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Effectiveness of irrigation methods in removing materials of different size and density from the apical… Kara, Aleem 2021

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EFFECTIVENESS OF IRRIGATION METHODS IN REMOVING MATERIALS OF DIFFERENT SIZE AND DENSITY FROM THE APICAL ROOT CANAL OF A MANDIBULAR MOLAR – AN IN VITRO STUDY  by  Aleem Kara  DMD, The University of British Columbia, 2014  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Craniofacial Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   February 2021  © Aleem Kara, 2021   ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled:  Effectiveness of irrigation methods in removing materials of different size and density from the apical root canal of a mandibular molar – An in vitro study  submitted by Aleem Kara in partial fulfillment of the requirements for the degree of Master of Science in Craniofacial Science   Examining Committee: Dr. Markus Haapasalo, Professor, Department of Oral Biological & Medical Sciences, UBC Supervisor  Dr. Ya Shen, Professor, Department of Oral Biological & Medical Sciences, UBC Supervisory Committee Member  Dr. Ricardo M Carvalho, Professor, Department of Oral Biological & Medical Sciences, UBC Supervisory Committee Member Dr. Vincent Lee, Assistant Professor of Teaching, Department of Oral Health Sciences, UBC External Examiner               iii Abstract   Objectives: Small pieces of material, typically from dental restorations, may accidently fall into the apical root canal obstructing treatment. The aim of this study was i) to compare the efficacy of irrigation by different methods in removing materials from the apical root canal and ii) to determine if the efficacy is affected by canal orientation and irrigant flow rate.  Methods: A transparent, 3D-printed, micro-CT scan-based resin mandibular molar tooth was selected. Four canals were confirmed for apical patency and their lengths measured. Apices were sealed with glue. Six materials of different density were passively placed into the apical part of the distal root canal: polyethylene, glass, titanium, stainless steel, gold alloy dust, and amalgam dust. Materials were removed using three irrigation methods at manufacturer recommended maximum irrigation times: GentleWave system (7 minutes), syringe needle irrigation with a 30 gauge open-ended and 30 gauge side-vented needle each at flow rates of 5 and 15 mL/min (2 minutes), and the ProUltra PiezoFlow ultrasonic irrigation system at flow rates of 0 and 15 mL/min (1 minute) resulting in seven experimental irrigation groups. For each material, each irrigation group was repeated 10 times, five in horizontal and five in vertical canal orientations using distilled water. Material removal was assessed using visual evaluation under a stereo microscope. The results were analyzed for the amount of different materials removed and for completely cleaned canals by each irrigation method using Fisher’s exact test. The percentage of materials removed was analyzed using one-way analysis of variance with Dunnett's test.  Results: The GentleWave system was the most effective at completely cleaning canals at maximum and 1-minute irrigation times. Combining all materials tested, all other irrigation methods removed the materials significantly less well than GentleWave. Mean percentage   iv removal by GentleWave was 98.6%, with overall values ranging from 67-100%. Canal orientation did not significantly affect the percentage of materials removed; however, higher flow rates did for open-ended needle and PiezoFlow.  Conclusions: The GentleWave system was more effective in removing materials from the root canal than other irrigation methods. Removal of material was affected by flow rates, but not by canal orientation.        v Lay Summary  The goal of this study was to assess the efficacy of irrigant flow by syringe needle irrigation, ultrasonic irrigation, and the GentleWave system in removing materials from the root canal of a lower tooth model. During root canal treatment, pieces of materials, typically from dental fillings, may fall into the root canal. These materials can prevent effective cleaning and disinfection of the root canal system. Traditional methods for removal are unpredictable and have potential for complications. In this study, a mandibular plastic tooth had materials of different size and density placed into the bottom of a root canal. Three irrigation methods at different flow rates were used to remove these materials. Visual evaluation through a stereo microscope was used to measure the amount of material present before and remaining after each type of irrigation. Combining the results from all materials tested, all other irrigation methods removed significantly less than GentleWave.       vi Preface This thesis is the principal work of Dr. Aleem Kara, as per the requirements of a Master of Science in Craniofacial Science with a Diploma in Endodontics. The research questions and study design were prepared by Dr. Aleem Kara, the supervisor Dr. Markus Haapasalo, and Dr. Ya Shen. Tooth model collection was carried out by Dr. Markus Haapasalo. Tooth selection, preparation of samples, and experimental testing was performed by Dr. Aleem Kara. Interpretation and statistical analysis of the results was analyzed by Dr. Aleem Kara with the assistance of Dr. Jolanta Aleksejuniene under the supervision of Dr. Markus Haapasalo. Writing of the thesis was prepared by Dr. Aleem Kara with editing by Dr. Markus Haapasalo and Dr. Ya Shen. Support and consultation were provided by Dr. Ricardo Carvalho. Laboratory assistance was provided by Dr. Abdullah Almegbel.       vii Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................ xi List of Figures ............................................................................................................................. xiii List of Symbols .......................................................................................................................... xvii List of Abbreviations ............................................................................................................... xviii Acknowledgements .................................................................................................................... xxi Dedication ................................................................................................................................. xxiii  Review of the Literature ............................................................................................1 1.1 Endodontic treatment ...................................................................................................... 1 1.1.1 Purpose .................................................................................................................... 1 1.1.2 Principles ................................................................................................................. 1 1.1.3 Outcome .................................................................................................................. 2 1.2 Endodontic complications ............................................................................................... 3 1.2.1 Classification of endodontic complications ............................................................ 3 1.2.2 Material obstructions .............................................................................................. 4 1.2.2.1 Types of material obstructions ............................................................................ 4 1.2.2.2 Management options for material obstructions ................................................... 7 1.2.2.3 Concerns with management options ................................................................. 12   viii 1.2.2.4 Prevention of material obstructions .................................................................. 13 1.3 Irrigation ....................................................................................................................... 14 1.3.1 Goals of irrigation ................................................................................................. 14 1.3.2 Fluid hydrodynamics ............................................................................................ 14 1.3.3 Irrigation methods ................................................................................................. 16 1.3.3.1 Syringe needle irrigation ................................................................................... 16 1.3.3.2 Ultrasonic irrigation .......................................................................................... 18 1.3.3.2.1 History of ultrasonic irrigation .................................................................... 18 1.3.3.2.2 Mechanism of action of ultrasonic irrigation .............................................. 18 1.3.3.2.3 Types of ultrasonic irrigation ...................................................................... 19 1.3.3.3 GentleWave ....................................................................................................... 22 1.3.3.3.1 GentleWave system .................................................................................... 22 1.3.3.3.2 Mechanism of action of GentleWave ......................................................... 22 1.3.3.3.3 Studies on GentleWave ............................................................................... 23 1.3.4 Effect of canal orientation on irrigation efficacy .................................................. 25 1.4 Endodontic research ...................................................................................................... 25 1.4.1 Microspheres ......................................................................................................... 25 1.4.2 3D printed resin in vitro tooth model .................................................................... 26  Aims and Hypotheses ...............................................................................................28 2.1 Rationale ....................................................................................................................... 28 2.2 Aims .............................................................................................................................. 28 2.3 Null hypotheses ............................................................................................................. 29  Materials and Methods ............................................................................................31   ix 3.1 Experimental design ...................................................................................................... 31 3.2 Tooth selection and preparation .................................................................................... 31 3.3 Pre-treatment tooth model preparation ......................................................................... 34 3.4 Material acquisition and preparation ............................................................................ 38 3.5 Pre-treatment material application ................................................................................ 45 3.6 Experimental groups ..................................................................................................... 48 3.7 Running the experiment and monitoring the removal of the materials ........................ 53 3.8 Visual evaluation and measurement of material removal ............................................. 59 3.9 Statistical analyses ........................................................................................................ 62  Results ........................................................................................................................64 4.1 The amount of different materials removed by each irrigation method ....................... 64 4.1.1 Removal of materials at recommended maximum irrigation times ...................... 64 4.1.2 Removal of materials at 1-minute of irrigation ..................................................... 73 4.2 The percentage of different materials removed by each irrigation method .................. 75 4.3 The percentage of materials removed based on canal orientation ................................ 87 4.4 The percentage of materials removed based on flow rate ............................................. 90 4.5 The time required for complete removal of materials .................................................. 99  Discussion ................................................................................................................105 5.1 Study importance ........................................................................................................ 