THE DEVELOPMENT OF AN EXTRACELLULAR VESICLE ISOLATION PROTOCOL FOR GINGIVAL CREVICULAR FLUID By ESTELA L. TRUZMAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2019 © 2019 Estela L. Truzman To my family and my husband for their infinite support, inspiration and love ACKNOWLEDGMENTS I would like to thank my mentors Dr. Shannon Holliday and Dr. Wellington Rody for their guidance and support throughout this process. I would also like to thank the faculty, staff and residents at the University of Florida’s Department of Orthodontics for their unwavering support throughout these past 3 years. Last, but not least, I would like to thank my wonderful parents and husband for always encouraging me to pursue my dreams. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION .................................................................................................... 13 Background ............................................................................................................. 13 Biomarkers in Orthodontics ..................................................................................... 16 Osteoclastogenesis Related Factors (RANKL/OPG) ........................................ 16 Biomarkers of Inflammation .............................................................................. 17 Extracellular Matrix Degradation Factors (MMPs) ............................................ 17 Dentin Breakdown Products ............................................................................. 18 Extracellular Vesicles (EVs) ............................................................................. 20 Biomarkers in Oral Fluids ........................................................................................ 26 Saliva................................................................................................................ 26 Gingival Crevicular Fluid (GCF) ........................................................................ 27 GCF Collection ....................................................................................................... 27 Gingival Washing Method................................................................................. 28 Capillary Tubing or Micropipettes ..................................................................... 28 Absorbent Filter Paper Strips ........................................................................... 29 Durapore Filter Membranes .............................................................................. 30 Specific Objectives or Hypotheses ......................................................................... 31 Objective .......................................................................................................... 31 Hypotheses ...................................................................................................... 31 2 MATERIALS AND METHODS ................................................................................ 33 Participants, Eligibility Criteria ................................................................................. 33 Study Design .......................................................................................................... 33 Sample Collection ................................................................................................... 33 PerioPaper Strip ............................................................................................... 34 Durapore Filter Membrane ............................................................................... 34 Microcapillary Tube .......................................................................................... 34 Control Samples Collection .............................................................................. 35 Sample Preparation ................................................................................................ 35 EV Isolation with Nanoparticle Tracking Analysis (NTA) ......................................... 35 5 Statistical Analysis .................................................................................................. 36 3 RESULTS ............................................................................................................... 41 4 DISCUSSION ......................................................................................................... 51 Main Findings ......................................................................................................... 51 Limitations ............................................................................................................... 55 5 CONCLUSIONS ..................................................................................................... 57 LIST OF REFERENCES ............................................................................................... 58 BIOGRAPHICAL SKETCH ............................................................................................ 63 6 LIST OF TABLES Table page 2-1 Outline of Inclusion and Exclusion Criteria ......................................................... 37 3-1 Raw Data with Average Concentration Obtained from the NanoSight Software (version 2.3, NanoSight) ...................................................................... 44 3-2 Volume of GCF Collected with PerioPaper and Average Concentration of Nanoparticles ...................................................................................................... 45 3-3 Average Nanoparticle Concentration of Control Samples .................................. 45 3-4 Comparison of Raw Data to Control Values ....................................................... 45 3-5 Adjusted Data- Average Control Values Subtracted from the Raw Data ............ 46 3-6 Concentration Difference between the three Collection Methods ....................... 46 3-7 Correlation between the adjusted values of the three methods .......................... 46 7 LIST OF FIGURES Figure page 1-1 Size distribution and concentration of nanoparticles recovered from control and GCF samples. Analysis performed on the NanoSight instrument available at UF-ICBR .......................................................................................... 32 2-1 PerioPaper, 2018. Courtesy of Dr. Estela Truzman. PerioPaper collection method on tooth #8 ............................................................................................. 38 2-2 Periotron 8000®, 2018. Courtesy of Dr. Estela Truzman GCF volume determination with the Periotron 8000® (Oraflow, Plainview, NY, USA) ............ 38 2-3 Durapore, 2018. Courtesy of Dr. Estela Truzman Volume of GCF collected with PerioPaper and concentration of nanoparticles ........................................... 38 2-4 Microcapillary tube, 2018. Courtesy of Dr. Estela Truzman Microcapillary tube collection method on tooth #8 ..................................................................... 39 2-5 Leuer port, 2018. Courtesy of Dr. Estela Truzman Installation of the leuer port on the NanoSight NS300 ............................................................................. 39 2-6 Leuer Port Connection, 2018. Courtesy of Dr. Estela Truzman Connection of the leuer port into the top-plate of the NanoSight NS300. .................................. 40 2-7 Top-plate, 2018. Courtesy of Dr. Estela Truzman 1 ml disposable syringe connected to a luer port on the top-plate of the NanoSight NS300..................... 40 3-1 Bar graph with raw data obtained from the NanoSight software. The average concentration of each method including the Standard deviation are shown ....... 47 3-2 Graph from the NanoSight showing the average nanoparticle concentration of a PerioPaper sample ...................................................................................... 47 3-3 Graph from the NanoSight showing the average nanoparticle concentration of a Durapore membrane sample ....................................................................... 48 3-4 Graph from the NanoSight showing the average nanoparticle concentration of a micropipette ................................................................................................. 48 3-5 Bar graph with adjusted data. The average control values were subtracted from the raw data for each sample. This number was multiplied by 15.7, which was the dilution media .............................................................................. 49 3-6 PerioPaper vs. Durapore Spearman correlation coefficient ................................ 49 3-7 PerioPaper vs. micropipette Spearman correlation coefficient ........................... 50 8 3-8 Durapore vs. micropipette Spearman correlation coefficient .............................. 50 9 LIST OF ABBREVIATIONS CBCT Cone Beam Computed Tomography CT Scan Computerized Tomography Scan DMP-1