Characterisation of resin-composite structure and collagen assembly at nanometre to micrometre length scales A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2018 Alvaro E. Munoz Schiemann School of Dentistry TABLE OF CONTENTS Abstract ........................................................................................................................................ 12 Declaration .................................................................................................................................. 13 Copyright statement ................................................................................................................. 14 Chapter I. Introduction and aims ....................................................................................... 15 I.1. Introduction ........................................................................................................................................... 16 I.2. Tooth structure .................................................................................................................................... 16 I.2.1. Enamel ................................................................................................................................. 17 I.2.2. Dentine ................................................................................................................................. 18 I.2.3. Pulp ....................................................................................................................................... 20 I.3. Dentine matrix .............................................................................................................................. 20 I.3.1. Collagen ............................................................................................................................... 20 I.3.1.1. Fibrillogenesis ......................................................................................................... 24 I.3.1.2. Mineralisation .......................................................................................................... 29 I.3.2. Non-collagenous proteins ............................................................................................ 31 I.3.2.1. Enzymes ..................................................................................................................... 34 I.3.2.1.1. Matrix metalloproteinases (MMPs) ......................................................... 34 I.3.2.1.2. Cysteine Cathepsins........................................................................................ 36 I.4. Dental Caries ................................................................................................................................. 38 I.4.1. Caries treatment ............................................................................................................... 39 I.4.1.1. Non-invasive ............................................................................................................ 39 I.4.1.2. Invasive treatment ................................................................................................. 39 I.4.1.2.1. Etch and rinse approach ............................................................................... 40 I.4.1.2.2. Self-etch approach .......................................................................................... 42 I.5. Tooth-resin interface ................................................................................................................. 43 I.5.1. Adhesion/micromechanical interlocking ............................................................... 43 I.5.1.1. Adhesion to Enamel............................................................................................... 44 I.5.1.2. Adhesion to Dentine .............................................................................................. 45 I.5.2. Failure of the adhesion .................................................................................................. 46 I.5.2.1. Hydrolysis of the resin adhesive ...................................................................... 46 I.5.2.2. Collagen degradation ............................................................................................ 47 I.5.3. Longevity of resin restorations .................................................................................. 49 I.5.4. Current strategies to prevent degradation of the hybrid layer ..................... 50 I.5.4.1. Adhesive-related strategies to prevent hybrid layer degradation ...... 51 I.5.4.2. Enzyme-related strategies to prevent hybrid layer degradation ........ 52 I.6. Resin-composites roughness and measurement techniques ..................................... 53 I.7. Clinical implications ................................................................................................................... 56 I.8. Atomic Force Microscopy ......................................................................................................... 57 2 I.8.1 Background ........................................................................................................................ 57 I.8.2 Configuration of the microscope ............................................................................... 59 I.8.3 Imaging modes .................................................................................................................. 62 I.8.3.1 Contact mode ........................................................................................................... 62 I.8.3.2 AC modes ................................................................................................................... 63 I.8.3.3 Kelvin Probe Force microscopy (KPFM) ....................................................... 64 I.9. Rationale ......................................................................................................................................... 66 I.10. Aims and Objectives .................................................................................................................... 67 Chapter II. “Optimisation of a collagen fibril model system” ................................ 68 II.1. Overview .......................................................................................................................................... 69 II.2. Materials and Methods .............................................................................................................. 69 II.2.1. Comparison of collagen models ............................................................................ 69 II.2.1.1. Model-1 Reconstituted Collagen ...................................................................... 69 II.2.1.2. Model-2 Extracted collagen ................................................................................ 70 II.2.2. Optimisation of AFM experimental conditions ............................................... 71 II.3. Results .............................................................................................................................................. 72 II.3.1. Comparison of models .............................................................................................. 72 II.3.1.1. Reconstituted collagen ......................................................................................... 72 II.3.1.2. Extracted collagen .................................................................................................. 75 II.3.2. Optimisation of AFM experimental conditions ............................................... 76 II.4. Discussion ........................................................................................................................................ 79 II.5. Conclusion ....................................................................................................................................... 83 Chapter III. “Influence of dental bonding procedures on collagen fibril nano- structure and nano-charge distribution” ......................................................................... 84 III.1. Overview .......................................................................................................................................... 85 III.2. Materials and Methods .............................................................................................................. 85 III.2.1 Sample preparation .................................................................................................... 85 III.2.2 AFM imaging ................................................................................................................. 86 III.2.3 Image post-processing and analysis .................................................................... 87 III.2.4 Statistical analysis ....................................................................................................... 88 III.3. Results .............................................................................................................................................. 88 III.3.1 Height .............................................................................................................................. 91 III.3.2 Width ............................................................................................................................... 95 III.3.3 D-Period .........................................................................................................................