105 5.2 Discussion of major findings ...................................................................................... 108 5.3 Novelty of the study .................................................................................................... 112 5.4 Limitations of the study .............................................................................................. 112 5.5 Future directions ......................................................................................................... 113   x  Conclusions .............................................................................................................114 Bibliography ...............................................................................................................................115    xi List of Tables  Table 3.1 Various measurements for each canal in mandibular resin molar tooth (L4) ............... 38 Table 3.2 Abbreviation, composition, density, and diameter for each microsphere bead used in the study (as per Cospheric Safety Data Sheet, 2019). Dentin density is approximately 1.79 to 2.21 g/cm3. .................................................................................................................................... 41 Table 3.3 Abbreviation, composition, approximate density, and diameter or length for each restorative dust particle used in study. Pure gold density is 19.3 g/cm3. ...................................... 42 Table 4.1 Number of tests (n) with material all removed, part removed, and not removed in different irrigation groups (Gr) at maximum irrigation times. Combined horizontal and vertical canal orientations. ......................................................................................................................... 66 Table 4.2 Number of tests (n) with material all removed, part removed, and not removed in different irrigation groups (Gr) at maximum irrigation times. Canal oriented horizontally. ........ 69 Table 4.3 Number of tests (n) with material all removed, part removed, and not removed in different irrigation groups (Gr) at maximum irrigation times. Canal oriented vertically. ............ 72 Table 4.4 Number of tests (n) with complete material removal (Yes) and not complete material removal (No) at a standardized 1-minute irrigation time in different irrigation groups (Gr). Combined horizontal and vertical canal orientations. ................................................................... 74 Table 4.5 Mean percentage (%) of materials removed with standard deviations at maximum irrigation times in different irrigation groups (Gr). Both horizontal and vertical canal orientations are combined. ................................................................................................................................ 79 Table 4.6 Mean percentage (%) of materials removed with standard deviations at maximum irrigation times in different irrigation groups (Gr). Canal oriented horizontally. ......................... 85   xii Table 4.7 Mean percentage (%) of materials removed with standard deviations at maximum irrigation times in different irrigation groups (Gr). Canal oriented vertically. ............................. 86 Table 4.8 Mean percentage (%) of materials removed compared between horizontal versus vertical canal orientation (all irrigation groups and materials combined; 210 tests per canal orientation). ................................................................................................................................... 87 Table 4.9 Mean percentage (%) of materials removed compared between horizontal versus vertical canal orientation. For each irrigation group while combining all materials (n = 30). ..... 89 Table 4.10 Mean percentage (%) of materials removed by syringe needle irrigation, open-ended (SNI OE) based on differences in flow rate (5 mL/min versus 15 mL/min). Maximum irrigation time of 2 minutes. ......................................................................................................................... 91 Table 4.11 Mean percentage (%) of materials removed by syringe needle irrigation, side-vented (SNI SV) based on differences in flow rate (5 mL/min versus 15 mL/min). Maximum irrigation time of 2 minutes. ......................................................................................................................... 94 Table 4.12 Mean percentage (%) of materials removed by PiezoFlow Ultrasonic (PF US) based on differences in flow rate (0 mL/min versus 15 mL/min). Maximum irrigation time of 1 minute........................................................................................................................................................ 97 Table 4.13 Mean time required, in seconds (s), for complete removal of materials with standard deviations. Combined horizontal and vertical canal orientations. .............................................. 101 Table 4.14 Mean time required, in seconds (s), for complete removal of materials with standard deviations. Canal oriented horizontally. ..................................................................................... 102 Table 4.15 Mean time required, in seconds (s), for complete removal of materials with standard deviations. Canal oriented vertically. .......................................................................................... 103   xiii List of Figures  Figure 1.1 Material obstructions – gold alloy ‘dust’ ....................................................................... 5 Figure 1.2 Material obstructions – amalgam .................................................................................. 6 Figure 1.3 Material obstructions – bur tip ...................................................................................... 6 Figure 1.4 Material obstructions – glass beads ............................................................................... 7 Figure 1.5 Techniques for removing materials in the root canal .................................................. 12 Figure 1.6 Computation fluid dynamic (CFD) models showing visualization of irrigant flow through i) streamlines and ii) particle tracking. ............................................................................ 15 Figure 1.7 Continuous flow ultrasonic irrigation .......................................................................... 21 Figure 3.1 Example of a transparent, 3D-printed, micro-CT scan-based resin molar tooth (lateral view) ............................................................................................................................................. 33 Figure 3.2 Example of maxillary and mandibular molar tooth models ........................................ 34 Figure 3.3 Mandibular molar (L5) showing an abbreviation for each canal (ML, MB, DL, DB) documented in a specific location on the external platform to assist with orientation purposes (occlusal view). ............................................................................................................................. 36 Figure 3.4 Mandibular molar with GP point placed in a canal to measure the canals length and diameter at the apical foramen. ..................................................................................................... 37 Figure 3.5 Mandibular molar (L4) – i) occlusal view, ii) lateral view, iii) mesial view, iv) distal view ............................................................................................................................................... 38 Figure 3.6 Fluorescent microscope image of i) amalgam ‘dust’ particles and ii) gold ‘dust’ particles. ........................................................................................................................................ 43 Figure 3.7 Photographs of different materials on Petri dish and spatula ...................................... 45   xiv Figure 3.8 Photographs of materials in apical root canal of tooth model ..................................... 47 Figure 3.9 Photograph of mandibular molar (L4) placed horizontally in sample holder ............. 48 Figure 3.10 Photograph showing GentleWave handpiece with yellow nozzle (size 9) placed into access cavity and pulp chamber of tooth L4. ................................................................................ 55 Figure 3.11 Irrigation needles used in the experimental groups ................................................... 56 Figure 3.12 Syringe needle irrigation setup .................................................................................. 57 Figure 3.13 ProUltra PiezoFlow ultrasonic needle setup .............................................................. 57 Figure 3.14 ProUltra PiezoFlow Ultrasonic Irrigation overall setup ............................................ 58 Figure 3.15 Photograph of mandibular molar (L4) placed vertically in sample holder (front view)....................................................................................................................................................... 59 Figure 3.16 Examples of samples for visual evaluation following irrigation. Photos show amalgam ‘dust’ particles before (ia-iva) and remaining particles (ib-ivb) after irrigation. Subsequently, percentage of material removed could be determined and then placed into an amount of material removed category. All photos taken at 10.4x magnification. ........................ 62 Figure 4.1 Interpretation guide for box and whisker plot (courtesy of Dr Jolanta Aleksejuniene)....................................................................................................................................................... 80 Figure 4.2 Percentage of material removed in irrigation groups (all materials and canal orientations combined; 60 tests per group) ................................................................................... 81 Figure 4.3 Percentage of blue PE removed in irrigation groups (combined canal orientations; 10 tests per group) .............................................................................................................................. 81 Figure 4.4 Percentage of glass removed in irrigation groups (combined canal orientations; 10 tests per group) .............................................................................................................................. 81   xv Figure 4.5 Percentage of titanium metal removed in irrigation groups (combined canal orientations; 10 tests per group) .................................................................................................... 82 Figure 4.6 Percentage of stainless steel metal removed in irrigation groups (combined canal orientations; 10 tests per group) .................................................................................................... 82 Figure 4.7 Percentage of gold alloy ‘dust’ removed in irrigation groups (combined canal orientations; 10 tests per group) .................................................................................................... 83 Figure 4.8 Percentage of amalgam filling ‘dust’ removed in irrigation groups (combined canal orientations; 10 tests per group) .................................................................................................... 83 Figure 4.9 Percentage of material removed by canal orientation (all irrigation groups and materials combined; 210 tests per canal orientation) .................................................................... 88 Figure 4.10 Percentage of material removed by canal orientation (horizontal or vertical) for each irrigation group at a respective maximum irrigation time (all materials combined; 30 tests per group) ............................................................................................................................................ 90 Figure 4.11 Percentage of material removed by syringe needle irrigation, open-ended (SNI OE) at flow rates of 5 mL/min and 15 mL/min (all materials combined; 60 tests per group) ............. 92 Figure 4.12 Percentage of material removed by syringe needle irrigation, open-ended (SNI OE) at flow rates of 5 mL/min and 15 mL/min for each material. ....................................................... 93 Figure 4.13 Percentage of material removed by syringe needle irrigation, side-vented (SNI SV) at flow rates of 5 mL/min and 15 mL/min (all materials combined; 60 tests per group) ............. 95 Figure 4.14 Percentage of material removed by syringe needle irrigation, side-vented (SNI SV) at flow rates of 5 mL/min and 15 mL/min for each material. ....................................................... 