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Thesis Final THESIS/DISSERTATION APPROVED BY 4-24-2020 Barbara J. O’Kane Date Barbara J. O’Kane, MS, Ph.D, Chair Margaret Jergenson Margret A. Jergenson, DDS Neil Norton Neil S. Norton, BA, Ph.D. Gail M. Jensen, Ph.D., Dean i COMPARISON OF PERIODONTIUM AMONG SUBJECTS TREATED WITH CLEAR ALIGNERS AND CONVENTIONAL ORTHODONTICS By: Mark S. Jones A THESIS Presented to the Faculty of The Graduate College at Creighton University In Partial Fulfillment of Requirements For the Degree of Master of Science in the Department of Oral Biology Under the Supervision of Dr. Marcelo Mattos Advising from: Dr. Margaret Jergenson, Dr. Neil S. Norton, and Dr. Barbara O’Kane Omaha, Nebraska 2020 i iii Abstract INTRODUCTION: With the wider therapeutic use of clear aligners the need to investigate the periodontal health status and microbiome of clear aligners’ patients in comparison with users of fixed orthodontic has arisen and is the objective of this thesis. METHODS: A clinical periodontal evaluation was performed, followed by professional oral hygiene treatment on a patient under clear aligner treatment, another under fixed orthodontics and two controls that never received any orthodontic therapy. One week after, supragingival plaque, swabs from the orthodontic devices, and saliva samples were collected from each volunteer for further 16s sequencing and microbiome analysis. RESULTS: All participants have overall good oral hygiene. However, our results showed increases in supragingival plaque, higher number of probing depths greater than 3mm, higher number of bleeding sites on probing, and a higher amount of gingival recession in the subject treated with fixed orthodontics. A lower bacterial count was observed colonizing the clear aligners, with less diversity than the other samples analyzed. Clear aligners exhibited a higher proportion of genus Porphyromona, which has a well-known periodontal pathogen, P. gingivalis. The genus Aggregatibacter had higher proportions on the subject with fixed orthodontics and the control with higher attachment loss. The species Aggregatibacter actinomycetemcomitans has been linked to aggressive forms of periodontal disease. CONCLUSION: Clear aligners showed improved periodontal status indices when compared to fixed orthodontics. Overall, fewer bacteria were found on the clear aligner when compared to fixed orthodontics. Genera with known periodontal pathogens were found on both orthodontics devices. iv Acknowledgements I would like to thank Dr. Marcelo Mattos for being my mentor throughout this process. The countless number of times I dropped by to ask questions or clarification he always welcomed me with open arms, thank you. I would like to thank Dr. Barbara O’Kane, Dr. Margaret Jergenson, Dr. Neil S. Norton, Dr. Sonia Sanchez, and Dr. Laura Brown for their advice and expertise throughout the project. I would also like to express my gratitude to Umesh Pyakuel for his technical assistance with the DNA extraction and interfacing with the UNMC sequencing core. Additionally, I would like to thank Jenifer Bushing and the UNMC sequencing core for their assistance. I would like to express my gratitude to Irina M. Velsko for her willingness to assist with the microbiome analysis. I would also like to thank Adrian Hankewycz and Saif Shah for their assistance with the clinical and data collection. Lastly, I would like to thank my parents for their never-ending support and encouragement. v vi Table of Contents Abstract .......................................................................................................................................... iii Acknowledgments ......................................................................................................................... iv Table of Content .................................................................................................................................... v Tables and Graphs ......................................................................................................................... vii Chapter 1: Introduction ................................................................................................................... 1 Chapter 2: Literature Review .......................................................................................................... 7 Chapter 3: Objectives .................................................................................................................... 14 Chapter 4: Materials and Methods ................................................................................................ 15 i. Subject Selection ................................................................................................... 15 ii. Clinical Evaluation of Periodontium ..................................................................... 15 a. Supragingival Plaque ................................................................................ 15 b. Probing Depth ........................................................................................... 16 c. Bleeding on Probing ................................................................................. 16 d. Clinical Attachment Level ........................................................................ 16 e. Gingival Recession ................................................................................... 16 iii. Oral Hygiene Treatment ....................................................................................... 16 iv. Samples Collection ............................................................................................... 17 v. Laboratory Analysis ............................................................................................. 17 a. DNA Extraction ....................................................................................... 17 vi. 16S Sequencing .................................................................................................... 19 a. PCR Amplification .................................................................................... 19 b. PRC Clean-up 1 ........................................................................................ 20 vii c. PCR Index ................................................................................................. 20 d. PCR Clean-up 2 ........................................................................................ 21 e. Library Denaturing and MiSeq Sample Loading ...................................... 22 vii. Microbiome Analysis ............................................................................................ 23 Chapter 5: Results ......................................................................................................................... 24 i. Demographic Data ................................................................................................ 24 ii. Clinical Evaluation of Periodontium ...................................................................... 24 a. Supragingival Plaque ................................................................................ 24 b. Probing Depth ........................................................................................... 25 c. Bleeding on Probing ................................................................................. 28 d. Clinical Attachment Level ........................................................................ 29 e. Gingival Recession ................................................................................... 30 iii. Microbiome Analysis ............................................................................................ 31 a. Quantitative Analysis ................................................................................ 31 b. Qualitative Analysis .................................................................................. 36 Chapter 6: Discussion ................................................................................................................... 45 Chapter 7: Conclusion ................................................................................................................... 59 Chapter 8: Bibliography ................................................................................................................ 61 viii Tables and Figures Tables Table 1. ........................................................................................................................................ 24 1. Demographic information on study subjects Table 2 ......................................................................................................................................... 32 1. The total number of OTUs and total number of bacterial species found in each sample Figures Figure 1a .................................................................................................................................... 25 1. Absolute number of surfaces with supragingival plaque. Figure 1b .................................................................................................................................... 25 1. Percentage of surfaces with supra gingival plaque. Figure 2 ...................................................................................................................................... 26 1. Average probing depth in mm. Figure 3a .................................................................................................................................... 27 1. The absolute number of probing depths greater than 3mm. Figure 3b ........................................................................................................................................
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