Structural Characterisation of Aggrecan in Cartilaginous Tissues and Tissue Engineered Constructs A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2017 Russell J Craddock The School of Biological Sciences Division of Cell Matrix Biology and Regenerative Medicine List of Contents List of Contents ................................................................................................................. 2 List of Figures ................................................................................................................... 6 List of Tables ..................................................................................................................... 8 List of Equations ............................................................................................................... 8 List of Conference Presentations and Publications ....................................................... 8 List of Abbreviations ........................................................................................................ 9 Abstract ........................................................................................................................... 10 Declaration ...................................................................................................................... 11 Copyright Statement ...................................................................................................... 11 Acknowledgements ......................................................................................................... 12 1 Chapter 1: General Introduction ............................................................................... 13 1.1 Overview .................................................................................................................... 14 1.1.2 Intervertebral Disc Biology ..................................................................................... 15 1.1.3 Intervertebral Disc Macrostructure.......................................................................... 16 1.1.3.1 Nucleus Pulposus ................................................................................................. 16 1.1.3.2 Annulus Fibrosus .................................................................................................. 16 1.1.3.3 Cartilage End-plates ............................................................................................. 17 1.1.4 Articular Cartilage ................................................................................................... 17 1.1.5 Structural differences between nucleus pulposus and articular cartliage ................ 18 1.1.6 Extracellular Matrix Molecules and Assemblies in Intervertebral Discs ................ 19 1.1.6.1 Collagen ................................................................................................................ 19 1.1.6.2 Proteogylcans ....................................................................................................... 20 1.1.6.3 Aggrecan .............................................................................................................. 21 1.1.6.3.1 Transcription of Aggrecan ................................................................................. 21 1.1.6.3.2 Aggrecan Core Protein Structure....................................................................... 22 1.1.6.3.3 Biosynthesis of Aggrecan .................................................................................. 24 1.1.6.3.4 Aggrecan Functionality and Protein Interactions .............................................. 27 1.1.6.3.5 Tissue-specific Variability in Aggrecan Structure ............................................ 27 1.1.6.3.6 Fragmentation/degradation of aggrecan ............................................................ 29 1.2 Hypothesis and aims of the project ............................................................................ 33 2 Chapter 2: General Methods ...................................................................................... 34 2.1 Cartilaginous tissue collection .................................................................................... 35 2.1.1 Native tissue sample collection ............................................................................... 35 2.1.2 Isolation and culture of nucleus pulposus cells ....................................................... 35 2.1.3 Encapsulation of nucleus pulposus cells in a type I collagen hydrogel .................. 36 2.1.4 Chondrogenic stimulation of encapsulated nucleus pulposus cells with GDF6/TGFβ3 ................................................................................................................... 36 2.2 Isolation and characterisation of aggrecan ................................................................. 36 2.2.1 Aggrecan isolation by dissociative caesium chloride centrifugation ...................... 36 2.2.2 Preparation of aggrecan for structural characterisation ........................................... 38 2.2.3 Structural Characterisation of aggrecan by atomic force microscopy and multi- angle light scattering ........................................................................................................ 41 2.3 Histological assessment of tissue composition and collagen fibril alignment ........... 45 2.3.1 Histological staining ................................................................................................ 45 2.3.2 Characterisation of collagen fibril ultrastructure by atomic force microscopy ....... 46 2.4 Measurement of micro-mechanical compressive stiffness by atomic force microscopy indentation .................................................................................................... 48 2.5 Relative gelatinase activity determined by in situ gelatinase zymography ................ 50 2.6 Computational modelling of aggrecan packing .......................................................... 51 2 2.7 Gene expression analysis using real-time quantitative-PCR ...................................... 52 2.7.1 Isolation of RNA from NP cell constructs .............................................................. 52 2.7.2 Reverse transcription of RNA ................................................................................. 53 2.7.3 Assessment of NP marker gene expression with real-time quantitative-PCR ........ 53 2.8 Statistical Analysis ..................................................................................................... 54 3 Chapter 3: Characterisation of aggrecan and collagen structure in young, healthy cartilaginous tissues ........................................................................................................ 55 3.1 Introduction .............................................................................................................. 56 3.1.2 Hypothesis and aims ................................................................................................ 57 3.2 Materials and methods ............................................................................................. 58 3.2.1 Experimental design ................................................................................................ 58 3.3 Results ........................................................................................................................ 59 3.3.1 The ultrastructure of intact aggrecan monomers was tissue-specific ...................... 59 3.3.2 Most isolated aggrecan molecules were fragmented and structurally heterogeneous ................................................................................................................... 59 3.3.3 Aggrecan fragmentation was not associated with aberrant structural or mechanical tissue remodelling or excessive protease activity ............................................................. 62 3.3.4 Aggrecan fragmentation was predicted to modulate molecular packing density .... 68 3.4 Discussion .................................................................................................................. 70 3.5 Conclusions ............................................................................................................... 75 4 Chapter 4: The effect of culture conditions on the nanostructure of aggrecan and mechanical properties in intervertebral disc tissue engineered constructs .............. 76 4.1 Introduction .............................................................................................................. 77 4.1.2 Hypothesis and aims ................................................................................................ 79 4.2 Materials and methods ............................................................................................. 80 4.2.2 Experimental design ................................................................................................ 80 4.3 Results ........................................................................................................................ 82 4.3.1 The effect of oxygen on aggrecan synthesis and structure ...................................... 82 4.3.1.1 Oxygen tension did not affect the proportion of intact aggrecan, but did affect molecular size ................................................................................................................... 82 4.3.1.2 Gene expression aggrecan was reduced in hypoxic conditions compared to normoxia