95 Figure 4.15 Percentage of material removed by PiezoFlow Ultrasonic (PF US) at flow rates of 0 mL/min and 15 mL/min (all materials combined; 60 tests per group) ......................................... 97   xvi Figure 4.16 Percentage of material removed by PiezoFlow Ultrasonic (PF US) at flow rates of 0 mL/min and 15 mL/min for each material. ................................................................................... 98 Figure 4.17 Time required, in seconds (s), for complete removal of each material using GentleWave. Both canal orientations combined. ........................................................................ 104      xvii List of Symbols  + Plus minus = Equals  > Greater than  < Less than # Number / Per % Percentage  o Degree oC Degree Celsius  a Type I error rate b Power µm Micrometer     xviii List of Abbreviations  3D  Three dimensional  A  Amalgam filling ‘dust’ Ag  Silver  ANOVA Analysis of variance Au  Gold CBCT  Cone beam computed tomography  CFD  Computational fluid dynamics  CFUI  Continuous flow ultrasonic irrigation cm  Centimeter  cm3  Cubic centimeter  Cu  Copper DB  Distobuccal DL  Distolingual  EDTA  Ethylenediaminetetraacetic acid g  Gram  G  Glass Gold  Gold alloy ‘dust’  GP  Gutta percha  Gr  Group GW  GentleWaveTM  Hg  Mercury   xix In  Indium IQR  Inter-quartile range L  Lower MB  Mesiobuccal Micro-CT Micro-computed tomography min(s)  Minute(s) mL  Milliliter ML  Mesiolingual  mm  Millimeter  n  Tests NaOCl  Sodium hypochlorite  OE  Open-ended OSU  Ohio State University p  Significance  Pd  Palladium PE  Polyethylene PF US  PiezoFlow ultrasonic  PMMA Polymethyl methacrylate PUI  Passive ultrasonic irrigation  RPM  Rotations per minute  s  Second sd  Standard deviation  sec  Second   xx Sn  Tin SNI  Syringe needle irrigation SS  Stainless steel metal SV  Side-vented  T  Titanium metal U  Upper WL  Working length  x  Times Zn  Zinc    xxi Acknowledgements  A special thank you is extended to Dr. Markus Haapasalo, who not only was my supervisor for this research project but also allowed me to pursue my passion in studying graduate endodontics at the University of British Columbia. Although research was relatively new to me and at times challenging, Dr. Haapasalo had the innate ability to make it both enjoyable and thought-provoking. Through Dr. Haapasalo’s passion and commitment to endodontic research and teaching, I truly see research in a new light and for that I am forever appreciative. Thank you for making such a positive impact on both an academic and personal level.   To my committee member Dr. Ya Shen, thank you greatly for always being available to answer all my many questions. The feedback you provided was always helpful, done efficiently, and delivered with a smile on your face. In addition, thank you to Dr. Ricardo Carvalho for being part of my supervisory committee and providing valuable insights along the progression of my project. Furthermore, I would like to thank Dr. Jolanta Aleksejuniene for her immense support with statistical analysis and interpretation of the results. The guidance you provided was always very valuable and allowed this research project to progress on time.  I would like to also thank Dr. Abdullah Almegbel and Dr. Zhejun Wang for their assistance with the laboratory work.  To the director of the graduate endodontic program, Dr. Jeffrey Coil, and to the director of the undergraduate endodontic program, Dr. Ahmed Hieawy, thank you for accepting me into the program and for being great mentors that I will always aspire to be like in my future endodontic career. To my fellow grad endodontic residents and clinic CDAs, thank you for the   xxii support and camaraderie during our time together. I am sure the memories we made together will be everlasting. Lastly, I would like to thank my family - my mom, Nuri, my dad, Nazir, and my brother, Fahim. Each one of you has instilled in me the values of hard work, compassion to others, and balancing academics with pleasure. Thank you for the love, support, and continuous encouragement to reach for my personal goals.     xxiii Dedication  The only person I can truly dedicate this work to is Amberene - my wife, my love, and my best friend. You encourage me to pursue my dreams, you embrace my desire to learn more, you support me throughout any ups and downs, and you are always there to listen patiently. This accomplishment is as much yours as it is mine. I am eternally grateful for all that you have given me.  With all my heart, thank you.     1  Review of the Literature  1.1 Endodontic treatment   1.1.1 Purpose The purpose of endodontic treatment is to retain the dentition by eliminating diseases of the dental pulp and periapical tissues. It is well established that these diseases, pulp necrosis and apical periodontitis, are caused by microorganisms and the by-products they release while infiltrating the dental pulp (Kakehashi et al., 1965; Bergenholtz, 1974). In 1965, the classic study by Kakehashi et al. (1965) on germ free Fisher rats showed that bacterial presence is the major determinant in inducing endodontic disease. Apical periodontitis is a host-induced inflammation and rapid destruction of the periapical tissues that follows bacterial infection of the root canal system (Nair, 2004). Overall, the ultimate goal of endodontic treatment is to prevent the progression of and treat apical periodontitis (Ørstavik & Pitt Ford, 2008). 1.1.2 Principles The principles of treating apical periodontitis involve eliminating infection from the root canal system and preventing re-infection (Nair, 2004). Root canal disinfection occurs through both mechanical and chemical methods using instruments, copious irrigating solutions, and intracanal medicaments (Haapasalo et al., 2010). It has been found that mechanical instrumentation with only saline irrigation can reduce intracanal bacteria by 100 to 1000-fold (Byström & Sundqvist, 1981). Instrumentation must be accompanied by irrigation to   2 mechanically create flow to flush out loose pulp tissue and dentin debris, as well as chemically decrease the amount of tissue remnants and microbial load (Chow, 1983; Haapasalo et al., 2010). Together, the combined process of mechanical and chemical disinfection can be called chemo-mechanical cleaning and shaping. It is deemed one of the most important stages as it includes disinfection of the root canal system and also prepares the root canal for obturation/filling (Johnson et al., 2016). However, even when the highest standards of cleaning and shaping are followed, there are still regions of the root canal system that cannot be disinfected (Siqueira et al., 2002). This can lead to endodontic failure (Nair, 2005). Therefore, the root filling is also important as it can entomb any surviving bacteria and create a seal to prevent ingress of bacteria attempting to re-infect the root canal system. Lastly, placement of a good quality coronal permanent restoration is critical to seal the dentin and prevent leakage of bacteria from the oral cavity into the root canal system (Ray & Trope, 1995).   1.1.3 Outcome The successful outcome of endodontic treatment is based on the criteria of fully cleaning, disinfecting, and sealing the entire root canal system. If attained, this can lead to resolution of clinical symptoms and a normal radiographic apex. However, successful endodontic therapy is challenged by factors such as resistant microorganisms that cannot be completely removed (Nair, 2004), a complex root canal morphology (Hess & Zürcher, 1925; Vertucci, 1984), and the possibility of endodontic complications (Peters, 2006). To overcome these challenges and improve outcome success, numerous contemporary devices are available that can agitate or activate irrigants (Gu et al., 2009). Previous studies have shown that agitation of irrigation   3 solutions improves the mechanical and chemical effectiveness of irrigation, resulting in improved treatment outcome (Moorer & Wesselink, 1982). For example, numerous studies have shown activation of irrigants using ultrasonic energy improves cleaning efficacy of the root canal system (van der Sluis et al., 2007; Jiang et al., 2011; Chen et al., 2014). However, the effectiveness in improving treatment outcome using newer generation irrigant activation devices, such as the GentleWave system, has not been thoroughly explored. Specifically, further studies on the ability of GentleWave to deal with endodontic complications and their impact on endodontic outcome are needed.   1.2 Endodontic complications   1.2.1 Classification of endodontic complications  Complications that occur during root canal treatment can be classified based on when they occur, either intra-treatment or post-treatment (Blicher et al., 2016). Intra-treatment complications can include missed canal anatomy, instrument separation, perforations, sodium hypochlorite accident, extrusion of materials beyond the tooth apex, thermal injury, air emphysema, allergic hypersensitivity reactions, adverse reactions to local anesthesia, paresthesia following root-end surgery, and obstructions of the root canal (Peters, 2009; Blicher et al., 2016). Post-treatment complications can comprise of flare-ups, persistent pain, persistent infections, coronal leakage, and vertical root fracture (Peters, 2009; Blicher et al., 2016).  Obstructions of the root canal are important to detect, manage, and treat as they can impede access to regions of the root canal system which may be infected, and proper disinfection   4 and sealing cannot be fully obtained. Obstructions of the root canal can be classified as either physiologic (natural) or iatrogenic. Physiologic obstructions of the root canal system can include pulp canal obliteration by tertiary dentin deposition, severe curvatures of the root canal, and the presence of calcified structures in the pulp. On the other hand, iatrogenic obstructions can be classified as intentional or unintentional (Chenail et al., 1987). Intentional iatrogenic obstructions may include obturation materials such as silver points and gutta percha, or posts (Chenail et al., 1987). Unintentional iatrogenic obstructions may occur due to separated instrument fragments, blockage by dentin debris, ledging leading to canal obstruction, and blockage by different materials.    1.2.2 Material obstructions   1.2.2.1 Types of material obstructions  Materials that can block canals include dental materials such as amalgam, gold, composite, and porcelain (Figures 1.1 and 1.2). These typically are part of coronal restorations which occasionally fall passively into the root canal during endodontic access cavity preparation. In addition, materials can also include small metal pieces like titanium and stainless steel separated from posts and dental instruments such as burs (Figure 1.3). In addition, there are reports of patients accidentally placing materials into root canals that have been left open for drainage resulting in possible blockage. Some materials include a toothpick, needles, pins, fingernails, screws, glass beads, and pencil lead (Chenail et al., 1987; Subbareddy & Mehta, 1990; Kalyan et al., 2010; Pinky et al., 2011) (Figure 1.4).   5  i)   ii)   Figure 1.1 Material obstructions – gold alloy ‘dust’  Gold alloy ‘dust’ (black arrows) over the orifice of the canal in a mandibular premolar after access cavity preparation for root canal treatment. i) 5.1x magnification, ii) 8.0x magnification.  i)  ii)      6 Figure 1.2 Material obstructions – amalgam  i) Radiograph showing mass of amalgam (black arrow) in the middle third of root canal of tooth #11. Image from Slutzky-Goldberg et al. (Slutzky-Goldberg et al., 2006), with permission from publisher (Quintessence Publishing). ii) Radiograph showing three pieces of amalgam (black arrows) in the root canal of tooth #45. Image from Al Ghamdi et al. (Al Ghamdi et al., 2017), with permission from publisher.       Figure 1.3 Material obstructions – bur tip Radiograph showing a separated bur tip (black arrow) in apical root canal of tooth #11. Image from Meidinger & Kabes (Meidinger & Kabes, 1985), with permission from publisher.    7 ia)  ib)   Figure 1.4 Material obstructions – glass beads ia) Radiograph showing an object (black arrows) in the middle third of root canals of teeth #11 and #21, ib) Picture after removal showing them to be glass beads. Images from Subbareddy & Mehta (Subbareddy & Mehta, 1990), with permission from publisher.   1.2.2.2 Management options for material obstructions  Multiple studies exist discussing management options for root canals obstructed by different materials. In general, these include bypassing the object(s), removing the object(s), or leaving the object(s) while performing standard non-surgical endodontic treatment coronal to the object(s) with the possibility of periapical surgery to be performed afterwards. Flotation of the foreign material using copious amounts of irrigating solution has also been advocated (Stewart, 1986).  