline containing 0.01% Tween 20 TGF Transforming growth factor b UDP Uridine diphosphate Xyl Xylose XylT Xylosyltransferase viii 1 Introduction ........................................................................................... 1 Arthritis ............................................................................................ 1 Osteoarthritis ................................................................................... 1 Diagnosis of OA ............................................................................... 4 Biological markers for osteoarthritis ........................................... 5 Current treatment of osteoarthritis ................................................... 6 Chondrocytes and cartilage ............................................................. 7 Chondrocyte progenitor cells ......................................................... 12 Cartilage extracellular matrix ......................................................... 12 Collagen ........................................................................................ 13 Proteoglycans ................................................................................ 14 Aggrecan .................................................................................. 14 Small Leucine-Rich Proteoglycans ........................................... 15 1.9.2.1 SLRP class I proteins ........................................................ 17 1.9.2.2 Decorin .............................................................................. 17 1.9.2.3 SLRP class II proteins ....................................................... 22 1.9.2.4 SLRP class III proteins ...................................................... 22 1.9.2.5 SLRP class IV proteins ...................................................... 22 1.9.2.6 SLRP class V proteins ....................................................... 23 Glycosaminoglycans ...................................................................... 23 Synthesis of chondroitin sulphate ............................................. 27 Post-translational modification of chondroitin sulphate ............. 32 Termination of chondroitin sulphate synthesis ......................... 39 Degradation of GAG chains ..................................................... 39 Chondroitinases ....................................................................... 40 Heparinases ............................................................................. 41 Hyaluronidases......................................................................... 42 Changes in GAG chain structure identified in disease states of cartilage .................................................................................................... 42 Aims of the thesis .......................................................................... 43 ix 2 Establishing HPLC disaccharide analysis methods and analysis of decorin GAG chain composition .............................................................. 44 Introduction .................................................................................... 44 Aims for this chapter: ................................................................ 46 Materials and Methods .................................................................. 47 HPLC analysis of disaccharides using CarboPac PA1 column 47 Labeling of chondroitin/dermatan sulphate disaccharides with 2- aminobenzamide (2-AB) ....................................................................... 48 Removal of excess 2-AB from the labeled disaccharide sample 48 HPLC analysis of 2-AB labeled disaccharides using YMC Pack Polyamine II column ............................................................................. 49 Labeling of chondroitin/dermatan sulphate disaccharides with 2- aminoacridone (AMAC)......................................................................... 49 Repurification of AMAC using hydrophobic interaction chromatography .................................................................................... 50 HPLC analysis of AMAC-labeled disaccharides using Kinetex C- 18 column ............................................................................................. 50 Cell culture ............................................................................... 51 Purification of decorin by anion exchange chromatography
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