BUILDING A SYNTHETIC PERIODONTAL LIGAMENT: COLLAGEN NANOSTRUCTURES ON TITANIUM By ELOISE PEYTON MILLER Submitted in partial fulfillment of the requirements For the degree of Master of Science Thesis Advisor: Dr. Steven Eppell Department of Biomedical Engineering CASE WESTERN RESERVE UNIVERSITY May 2020 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Eloise Miller candidate for the degree of Master of Science in Biomedical Engineering*. Committee Chair Steven Eppell Committee Member Anirban Sen Gupta Committee Member Leena Palomo Committee Member Jonathan Pokorski Date of Defense March 10, 2020 *We also certify that written approval has been obtained for any proprietary material contained therein. ii Table of Contents Table of Contents .............................................................................................. iii List of Tables ................................................................................................... vii List of Figures ................................................................................................. viii Abstract ............................................................................................................. 1 Chapter 1: Introduction and Background ............................................................ 2 1.1 Dental Implants ................................................................................. 2 1.2 Periodontal Ligament ........................................................................ 5 1.3 Artificial Ligament Systems .............................................................. 8 1.4 Collagen Self-Assembly: Fibrillogenisis .......................................... 10 1.5 Non-Specific Covalent Bonds Between Proteins and Surfaces ......... 14 1.6 Carbonyl Formation and Oxyamine Conjugation ............................. 15 Pyridoxal-5-Phosphate as a Transamination Reagent ........................... 15 Rapoport’s Salt as a Transamination Reagent ...................................... 25 Summary of Carbonyl Formation and Oxyamine Conjugation............. 26 1.7 Silane Surface Reactions ................................................................. 27 Chapter 2: Study Design .................................................................................. 29 2.1 Study Goal ...................................................................................... 29 2.2 Experimental Design ....................................................................... 30 2.3 Developing the Experimental Protocol ............................................ 31 Step 1: Silanization of Titanium .......................................................... 31 Step 2: Ketone-Collagen Production.................................................... 42 Step 3: Binding Ketone-Collagen to Oxyamine-Silanized Titanium .... 55 Step 4: Nucleation and Growth of Collagen Fibrils on Titanium .......... 60 Summary of Developed Protocol ......................................................... 65 Chapter 3: Manuscript ...................................................................................... 67 iii 3.1 Preface ............................................................................................ 67 3.2 Manuscript ...................................................................................... 67 3.2.1 Abstract ...................................................................................... 67 3.2.2 Introduction ................................................................................ 68 3.2.3 Results and Discussion ............................................................... 70 3.2.4 Conclusions ................................................................................ 79 3.2.5 Methods ..................................................................................... 80 Chapter 4: Conclusions and Future Work ......................................................... 84 4.1 Conclusions ..................................................................................... 84 4.2 Future Work .................................................................................... 85 Loading Fibril Surfaces ....................................................................... 87 Animal Modeling ................................................................................ 89 4.3 Other Potential Applications ............................................................ 91 Space-safe collagen adhesive .............................................................. 91 Dynamic Testing of Fibrils Synthesized In-vitro ................................. 92 Protocols ..................................................................................... 94 Titanium Slides......................................................................................... 94 Necessary Components ....................................................................... 94 Protocol for obtaining titanium squares: .............................................. 95 To remove titanium from holders ........................................................ 95 Cleaning Procedures ................................................................................. 95 Set up the Sonicator ............................................................................ 96 Sonicating Titanium ............................................................................ 96 Washing holders with soapy water ...................................................... 98 Sonicating Holders .............................................................................. 98 Protocols – Chemical Steps to Produce Fibrils on a Titanium Surface ..... 100 Ketone-Collagen ............................................................................... 100 iv Plasma Treatment.............................................................................. 106 Oxyamine Surfaces ........................................................................... 107 Oxyamine-Ketone Collagen Surfaces (oxime linkage reaction) ......... 112 Nucleation and Growth of fibrils (fibrillogenesis) ............................. 114 Buffers and Rinsing Solutions................................................................. 116 Safety................................................................................................ 116 Transamination MES Buffer: pH 6.5 with NaCl ................................ 117 Oxime Linkage MES Buffer: pH 5.5 with MgCl2 .............................. 118 KeCol Rinsing Solution: 150 mM MgCl2 (“ketone-collagen”)........... 119 PBS: Phosphate Buffered Saline........................................................ 120 Determining Efficacy .............................................................................. 120 UV-Vis Spectroscopy: Oxyamine-TAMRA reaction and analysis ..... 120 XPS .................................................................................................. 124 Immunostaining: Immunofluorescent Imaging .................................. 126 SEM ................................................................................................. 130 3D-Printed Holders ................................................................... 133 Ordering Oxyamine Molecule ................................................... 135 Use of Selected Tools ................................................................ 136 Using the Scale ....................................................................................... 136 Necessary Components: .................................................................... 136 Protocol: ........................................................................................... 136 Using the pH Meter ................................................................................ 137 Calibration ........................................................................................ 138 Use ................................................................................................... 139 Squeeze Bottles ...................................................................................... 139 Soap .................................................................................................. 140 Alcohols – Methanol, ethanol, isopropanol ........................................ 140 v Waste – How to dispose of used solvents and chemicals ......................... 140 Alcohol used for cleaning.................................................................. 140 Oxyamine-contaminated solvent ....................................................... 141 Acetic acid buffer, PBS, collagen, ketone collagen, soapy water, water ............................................................................................................ 141 UV-Vis Spectroscopy, NanoDrop 1000 ..................................... 142 Why not use a Bradford Assay? .............................................................. 143 Sample Preparation ................................................................................. 143 Concentration for NanoDrop ............................................................. 143 Dilution for NanoDrop ...................................................................... 143 Using the Nanodrop 1000 ....................................................................... 144 ImageJ processing of Immunofluorescence images .................... 150 ImmunoFluorBrightness.m ........................................................ 152 Plasma Cleaner Operation