Fors & Berg (1986) were one of the first to create what they called a ‘therapeutic schedule’ indicating how to manage a material obstruction. It was based on important prognostic factors   8 such as the initial endodontic diagnosis, the location of the obstruction in the root canal, the adaptation between the material and canal wall, the sealing ability of the obstruction, and if there was presence of necrotic pulp tissue apical to the obstruction. The authors concluded that an obstruction should always be removed if present in the coronal third of the root canal (Fors & Berg, 1986). If present in the middle third and there is necrotic pulp tissue apical to the object(s), then attempts to bypass or remove the object(s) should be performed (Fors & Berg, 1986). Lastly, the authors recommend leaving the object(s) within the canal if it seals well to dentine and there is non-infected vital tissue apical to the object(s) (Fors & Berg, 1986). If this is not the case, the authors advocate apical surgery with application of a well-sealed retrograde filling (Fors & Berg, 1986).  According to Friedman et al. (1990), if an obstructing material cannot be grasped, then bypassing should first be attempted before trying to remove. Friedman et al. (1990) stated that an attempt to bypass the obstruction by carefully using small stainless steel hand files can possibly loosen the obstruction. Removal can then be attempted with more ease using copious irrigation and one or a combination of the methods described below.  The method and success of removal depends on the obstruction’s composition, adherence to root canal wall, length and size of the obstruction, location in the canal, and diameter and curvature of the root canal (Hülsmann, 1993; Al Ghamdi, 2017). Multiple classical and current methods in the literature have been proposed to remove material obstructing root canals. Initially, attempts using chemical agents were used to intentionally corrode the metal objects (Hülsmann, 1993). However, they were unsuccessful for objects that bound to the root canal wall. Manual retrieval techniques have also been advocated (Hülsmann, 1993). If the material is accessible in the root canal, these may include Stieglitz forceps (Figure 1.5i) or a Mosquito   9 hemostat. If space is available between the material and canal wall, other manual techniques include using individual endodontic hand files (K-files, H-files) or the braiding technique. The braiding technique involves placing multiple Hedström files around the object, twisting the files to engage the object, then pulling coronally to remove the object engaged within the files (Figures 1.5iia and 1.5iib). Gates Glidden burs (Dentsply Maillefer, Tulsa, OK) (Figure 1.5iii) have also been advocated to break down larger pieces of material to ease their removal using other methods (McCullock, 1993). Hollow microtube delivery systems accompanied by mechanical or adhesion techniques have also been proposed (Friedman et al., 1990; Hülsmann, 1993). For example, the Masserann technique (Masserann Kit, Medidenta International Inc., Woodside, NY) involves using a trephan bur to cut the dentin and expose the obstruction. An extractor tube is then used to retrieve the obstruction (Pai et al., 2013) (Figure 1.5iv). Loosening the obstruction using energy from ultrasonic instruments, through direct contact or indirectly through irrigant flow, has also been advocated (Meidinger et al., 1985; Nehme, 2001). If straight-line access is available and in the absence of other methods above, Pai & Arora (2018) advocated a novel technique using a syringe needle with a sharp beveled edge to cut dentin and dislodge the obstruction (Figure 1.5v). Usually, multiple methods mentioned above need to be used in combination to remove the obstructing material (Nehme, 2001).     10 i)   iia)          iib)      11 iii)  iv)   v)         12 Figure 1.5 Techniques for removing materials in the root canal i) Stieglitz forceps. iia) Diagram of Hedström braiding technique. Image from Hülsmann (Hülsmann, 1993), with permission from publisher. iib) Intraoral clinical picture showing the Hedström braiding technique. Image from McCullock (McCullock, 1993), with permission from publisher (Quintessence Publishing).  iii) Gates Glidden burs. iv) Masserann Kit showing example of a trephan bur and extraction tube. Image from Hülsmann (Hülsmann,        1993), with permission from publisher. v) Sharp, beveled syringe needle. Metallic obstruction (black arrow) removed from root canal. Based on      model suggested in Pai & Arora, 2018.   1.2.2.3 Concerns with management options    There are multiple concerns with the current techniques used to bypass or remove material obstructions. Hülsmann (1993) reports there is a risk of extruding chemical agents into the periapical tissues, with a possibility of a cytotoxic reaction. Forceps or hemostats are unable to access an obstruction when it is in the apical portion of the root canal or present around a curvature (Hülsmann, 1993). In addition, it has been reported that using files, burs, specialized retrieval kits, and ultrasonics may remove excessive amounts of radicular dentin. This could lead to ledge formation, over-enlargement and transportation of the root canal, root perforation, and future vertical root fracture (Friedman et al., 1990; Hülsmann, 1993; Al Ghamdi, 2017). As well, Pai et al. (2013) reported these techniques can be very time-consuming, taking 20 minutes to several hours for the Masserann technique. Nehme (2001) reported that using ultrasonics for long periods of time without intermittent water cooling can lead to PDL necrosis due to excessive heat   13 generation. There is also the concern of separating the instrument being used to bypass or remove the obstruction (Friedman et al., 1990). It has been reported that management options causing extrusion of material out of the tooth apex can potentially result in inflammation and infection. Costa et al. (2006) reported pushing a foreign body through a root canal into the maxillary sinus resulted in long-term maxillary sinusitis. There have also been reports of actinomycosis in the maxilla due to a piece of jewelry extruding out the apex of a maxillary central incisor (Pinky et al., 2011; Alrahabi et al., 2014).    1.2.2.4 Prevention of material obstructions  There are several recommendations in the literature about how to prevent dental materials from falling into the root canal system causing obstruction. Al Ghamdi (2017) advises removal of the coronal restoration completely or from the walls of the endodontic access and a new build-up placed prior to access cavity preparation if the restoration appears old and needs replacement. It has also been stated that removal of the restoration can allow the operator to visualize for any cracks or fractures, determine restorability of the tooth, better assess for crown lengthening, improve tooth isolation, and improve visibility (Al Ghamdi, 2017). In addition, a high-volume suction should always be placed nearby during removal of the coronal restoration (Al Ghamdi, 2017).       14 1.3 Irrigation   1.3.1 Goals of irrigation        The goal of irrigation during endodontic treatment is to both mechanically debride (flushing) and chemically disinfect the entire root canal system (Haapasalo et al., 2010). Mechanical and chemical functions are dependent on the irrigant and its use in combination with various delivery and activating systems. The mechanical function involves creation of a flow to flush microorganisms, pulp tissue remnants, dentin debris, smear layer and biofilm from the root canal system. The chemical action involves reducing instrument and dentin friction through acting as a lubricant, improving the cutting effectiveness of endodontic files, dissolving pulp tissue (inorganic and organic) and biofilm, and inactivating microorganisms and their by-products. These functions are all to be performed within the confines of the root canal system because of the cytotoxic potential of the irrigating solutions if extruded into the periapical tissues (Zehnder, 2006; Haapasalo et al., 2010; Haapasalo et al., 2014).   1.3.2 Fluid hydrodynamics  In order to eliminate debris, microorganisms, and pulp tissue from the root canal system through irrigation, various irrigant agitation techniques and devices have been developed (Gu et al., 2009). Their purpose is to optimize fluid flow, thereby allowing the irrigant to access complex anatomical areas of the root canal system that were not accessed by mechanical instrumentation. Irrigation hydrodynamics involves the study of fluid flow patterns, velocity,   15 depth of penetration, exchange of irrigants, as well as the forces created by these irrigants within the root canal system.  The direct measurements of fluid flow patterns in the root canal system were traditionally difficult to do in endodontic research. However, more recently, 3-dimensional fluid flow can be studied through a novel approach called computational fluid dynamics (CFD) (Boutsioukis et al., 2009; Gao et al., 2009). CFD has been used to simulate irrigant flow in a root canal model and subsequently evaluate parameters such as apical fluid pressure, pressure and shear stress on the root canal wall, and velocity. In addition, CFD can create streamlines and track simulated particles, thus allowing the irrigant flow to be visualized in the root canal model (Haapasalo et al., 2010) (Figure 1.6).   i)    ii)    Figure 1.6 Computation fluid dynamic (CFD) models showing visualization of irrigant flow through i) streamlines and ii) particle tracking.  Images from Haapasalo et al. (Haapasalo et al., 2010), with permission from publisher (Elsevier Inc.).   16 1.3.3 Irrigation methods   1.3.3.1 Syringe needle irrigation   Irrigation of the root canal system is most traditionally performed by attaching a needle to a plastic syringe. The sizes of plastic syringes most commonly used for irrigation range from 1 to 20 mL. There are also various needle tip designs varying in their gauges, portals of exit (open-end, closed-end), venting areas, and modifications (notched, beveled) (Shen et al., 2010). Syringe needle irrigation (SNI) occurs through positive pressure. Irrigant is extruded through the needle apically into the root canal system while pressure is being applied to the syringe barrel.  The efficacy of SNI is affected by multiple variables. These can include the preparation size and taper of the apical root canal, needle placement depth, needle size and design, type of irrigant solution, and irrigant volume (Hu et al., 2019).  In 1983, Chow (1983) stated that if irrigation is to be mechanically effective in removing debris from the apical root canal, then the irrigant must “(a) reach the apex, (b) create a current (force); and (c) carry the particles away”. In Chow’s (1983) study, it was found that there is minimal displacement of particles beyond the needle tip. Therefore, he concluded irrigation effectiveness is based on depth of needle insertion. Both Ram (1977) and Chow (1983) concluded that smaller size needles and larger apical preparations allow needles to reach the apex and more effectively remove debris. Using a CFD model, Boutsioukis et al. (2010b) confirmed larger apical preparation sizes and deeper needle insertion allow greater irrigant replacement, greater shear stress on the canal wall, and higher apical pressures.    17 It has been found that different needle tip designs cause different irrigant flow patterns in the root canal (Boutsioukis et al., 2010a; Shen et al., 2010). Open-ended needles result in more irrigant replacement apical to the needle tip than close-ended needles, however they also create higher apical pressures (Boutsioukis et al., 2010a). Flow pattern, flow velocity, and apical wall pressure are all affected by needle tip design (Shen et al., 2010). Side-vented needles cause the lowest apical pressure whereas beveled needles cause the highest (Shen et al., 2010).  In a 1975 study, Baker et al. (1975) found that irrigation efficacy is influenced more by the volume of the irrigant than the type of irrigation solution used. Therefore, it is advocated that irrigant solution be continuously replenished when performing SNI as it encourages more debris removal, canal disinfection, and irrigant penetration into dentinal tubules (Bronnec et al., 2010).  