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Investigations Into the Removal and Destruction of Bacterial Biofilms by Sodium Hypochlorite Irrigant Delivered Into an in Vitro Model

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Investigations Into the Removal and Destruction of Bacterial Biofilms by Sodium Hypochlorite Irrigant Delivered Into an in Vitro Model Investigations into the removal and destruction of bacterial biofilms by sodium hypochlorite irrigant delivered into an in vitro model A thesis submitted to the University College of London in fulfilment of the requirements for the degree Doctor of Philosophy By Saifalarab Mohmmed BDS (Iraq), MSc (Iraq) University College London 2017 UCL Eastman Dental Institute Biomaterials and Tissue Engineering Research Division University College London 1 Declaration DECLARATION I, Saifalarab Mohmmed confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Saifalarab Mohmmed 2 Abstract ABSTRACT Aims: To investigate the influence of canal design (closed, open), irrigant concentration, agitation, canal complexity, and biofilm type on the efficacy of sodium hypochlorite to remove biofilm. To examine the extent of biofilm destruction following irrigation protocols. Methodology: Standardized in vitro models were developed (Endo-Vu block, flow cell, and 3D printing root canal models). The canal consisted of two halves of an 18 mm length, size 30 and taper 0.06, with or without a lateral canal of 3 mm length, and 0.3 mm diameter. Biofilms were grown for 10 days, and stained using crystal violet. The model was attached to an apparatus and observed under a fluorescent microscope. Following 60 s of 9 mL NaOCl irrigation using syringe and needle, the irrigant was either left stagnant or agitated using gutta-percha, sonic and ultrasonic methods for 30 s. Images were captured every second using an external camera. The residual biofilm percentages were measured using image analysis software. The SPSS software was used for statistical analysis. The residual biofilms were observed using confocal laser scanning, scanning electron, and transmission electron microscope. Results: The removal of biofilm by NaOCl was more extensive in the open than in the closed canal. The concentration and extent of the needle had an influence on the amount of the residual biofilm. Ultrasonic agitation increased the biofilm removal from the main canal (90.13%) and lateral canal (66.76%). Extensive destruction of residual biofilm was observed in the ultrasonic groups. More residual multi-species biofilm than single species biofilm was identified (p = 0.001). Conclusion: The 3D-printing model provides a reliable method to investigate irrigation procedure. The closed canal adversely affect the efficacy of NaOCl. Concentration and position of the needle affect the efficacy of NaOCl. The results recommend the ultrasonic method for NaOCl agitation. The multi-species biofilm was more resistant than the single species biofilm. 3 Acknowledgement ACKNOWLEDGEMENTS I would like to express my sincere gratitude and thanks to: My primary supervisor Professor Jonathan c. Knowles for his encouragement, and professional contribution to improve my academic education. My secondary supervisor Dr Morgana E. Vianna, for her endless support, teaching, advice, and help throughout the study. The Ministry of Higher Education and Scientific Research of Iraq for funding my PhD study as a part of educational scholarship programme. Dr Nicky Mordan for her invaluable assistance, and help with the image analyses. Dr Stephen Hilton and Dr Matthew Penny for their assistance in printing of the root canal model. Dr Adam Roberts for his supervision and assistance during PCR amplification procedure to identify the bacterial strains. Professor Kishor Gulabivala as this study is based on his ideas and concepts originated, developed and under his investigation. Dr. Paula Ng for her advice in the experiments related to chapter 3 of this study. Dr Graham Palmer and Dr George Georgiou for their help with the iodometric titration of NaOCl and ordering materials. Dr Haitham Hussain for providing assistance in the microbiology laboratories. Dr Aviva Petrie and Mr David Boniface for their assistance in reviewing the statistical analyses. Mrs Angela Cooper for her advice and support to improve my skills in academic writing. Mr Jonathan Brittain; Acteon UK Southern Territory Manager for loaning the Satelec® P5 ultra-sonic device. Finally, I would like to thank my mother, brother, and wife for their support and encouragement throughout my study. 4 Contents CONTENTS DECLARATION ............................................................................................................ 2 ABSTRACT .................................................................................................................. 3 ACKNOWLEDGEMENTS ............................................................................................. 4 CONTENTS .................................................................................................................. 5 List of figures .............................................................................................................. 11 List of tables ............................................................................................................... 20 PUBLICATIONS ......................................................................................................... 23 PRESENTATIONS ..................................................................................................... 23 Chapter 1 .................................................................................................................... 24 Introduction ................................................................................................................. 24 1.1. Introduction ...................................................................................................... 24 1.2. Literature review ............................................................................................... 26 1.2.1. Composition of the root canal microbiota and pathogenesis of apical periodontitis ......................................................................................................... 26 1.2.2. The biofilm concept .................................................................................... 30 1.2.3. Formation of biofilm ................................................................................... 31 1.2.4. Resistance of biofilm to antimicrobial agent ............................................... 34 1.2.5. Role of root canal treatment in management of an intracanal biofilms........ 37 1.2.6. Root canal irrigation solutions .................................................................... 38 1.2.7. Physical and chemical efficacy of NaOCl in root canal treatment ............... 39 1.2.8. Fluid dynamics concept of irrigation ........................................................... 42 1.2.9. Factors influencing the penetration of an irrigant within the root canal system ............................................................................................................................ 45 1.2.10. Overcoming the problems of irrigant penetration ...................................... 46 1.2.11. Agitation of irrigant solution ...................................................................... 48 1.2.12. Investigations of efficacy of irrigants against root canal microbiota .......... 50 1.3. Statement of the problem ................................................................................. 54 1.4. Null hypothesis ................................................................................................. 56 1.5. Aim of the study ............................................................................................... 56 Chapter 2 .................................................................................................................... 57 Development and optimization of the materials used in the study ............................... 57 2.1. Introduction ...................................................................................................... 57 2.2. Investigation to test potential substratum materials for development of an in vitro biofilm model. .......................................................................................................... 58 2.2.1. Justification of the investigation.................................................................. 58 5 Contents 2.2.2. Materials and Methods ............................................................................... 59 2.2.3. Results ....................................................................................................... 67 2.2.4. Discussion ................................................................................................. 75 2.2.5. Conclusion ................................................................................................. 79 2.3. The effect of needle position on the velocity distribution of irrigant in the root canal system ........................................................................................................... 80 2.3.1. Justification of the investigation.................................................................. 80 2.3.2. Materials and methods ............................................................................... 81 2.3.3. Results ....................................................................................................... 83 2.3.4. Discussion ................................................................................................. 85 2.3.5. Conclusion ................................................................................................
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