Although SNI is the most common form of irrigation in endodontics, it has several limitations. It has a limited ability to penetrate into all aspects of a tooth’s complex root canal anatomy, such as fins and isthmuses. Studies show that irrigant does not progress further than 1 to 1.5 mm apical to a side-vented needle tip at flow rates ranging from 0.02-0.79 mL/sec (Boutsioukis et al., 2009) and 2 to 3 mm apical to open-ended and side-vented needle tips at flow rates beyond 4 mL/min (Park et al., 2013). Some irrigants can be cytotoxic to the periapical tissues, therefore irrigant extrusion is a potential concern. To reduce the risk of irrigant extrusion, it has been advocated to use side-vented needles rather than open-ended needles and not to bind the needle tip into the root canal wall (Chen et al., 2014). In fact, Hu et al. (2019) performed a recent study finding that moving the needle manually up and down during irrigation allows an improved flushing effect and less risk of apical extrusion. In addition, the formation of bubbles of air in the root canal can block irrigant from advancing into the apical root canal system. This phenomenon is called the vapor lock effect and was confirmed in a study by Tay et al. (2010). To   18 overcome these limitations, newer irrigation methods have been developed which use energy to agitate the irrigation solution.  1.3.3.2 Ultrasonic irrigation   1.3.3.2.1 History of ultrasonic irrigation  The introduction of ultrasonics for root canal treatment was first recommended by Richman in 1957. Ultrasonic activation involves attaching files, wires, irrigation needles, reamers or broaches to an ultrasonic dental unit. Upon activation, the attachment oscillates and transfers acoustic vibrations to irrigants in the root canal at ultrasonic frequencies of 25 to 35 kHz. The transmitted vibrations are used to maximize the cleaning and disinfection of the root canal system.    1.3.3.2.2 Mechanism of action of ultrasonic irrigation  There has been much controversy over ultrasonics true mechanism of action in the root canal system. Martin (1976) was the first to propose that ultrasounds mechanism of action was through mechanical vibrations increasing the antibacterial properties of the irrigating solution and through cavitation effects. Cavitation involves the generation of bubbles which grow and eventually implode or collapse (Ashokkumar, 2011; van Wijngaarden, 2015). This results in the release of shock waves and turbulence that can disrupt biofilm and rupture bacterial cell walls (Martin, 1976; Layton et al., 2015). Ahmad (1987a) performed a study to validate the claims by   19 Martin and found that ultrasonic cleaning of the root canal system did not occur through cavitation, but through acoustic microstreaming. Acoustic microstreaming involves the generation of rapid fluid movement in a vortex motion which generates shear stresses on the root canal wall, ultimately enhancing disinfection and debridement (Ahmad et al., 1987a; van der Sluis et al., 2007; Layton et al., 2015). In a follow-up study, Ahmad et al. (1987b) also found that smaller files (size #15) and higher ultrasonic power settings result in more acoustic microstreaming. In a more recent study, Jiang et al. (2011) confirmed that the higher ultrasonic setting resulted in increased velocity of the irrigant and overall improved cleaning efficacy of the root canal system. Others have claimed the cavitation effect does occur, but only along certain portions of the oscillating file and only if contact with the root canal wall does not inhibit the files motion (Lumley et al., 1988; Roy et al., 1994; van der Sluis et al., 2007). Ultrasonic irrigation has been shown to be more effective in removing tissue and debris, disrupting root canal biofilm, and improving irrigant penetration into dentinal tubules than SNI (Chen et al., 2014).   1.3.3.2.3 Types of ultrasonic irrigation The ultrasonic technique described above has become commonly known as passive ultrasonic irrigation (PUI). The file attached to an ultrasonic handpiece is immersed in the irrigation solution within the root canal and then ultrasonically activated to transfer acoustic energy (van der Sluis et al., 2007). There are two flushing methods with PUI, intermittent flush and continuous flush. The intermittent flush technique involves using a syringe needle to deliver irrigant into the root canal,   20 then activating the irrigant with the oscillating ultrasonic file, then flushing the root canal with fresh irrigant to remove loose debris and tissue remnants. A commercially available system that uses the intermittent PUI technique are the Irrisafe ultrasonic tips (Satelec Acteon, Merignac, France). In contrast, the continuous flow method delivers a continued supply of fresh irrigant directly through an ultrasonically activated needle. It is known as the continuous flow ultrasonic irrigation (CFUI) technique.  Although there are reports in the literature that the intermittent flush technique results in increased canal and isthmus cleanliness, less remaining debris, and better canal disinfection than SNI, there are many potential limitations (Jiang et al., 2012; Chen et al., 2014; Topcuoglu et al., 2015). A study by Jiang et al. (2010) showed that complete debris removal from the root canal system is not possible and the direction of movement of the oscillating file has importance. In ovoid shaped canals, the oscillating file is more effective if directed into recesses or grooves to maximize irrigant movement and debris removal. In addition, the intermittent flush technique does not allow a continuous replenishment of fresh irrigant. Irrigant solutions, such as sodium hypochlorite, have been shown to lose their tissue dissolving and antibacterial effects after a few minutes (Siqueira et al., 2000). Intermittent flushing also limits the volume of irrigant flowing through the canal system and cannot dissipate heat produced by ultrasonic irrigation. To overcome these limitations of intermittent flushing, a group out of Ohio State University (OSU) in 2007 created an irrigation needle that can simultaneously be ultrasonically activated and apically expel a continuous supply of fresh irrigant. This CFUI prototype (Figure 1.7) became commercially available as the ProUltra PiezoFlow ultrasonic irrigation system (Dentsply Tulsa Dental Specialties, Johnson City, TN). The OSU group performed multiple studies assessing the   21 effectiveness of adding a final one-minute rinse and agitation with CFUI. In their first study, they found CFUI resulted in significantly less necrotic debris and biofilm in canals and isthmuses compared to using SNI alone (Burleson et al., 2007). In a subsequent study, they found CFUI had greater antibacterial efficacy than SNI alone and reported a significant reduction in positive cultures by CFUI (Carver et al., 2007). Jiang et al. (2012) compared six different final irrigation techniques and found CFUI as a final rinse to be significantly better at removing dentinal debris from apical irregularities compared to the intermittent flush and SNI techniques. SNI was found to be the least effective. Altogether, the addition of ultrasonic irrigation to endodontic treatment has shown to improve the cleaning and disinfection of the root canal system.    Figure 1.7 Continuous flow ultrasonic irrigation Diagrammatic representation (left) and picture of prototype (right) of continuous flow ultrasonic irrigation from the Ohio State University group. Image from Burleson et al. (Burleson et al., 2007), with permission from publisher.     22 1.3.3.3 GentleWave  1.3.3.3.1 GentleWave system GentleWave (Sonendo Inc, Laguna Hills, CA) is a novel irrigation system using optimized fluid dynamics and acoustics, developed to clean and disinfect the complexities of the root canal system with no to minimal instrumentation. The GentleWave System is made up of a console and handpiece. The console functions to degas the irrigating solutions, and to create high speed flow of the solutions at 45 mL/min. After a custom tip is attached to the handpiece, it is then placed into the endodontic access cavity to sit 1mm above the pulp chamber floor. To ensure there is no leakage of irrigant into the oral cavity, a block out resin (Kool-Dam; Pulpdent, Watertown, MA) is used to create a seal between the head of the handpiece and tooth’s occlusal surface. Different degassed irrigants spray from the handpiece tip for a pre-set amount of time into the pulp chamber. Upon friction of the liquid hitting the nozzle tip at high speed, the irrigation fluid increases in temperature to 45oC (Haapasalo et al., 2014). The handpiece also has built-in 5-point vented suction holes that allow simultaneous removal of excess irrigation fluid and other material, as well as participates in creating negative pressure within the root canal system and pulp chamber.   1.3.3.3.2 Mechanism of action of GentleWave The GentleWave mechanism of action uses an interplay between multisonic energy, advanced fluid dynamics, and hydrodynamic cavitation. Degassed treatment fluid from the handpiece contacts stagnant fluid in the pulp chamber resulting in a powerful shear force. This causes hydrodynamic cavitation which is the formation of many microbubbles as a cavitation   23 cloud. These bubbles eventually implode creating acoustic sound waves over a broad frequency spectrum that travel through the treatment fluid apically into the entire root canal system (Molina et al., 2015). A slight negative pressure in the apical portion of the canals is also induced through the creation of a vortical flow in the procedure fluid. The interplay between the multisonic energy waves and fluid dynamics gives the term Multisonic Ultracleaning (Sigurdsson et al., 2018).   1.3.3.3.3 Studies on GentleWave There are multiple studies on the GentleWave system looking at its cleaning and disinfection efficacy, safety, ability to remove materials within the root canal system, and outcomes.  Haapasalo et al. (2014) performed an in vitro study showing GentleWave had the ability to dissolve bovine muscle tissue in a simulated model at a significantly faster rate (more than 8x) than conventional irrigation devices, including PUI. Ex vivo studies on extracted teeth have shown the GentleWave system resulted in greater reduction of pulp tissue debris (Molina et al., 2015), intracanal bacterial DNA (Zhang et al., 2018), smear layer, and intraradicular biofilm (Choi et al., 2019) compared to SNI or ultrasonic agitation. GentleWave has also been shown to allow NaOCl to penetrate deeper into dentinal tubules throughout the root canal system, including the apical region (Vandrangi, 2016). In a recent study, Chen et al. (2020) showed the GentleWave systems ability to partially or completely remove calcifications, such as pulp stones, from the root canal system of uninstrumented canals.   In vitro studies on the GentleWave systems safety have shown it generates a negative apical pressure (Haapasalo et al., 2016) and zero apical extrusion of irrigant (Charara et al.,   24 2016). Compared to conventional SNI, GentleWave can provide improved safety when irrigating the apical regions of the root canal. It has also been shown that while GentleWave cleans and disinfects, there is minimal dentin erosion (Wang et al., 2016) and the integrity of the original root canal wall anatomy is maintained (Wang et al., 2018).  Using extracted teeth, the GentleWave system has been shown to successfully remove separated instruments in an in vitro experiment (Wohlgemuth et al., 2015). In addition, a study showed GentleWave’s ability to remove calcium hydroxide paste within 90 seconds using only water irrigation (Ma et al., 2015), whereas several other studies have confirmed that no other irrigation method so far has been able to remove all calcium hydroxide from the root canal system of teeth. High levels of clinical success have also been reported using the GentleWave system (Sigurdsson et al., 2016a; Sigurdsson et al., 2016b). In a prospective clinical study, Sigurdsson et al. (2018) reported a 97.7% success rate at 12-month re-evaluation after treatment with the GentleWave procedure. Success was defined as healed or healing based on the periapical index (PAI) score (Orstavik et al., 1986). All teeth in this study had pulpal necrosis and presence of periapical radiolucencies. Using a visual analogue scale, Sigurdsson et al. (2016b, 2018) also reported in two studies that patients reported only mild to moderate pain in the first two days after initial GentleWave treatment. However, at 14 days, 6 months, and 12 months after initial therapy, there were no reports of patients experiencing pain. In a recent randomized control trial study, it was also found that postoperative pain incidence or intensity was similar between GentleWave treatment and conventional treatment using SNI and ultrasonic activation (Grigsby Jr et al., 2020). Longer follow-up times and other in vivo studies are needed to improve the validity of the findings on healing outcomes and post-operative pain after GentleWave treatment.    25  1.3.4 Effect of canal orientation on irrigation efficacy There is limited literature reporting if the efficacy of irrigation flow is affected by the orientation of the root canal due to gravity. Orientation refers to the root canal of a maxillary or mandibular tooth being oriented horizontally or vertically, respectively, when the patient is laying supine during root canal treatment. The effect of gravity has importance because in order for irrigant flow to exert its favorable actions of debris removal, it must penetrate the entire root canal system (Chow, 1983). However, patient safety is also a concern if the irrigant extrudes into the periapical tissues (Haapasalo et al., 2010). Uzunoglu et al. (2015) performed an in vitro study whereby they assessed if gravity affected the amount of irrigation extrusion from the apex. They compared different irrigation methods on teeth placed vertically as in the mandible, and on teeth placed at a 45o angle as in the maxilla. The authors reported significantly more irrigant extrusion in the mandibular vertically oriented teeth (Uzunoglu et al., 2015). However, even though the maxillary teeth were positioned at an angle against the force of gravity, irrigant extrusion was still observed (Uzunoglu et al., 2015).    1.4 Endodontic research   1.4.1 Microspheres  Microspheres have not been used in clinical endodontics or for endodontic research purposes thus far. Microspheres are microparticles which have a round, spherical shape. Their external diameter can typically range in size from 1 to 1000 µm. There are many types based on the raw materials the microspheres are manufactured from. The raw materials can be natural or   26 synthetic. Most commonly these include glass, polymers, ceramics, and metals. In the life science industry, there are numerous applications of microspheres. These can include, but are not limited to, delivering medications, calibrating and testing medical devices, tracers for fluid or air flow visualization, and acting as spacers or fillers. Microspheres can be customized based on their diameter, density, color, fluorescence, opacity, electrostatic charge, and magnetism. These customized parameters allow each type of microsphere a unique advantage and the ability to be modified for a specific application (Lipovetskaya, 2011; Cospheric LLC, n.d., https://www.cospheric.com/resources_technical_data/microspheres_microparticles.htm accessed on October 12, 2020).    1.4.2 3D printed resin in vitro tooth model Historically, endodontic education for pre-clinical training exercises and endodontic research has relied upon extracted human teeth, human cadavers, resin blocks, and commercially available artificial resin teeth (Nassri et al., 2008). Although extracted teeth remain the most realistic for learning, there are limitations such as availability, lack of standardized root canal anatomy, time wastage selecting appropriate teeth, ethical concerns, and cross-infection. The use of human cadavers also presents similar concerns as using extracted teeth. Resin plastic blocks with a simulated canal allow visualization of treatment steps, however there is lack of realism due to differing material physical properties compared to dentin and the deficiency of an entire root canal system (Spenst & Kahn, 1979). Artificially fabricated teeth are more realistic, standardized, and allow direct visualization if transparent. However, they have disadvantages   27 such as being costly, limited in number of tooth types and canal anatomy, and may require long delivery times (Nassri et al., 2008; Reymus et al., 2019).  More recently, 3D printing technology has evolved and can be used for various endodontic applications. These can include guides for endodontic access opening and root-end surgery, printed replicas to estimate an accurate size for autotransplantation, and 3D printed resin tooth models (Anderson et al., 2018; Shah & Chong, 2018). There are various types of tooth models that can be used for different areas of endodontic education such as surgery, pre-clinical simulation, regenerative endodontics and research simulation (Anderson et al., 2018). The 3D printed resin tooth models are manufactured by digitizing an extracted human tooth through a CBCT or micro-CT scan, applying appropriate software, then finally reproduced using a 3D printer system (Shah & Chong, 2018; Reymus et al., 2019). They allow the benefit of standardization and realism for pre-clinical endodontic simulation exercises and pre-clinical research because multiple identical prototypes of the same tooth can be printed (Anderson et al., 2018). The 3D printed resin tooth model also has limitations such as higher cost, lack of availability, and dissimilar physical properties to dentin (Reymus et al., 2019).        28  Aims and Hypotheses  2.1 Rationale During root canal treatment, small pieces of dental and other materials, typically part of coronal restorations, may accidently fall into the root canal. Depending on the size of such particles and size of the root canal, the particles may reach to the most apical part of the canal. This may prevent not only the progression of endodontic instruments to the required apical length for cleaning and shaping, but also effective disinfection of the apical root canal. As a result, obturation and sealing of the system can be negatively affected. Therefore, in the event materials fall into the root canal, it is important for the dentist to detect such obstructions and take an effort to remove them as they may compromise the outcome of endodontic treatment. Various traditional methods have been recommended for removal of these materials from all levels of the root canal. However, these methods have concerns such as removing excess amount of radicular dentin, being time-consuming, and pushing material out of the canal apex (Friedman et al., 1990; Hülsmann, 1993; Pai et al., 2013; Al Ghamdi, 2017). No study thus far has evaluated and compared the ability of different irrigation methods to remove pieces of dental and other materials from the apical aspect of a root canal.     2.2 Aims 1. To examine and compare to a multisonic cleaning system, GentleWave, the success by conventional irrigation methods of syringe needle irrigation and ProUltra PiezoFlow ultrasonic irrigation in removing particles of dental and other materials from   29 the apical part of a distal root canal of a 3D printed, micro-CT scan-based resin mandibular molar.   2. To examine if the orientation of the canal, horizontally or vertically, has an effect on the ability of the different irrigation methods to remove materials from the apical root canal.  3. To examine if the type of material has an effect on the success of removing it from the apical root canal.   4. To examine if a flow rate of an irrigation method has an effect on the ability to remove materials from the apical root canal.  2.3 Null hypotheses 1. The different irrigation methods compared to the GW system do not differ in their ability to remove dental and other materials from the apical part of a root canal.  2. The canal orientation does not influence the ability of different irrigation methods to remove dental and other materials from the apical part of a root canal.  3. The success of removing particles of materials does not depend upon the type of the material.     30 4. Flow rate of an irrigation method is not associated with a larger percentage of material removed.   31  Materials and Methods   3.1 Experimental design  This in vitro study was designed to compare the efficacy of irrigant flow of the multisonic GW system compared to syringe needle irrigation and ultrasonic irrigation in the apical aspect of a distal root canal. The parameters measured and analyzed were: (1) the amount of dental and other material removed from the apical root canal, as expressed by the categorical amount of material removed and the percentage of material removed, and (2) the time required for complete removal of the material.   3.2 Tooth selection and preparation Ten transparent, 3D-printed, micro-CT scan-based resin molar teeth were selected (Figure 3.1). The tooth models were printed using the Objet Eden260V 3D printer system (Stratasys, Rehovot, Israel). They were manufactured from VeroClear, which is a rigid transparent 3D printing resin material (Stratasys, Rehovot, Israel). According to the manufacturer, VeroClear is similar to polymethyl methacrylate (PMMA), also known as acrylic. It can be used as an alternative to glass and is ideal for concept modeling or prototyping clear products such as eyewear, light covers, and medical devices. Due to its see-through transparency, VeroClear allows visualization of internal components and features.  Tooth models were chosen for this in vitro study in order to visualize irrigant flow dynamics and to measure and analyze the parameters mentioned above. Each tooth model was given a number for documentation purposes which was written on the external platform with a red felt marker. These included three maxillary molars with three roots and four canals   32 (designated U1 to U3), and seven mandibular molars with two roots and four canals (designated L1 to L7) (Figure 3.2). The canals of each tooth were not previously instrumented. Each tooth model was provided with a flat, circular resin platform attached to the most coronal portion of the crown (Figure 3.2). Within the center of each platform, a circular and flat hollow space was present which also had the central presence of an access cavity to the tooth’s pulp chamber (Figure 3.2). The circumferential diameter and depth of the hollow space corresponded to the circumferential diameter of the nozzle placed into a GentleWaveTM molar handpiece (Sonendo Inc., Laguna Hills, CA). All resin teeth were inspected under a stereo microscope (Zumax OMS2350 LED Dental Microscope, Executive Dental Supply Ltd., Burnaby, BC, Canada) at magnifications of 2.8x, 4.2x, 6.9x, and 10.4x. In each model, canal orifices were located, and all canals were checked to ensure patency by advancing a size #6 stainless steel K-file (Dentsply Maillefer) approximately 1 mm past the apical foramen, however no filing was performed. The apical foramen was defined as the point the canal apically met the external surface of the resin root. Teeth models were excluded if the transparency of the tooth did not allow visual inspection of each canal from their entire coronal to apical extent, there was presence of cracks in the resin, there was lack of patency, or canals were unnegotiable. After exclusion, five molars (two maxillary, three mandibular) remained for preparation.     33  Figure 3.1 Example of a transparent, 3D-printed, micro-CT scan-based resin molar tooth (lateral view)     (a) Maxillary Molar                  (b) Mandibular Molar i)          i)   ii)                             ii)   1 2 3 3 2 1   34 Figure 3.2 Example of maxillary and mandibular molar tooth models (a) Example of a maxillary molar (U3) showing i) DB and P canals (lateral view), ii) circular resin platform (1), hollow space (2), and pulp chamber access cavity (3) (occlusal view). (b) Example of a mandibular molar (L5) showing i) MB and ML canals (lateral view), ii) circular resin platform (1), hollow space (2), and pulp chamber access cavity (3) (occlusal view).  3.3 Pre-treatment tooth model preparation  All pre-treatment preparation was performed under the stereo microscope at magnifications mentioned above. Using a black felt marker, an abbreviation for each canal was documented in a specific location on the external platform to assist with orientation purposes (Figure 3.3).  To create a standardized starting point, root canals of each tooth were cleaned by the GentleWave system for 2 minutes and 30 seconds using only sterile water stored at room temperature. Subsequently, each canal was dried with paper points (Diadent Group International, Burnaby, BC, Canada) and allowed to air dry at room temperature for 24 hours.  After 24 hours, patency of each canal was re-checked as described above. Each canal’s length was measured from the external surface of the resin platform to the apical foramen, the bottom surface of the hollow space to the apical foramen, and the canal orifice to the apical foramen using a size 15/.04 taper GP point (Diadent Group International, Burnaby, BC, Canada) held by cotton pliers (DPU17 Pliers, Hu-Friedy, Chicago, IL). Once the GP point was removed from the canal, its length (mm) was measured using a Miltex finger ruler (Integra Miltex, Princeton, NJ) and recorded. Each canal’s diameter (mm) at the apical foramen was measured using a passively fitting size 15/.04, 20/.04, or 25/.04 taper GP point (Diadent Group International, Burnaby, BC, Canada). The best GP point that passively fit to the apical foramen   35 or just apical to the apical foramen was placed into the canal (Figure 3.4). If the GP point extended past the apical foramen, the extended portion was cut to the external surface of where it exited the resin root using a scalpel blade (Miltex #15C Sterile Carbon Steel Surgical Scalpel Blade, Integra Miltex, Princeton, NJ). Once removed from the canal, the most apical diameter of each GP point was measured using an electronic digital caliper (Mitutoyo 500-196-30 Absolute Digimatic 6 inch/150 mm Stainless Steel Digital Caliper, Global Industrial, Canada) and recorded. From the remaining five molars, one mandibular resin molar (labelled L4) was arbitrarily chosen to perform all study experiments (Figure 3.5). Only one tooth model was chosen as this would allow the root canal morphology between all irrigation groups to be standardized. Specifically, standardization of the root canal’s taper, shape, diameter, length and configuration. Therefore, these potential confounding factors could be controlled. The chosen resin molar (L4) had one mesial root and one distal root. The mesial root had two canal orifices and two separate apical foramina. The mesial root canals had a Vertucci type VI classification - two canals which joined into one canal in the middle root third and separated into two canals in the apical root third (Vertucci, 1984). The distal root had two canal orifices and one common apical foramen. The distal root canals had a Vertucci type II classification – two canals which joined into one canal in the middle root third (Vertucci, 1984). In the distal root, the presence of anastomoses could also be visualized between both distal canals in the coronal and middle third, however the presence of lateral or accessory canals could not be visualized. Each canal’s diameter at the apical foramen and each canal’s length measurement is shown in Table 3.1. The working length for both distal canals was defined as 1 mm less than the length from the external surface of the resin platform to the apical foramen, therefore 26.0 mm. To simulate the clinical situation, a   36 closed system was created by sealing all apical foramina of both the mesial and distal roots using transparent white hot glue (AdTech Crystal Clear Hot Glue Gun Sticks, Hampton, NH). To prevent glue from entering the canals apically, a well-fitted GP point was first placed up to the apical foramen of each canal. Care was taken to ensure the glue did not obstruct visual examination of the apical aspect of the distal root canal.    Figure 3.3 Mandibular molar (L5) showing an abbreviation for each canal (ML, MB, DL, DB) documented in a specific location on the external platform to assist with orientation purposes (occlusal view).    37  Figure 3.4 Mandibular molar with GP point placed in a canal to measure the canals length and diameter at the apical foramen.  i)                    ii)     38 iii)                  iv)  Figure 3.5 Mandibular molar (L4) – i) occlusal view, ii) lateral view, iii) mesial view, iv) distal view  Tooth Canal Canal diameter at apical foramen (mm) Length from external surface of resin platform to apical foramen (mm) Length from bottom surface of hollow space to apical foramen (mm) Length from canal orifice to apical foramen (mm) L4 MB 0.15 27.0 21.0 14.0  ML 0.15 27.5 21.5 13.5  DB 0.25 27.0 21.0 12.5  DL  0.25 27.0 21.0 12.5  Table 3.1 Various measurements for each canal in mandibular resin molar tooth (L4)  3.4 Material acquisition and preparation  Two different methods were used to obtain particles of dental and other materials that may fall into the apical part of root canals.   The first method involved ordering manufactured microsphere beads (Cospheric LLC, Santa Barbara, CA). Different beads were chosen based on: (1) similarity to dental restorations or to pieces of dental burs, (2) diameters of approximately 200 μm, which is similar to the diameter at the apical end of a distal canal in a mandibular molar in a younger age group (18 to   39 25 years old) (Kuttler, 1955), (3) densities less than, similar to, and greater than 1.79-2.12 g/cm3, which is the density of dentin and pulp stones that have been found to be removable in uninstrumented distal canals using the GW system (Chen et al., 2020), and (4) ability to visualize through a stereo microscope for removal. The four microsphere beads chosen included blue polyethylene (PE), soda lime glass, titanium metal, and stainless steel metal. The designated abbreviation, composition, density, and range of diameters is shown in Table 3.2. This information was provided from the manufacturer (Cospheric LLC, Santa Barbara, CA).  The second method involved manually grinding dental materials from coronal restorations into dust particles. From the dental clinic at the University of British Columbia’s Faculty of Dentistry, multiple teeth containing amalgam restorations and one molar tooth containing a gold alloy full coverage crown were collected. All teeth were extracted for reasons not related to this study and were found stored in 0.5% NaOCl (one part water in nine parts bleach). All teeth were rinsed with filtered tap water. Amalgam was removed from the extracted teeth using a new high speed 557 carbide bur (Patterson Dental, Richmond, BC, Canada) placed in an air-driven high-speed dental handpiece (A-dec/W&H, Synea 400 series TG-98L, Newberg, OR) at a maximum RPM of approximately 200,000 under no water cooling. The amalgam dust was collected on a Petri dish (FisherbrandTM Sterile Polystyrene Petri Dish, Fisher Scientific, Waltham, MA). The gold crown was removed from the extracted tooth by sectioning using a new high speed 557 carbide bur placed in an air-driven high-speed dental handpiece under no water cooling. Once removed, a large piece of gold crown was held firmly with a dental hemostat (H4 Straight Dental Mosquito Hemostat, Hu-Friedy, Chicago, IL) while new slow speed round carbide burs (sizes 4LA, 6LA, 8LA) (Patterson Dental, Richmond, BC, Canada) placed in an air-driven handpiece (as above) under no water cooling were used to create gold   40 alloy dust. The gold dust was collected on a Petri dish. As it represents a clinical in vivo situation, the fabricated dust particles were not all the same size. The dust particle sizes were measured using a fluorescence microscope (Nikon Eclipse 80i, Nikon Corporation, Tokyo, Japan) attached to software (NIS Elements Imaging Software, Melville, NY) with an internal scale option. The designated abbreviation, general composition, approximate density, range of diameters and range of lengths is shown in Table 3.3.  Since the amalgam dust particles appeared round (Figure 3.6i), a diameter measurement is provided (Table 3.3). To determine the approximate density of the amalgam dust particles used in this study, a common dental amalgam used in the UBC Dentistry Clinic, Dispersalloy® (Dentsply Milford, Milford, DE), was used as a reference. It is an admixed amalgam containing both lathe-cut rods and silver-copper spheres. According to the manual on directions for use of Dispersalloy® Dispersed Phase Alloy Capsules (Dentsply International 2007, Charlotte, NC) and the materials Safety Data Sheet (Dentsply Milford 2016), Dispersalloy® dental amalgam is composed by combining 50% liquid mercury and 50% metallic alloy powder in a 1:1 ratio by weight. The liquid mercury has a density of 13.6 g/cm3. The alloy powder by weight percentage consists of 69% silver (Ag), 18% tin (Sn), 12% copper (Cu), and 1% zinc (Zn). The alloy powder has a density of 9.6 g/cm3. According to the manufacturer, when the mercury and alloy powder are combined, Dispersalloy® has a density of approximately 13.9 g/cm3. Since the gold dust particles appeared long and flake-like (Figure 3.6ii), a length measurement is provided (Table 3.3). As per the manufacturer (Protec Dental Laboratories Ltd., Vancouver, BC, Canada), the type of alloy used to fabricate the gold crown was a semi-precious (noble metal) silver and palladium-based, yellow, low gold alloy called Solarcast 20 (Noblecap® crown, ISO 13485 certified alloy). As per the Solarcast 20 Safety Data Sheet from 2018 (Ivoclar   41 Vivadent®, Mississauga, ON, Canada), it is composed of 40% silver (Ag), 15% palladium (Pd), 20% gold (Au), 10% copper (Cu), 11% indium (In), 2% tin (Sn), and 2% zinc (Zn). Its density at 20oC is 10.8 g/cm3.  The four microsphere beads and two manually ground dust particles were photographed on a Petri dish and on a spatula at various high magnifications using a camera (Sony Alpha a7R II Mirrorless Digital Camera, Bangkok, Thailand) attached to the stereo microscope (Figure 3.7). When not being used for experimental purposes, all six materials were stored in their own sealed clear plastic scintillation vials (Fisherbrand™ 20 mL Borosilicate Glass Scintillation Vials, White Polyethylene Caps, Fisher Scientific, Waltham, MA) at room temperature.   Microsphere bead Abbreviation Composition Density (g/cm3) Diameter (um) Blue polyethylene PE Polyethylene (>70%) Proprietary additive (<30%) 1.00 212-250 Soda lime glass G Glass oxide (>99.9%) ~2.5 190-197 Titanium metal T Titanium (>99.9%) 4.5 180-220 Stainless steel metal SS Stainless steel (>99.9%) 7.8 190-220  Table 3.2 Abbreviation, composition, density, and diameter for each microsphere bead used in the study (as per Cospheric Safety Data Sheet, 2019). Dentin density is approximately 1.79 to 2.21 g/cm3.         42 Restorative dust particle Abbreviation Composition Density (g/cm3) Diameter (um) Length (um) Amalgam filling ‘dust’ A Liquid (~50%): - Mercury (Hg) Alloy Powder (~50%): - Silver (Ag) - Tin (Sn) - Copper (Cu) - Zinc (Zn) ~13.9 2.5-25 Not applicable Gold alloy ‘dust’ Gold 40% Silver (Ag) 15% Palladium (Pd) 20% Gold (Au)  10% Copper (Cu) 11% Indium (In) 2% Tin (Sn) 2% Zinc (Zn) 10.8 Not applicable 5-25  Table 3.3 Abbreviation, composition, approximate density, and diameter or length for each restorative dust particle used in study. Pure gold density is 19.3 g/cm3.  i)    43 ii)  Figure 3.6 Fluorescent microscope image of i) amalgam ‘dust’ particles and ii) gold ‘dust’ particles.  Both taken at 40x magnification. Green lines represent scale bar and number represents corresponding size (diameter in (i) and length in (ii), respectively).  i)                 44 ii)             iii)            iv)            45 v)                       vi)                  Figure 3.7 Photographs of different materials on Petri dish and spatula i) blue polyethylene beads, ii) soda lime glass beads, iii) titanium metal beads, iv) stainless steel beads, v) gold alloy ‘dust’ particles, and vi) amalgam ‘dust’ particles on a Petri dish (left column) and on a spatula (right column) at various magnifications (2.8x to 10.4x).  3.5 Pre-treatment material application  All pre-treatment material application was performed under the stereo microscope at the magnifications mentioned above. Each of the six materials were placed into the apical aspect of the distal root canal by the same steps described below. The distal canals were confirmed dry by placing pre-measured fine paper points (Diadent Group International, Burnaby, BC, Canada) up to the apical foramen. The MB and ML canal orifices were blocked using coarse paper points to   46 not allow materials to fall into these canals. A size 25/.04 taper GP cone was dipped in alcohol (Isopropanol 70%) for approximately 1 second to allow the material to adhere to the GP surface. The GP cone with material attached was carefully placed over both distal canal orifices and shaken to allow the material to fall passively into the canal. If the material did not passively reach the apex, a dry size 15 to 25/.04 GP point was used to gently push the material more apically or the resin tooth was vibrated from its lateral surface using a dental lab vibrator (VWR Standard Mini Vortexer, Scientific Support, Hayward CA) at a medium speed of 5. The application steps were repeated until the apical 3 to 8 mm of the distal canal was filled with the respective material. The two paper points in the mesial canal orifices were removed. Photographs of mandibular molar L4 filled with each of the six materials is shown in Figure 3.8.  The tooth model and therefore canal was then placed horizontally (long axis of canal laying parallel to the benchtop) into a sample holder (Tornim - Torno Academico, Industria Brasileira, Brazil) as shown in Figure 3.9. Its placement allowed visualization of the material in the distal canal directly through the stereo microscope or on an external connected monitor (ASUS Designo MX239H LED IPS Monitor, Asustek Computer Inc, Taipei, Taiwan). Depending on the material, a white 2 x 2 inch gauze was sometimes placed under the sample to provide better contrast and visualization of the material (see Figure 3.8vi for example). The length of apical canal filled with material was measured visually using a periodontal probe (CP-12 SE QulixTM color-coded probe, Hu-Friedy, Chicago, IL) and recorded. This was defined as the vertical length of material visually measured from the apex of the distal canal extending to its furthest coronal level. A picture of the filled canal was taken with the camera attached to the stereo microscope.     47 i)    ii)           iii)   iv)    v)   vi)  Figure 3.8 Photographs of materials in apical root canal of tooth model Photographs of i) blue polyethylene beads, ii) soda lime glass beads, iii) titanium metal beads, iv) stainless steel beads, v) gold alloy ‘dust’ particles, and vi) amalgam ‘dust’ particles in the apical portion of the distal root canal of the mandibular molar (L4) tooth model. Note all apical foramina have been sealed with white glue. All photos taken at 10.4x magnification.      48 i)             ii)  Figure 3.9 Photograph of mandibular molar (L4) placed horizontally in sample holder i) top view (taken from above the sample), ii) side view. Left photo taken at 2.8x magnification and right photo taken at no magnification.   3.6 Experimental groups  Each dental and other material was removed using different irrigation methods (total of seven experimental irrigation groups). All experiments were performed with the canal in a horizontal and vertical orientation. Irrigation in all experimental groups was performed until manufacturer recommended maximum irrigation times (GW and PF US) or estimated cumulative maximum irrigation times (SNI). All irrigation was performed using only distilled water that was stored at room temperature.  Sample size calculation An online sample size calculator was used to determine the sample size for this study (https://www.stat.ubc.ca/~rollin/stats/ssize/n2.html accessed on October 07, 2020). Sample size was calculated based on means and standard deviations from a pilot study (mean 1= 1.0; mean   49 2= 0.8, common standard deviation= 0.15, a= 0.05; b= 0.80). This sample size calculation showed that a minimum of 9 irrigations (tests) per group was required. For each material, it was decided that 10 irrigations per group and 5 irrigations per canal orientation (horizontal or vertical) would be performed for a total of 420 irrigations (60 per each group).   Group 1: Multisonic GentleWave System (Sonendo Inc.) After sizing, a yellow nozzle (size 9) was chosen and placed into the GW handpiece hub as it allowed the tip to be approximately 1 mm from the pulp chamber floor (Figure 3.10). The nozzle and handpiece were used for four tests before being discarded and replaced with new ones. As per the manufacturer recommendations, the GW machine was primed at the start of each day. In addition, a 60 second leakage test was performed on a separate resin tooth prior to running each test on resin tooth L4. The GW handpiece was then placed into the access cavity and the irrigation protocol set for a vital tooth was then started. Maximum irrigation treatment time was 7 minutes based on manufacturer recommendations for a vital tooth (Sonendo Inc.). This was to simulate a 3% NaOCl cycle for 4 minutes and 30 seconds, followed by a distilled water flush for 30 seconds, an 8% EDTA cycle for 1 minute and 30 seconds, and lastly a distilled water flush for 30 seconds. The handpiece tip delivers the distilled water into the pulp chamber as a degassed fluid at a high flow rate (45 mL/min). Friction between the high flow rate liquid and the nozzle tip creates increases of temperature to 45oC. The handpiece also has built-in suction holes that allow simultaneous removal of excess irrigation fluid and any other material from the root canal system and pulp chamber. GW has been shown to create negative pressure at the apical foramen resulting in prevention of irrigant extrusion and improved safety (Haapasalo et al., 2014; Haapasalo et al., 2016; Charara et al., 2016).   50 Group 2: Syringe Needle Irrigation (SNI): open-ended, Flow rate 5 mL/min Conventional positive pressure irrigation was performed using a 25 mm length, 30 gauge blunt open-ended irrigation needle (NaviTip, Ultradent Products Inc., St. Louis, MO) (Figure 3.11). The needle was attached by a Luer-lock connection to clear laboratory tubing of 4.8 mm total outside diameter and 3.2 mm hollow inside diameter (TygonTM R-3603 Polyvinyl Chloride-based, Fisher Scientific, Waltham, MA). A digitally controlled peristaltic pump (Reglo Digital MS-2/8; Ismatec, Wertheim, Germany) was used to deliver the irrigant at a constant flow rate of 5 mL/min. This specific flow rate was chosen as it is clinically relevant (Boutsioukis et al., 2009). The pump was set to automatically stop delivery of the distilled water at a maximum irrigation time of two minutes. The peristaltic pump was calibrated at the beginning of each day it was being used. The flow rate was ensured correct by measuring the volume of the solution delivered through the pump in a specified period of time. The needle was only replaced if its shaft disconnected in any way from the hub. If the needle was replaced, calibration was performed again. Prior to the irrigation procedure, no intentional bend or angulation was created into the needle's shaft. At the start of the procedure, the needle was placed in either the DB or DL canal approximately 0.5 mm coronal to the material. Once the irrigation procedure began, the needle was extended apically up to a maximum of 24 mm (2 mm coronal to WL). This was indicated by a pre-measured rubber stopper placed on the needles shaft. During irrigation, the needle was withdrawn from the canal coronally up to its orifice while performing a 2 to 3 mm up-and-down motion. The needle was randomly inserted into both distal canals. Expelled distilled water and material was suctioned by placing a high-volume surgical suction tip of 1/16th inch diameter (Blue – Extra Small Size, Sky Dental Supply Inc., Vernon, CA) attached to a portable emergency suction pump (Vacu-Aide QSU Quiet Suction Unit, Devilbiss Healthcare,   51 Mannheim, Germany) at the height of the occlusal access, but not into the tooth model’s pulp chamber.   Group 3: Syringe Needle Irrigation (SNI): open-ended, Flow rate 15 mL/min Same as Group 2, except flow rate increased to 15 ml/min.  Group 4: Syringe Needle Irrigation (SNI): side-vented, Flow rate 5 mL/min Same as Group 2, except conventional positive pressure irrigation was performed using a 25 mm length, 30 gauge closed-ended side-vented irrigation needle (ProRinse, Dentsply Sirona Canada, Woodbridge, ON, Canada) (Figure 3.11).   Group 5: Syringe Needle Irrigation (SNI): side-vented, Flow rate 15 mL/min Same at Group 2, except used side-vented needle (same as Group 4) and flow rate increased to 15 ml/min (Figure 3.12).  Group 6: ProUltra PiezoFlow Ultrasonic Irrigation, Flow rate 0 mL/min [no flow] This irrigation system delivers continuous flow ultrasonic irrigation. The ProUltra PiezoFlow Ultrasonic Irrigation system (Dentsply Tulsa Dental Specialties, Johnson City, TN) uses a rigid, 24 mm length, 25 gauge open-ended needle (Figure 3.11). The needle is equipped with an attachment for an irrigation source and also attaches to an ultrasonic handpiece. The manufacturer recommends setting WL by trial fitting the needle in the canal to determine the depth at which it binds with the canal walls. From this binding point, it is advised to pull the needle back coronally 1 mm and set a stopper at this depth. In addition, the needle should not be   52 taken deeper than 75% of WL and never to the apex of the canal. With the needle inserted into the straighter DB canal, it bound with the canal walls at 16 mm (Figure 3.13i). The occlusal reference point used for this group was the bottom surface of the hollow space because it allowed more favorable straight-line access (Figure 3.13ii). Therefore, the irrigation system was used at 15 mm (1 mm coronal to binding) (Figure 3.13iii). The needle was attached to the handpiece accompanying the Piezo Pilot ultrasonic unit (Vista Dental Products, Racine, WI) and set to power level 5.  For this group, to simulate passive ultrasonic irrigation without flushing, the needle was not attached to a pump. Therefore, no continuous flow was provided. No intermittent flushing with SNI was performed.  The irrigation protocol for this group included: Filling both distal canals up to their orifices with distilled water irrigant using a syringe needle, placing the PiezoFlow needle only into the DB canal up to 15 mm depth, and continuously activating the ultrasonic for maximum 1 minute while moving the needle 3 to 5mm in an up-and-down motion. A stopwatch (Apple Inc.TM, Cupertino, CA) was used to time the 1 minute. If any distilled water and material was expelled, it was suctioned by placing a high-volume suction tip at the same level described above. The needle was brand new for the first test and the same one was used for all subsequent tests.  Group 7: ProUltra PiezoFlow Ultrasonic Irrigation, Flow rate 15 mL/min Continuous flow ultrasonic irrigation was performed. The needle was attached to both the ultrasonic mentioned above and the irrigation source. The ultrasonic unit was set at power level 5. The needle with its accompanying tubing was attached by a Luer-lock connection to the same   53 clear laboratory tubing mentioned above. A digitally controlled peristaltic pump was used to deliver the irrigant at a constant flow rate of 15 mL/min. This specific flow rate is recommended by the manufacturer (Dentsply Tulsa Dental Specialties). The pump was set to automatically stop delivery of the distilled water at a maximum irrigation time of one minute based on manufacturer recommendations (Dentsply Tulsa Dental Specialties). The peristaltic pump was calibrated at the beginning of each day it was being used the same way as mentioned above (Figure 3.14).  The irrigation protocol for this group included: Placing the needle only into the DB canal up to 15 mm with no irrigant previously in the canal, beginning suction at the access opening as above, starting irrigant delivery at 15 mL/min, and continuously activating the ultrasonic for 1 minute while moving the needle 3 to 5 mm in an up-and-down motion. The same one needle was used from Group 6.  3.7 Running the experiment and monitoring the removal of the materials  Each irrigation method was monitored under a stereo microscope at various magnifications and on an external monitor connected to the microscope video camera. The presence of a closed system was continuously monitored. At the conclusion of an irrigation method, the length of apical canal remaining with material was visually measured using the same periodontal probe as above and recorded. This was defined as the vertical length of material remaining from the apex of the distal canal extending to its furthest coronal level within the initial length of apical canal filled. If material was visualized coronal to this level, it was considered “risen” and recorded. A picture of the material remaining in the canal was taken with the camera attached to the stereo microscope.    54 A stopwatch (Apple Inc.TM, Cupertino, CA) was started at the beginning of every test group. If material was completely removed from the entirety of the tooth model (both distal canals and pulp chamber) prior to maximum procedure time, the procedure and stopwatch were stopped. Some material that had “risen” into the coronal aspect of the distal canals or into the pulp chamber was not included. The exact time for complete removal was recorded in seconds. After a test, if the material was completely removed, all canals were dried with high volume suction placed in the pulp chamber and fine to course paper points placed in all 4 canals to the apical foramen. Subsequently, a new round of material was applied as per the steps mentioned above. If all material was not completely removed, the remaining material was completely removed with a combination of using GW irrigation and loosening with sizes #6 to #20 stainless steel K-files. To ensure the length and apical diameter of the distal root canal had not changed over multiple tests, the above measurements were checked and confirmed to have not changed after every five irrigation tests. In addition, the resin tooth was examined visually under the stereo microscope after every irrigation test for any changes in transparency or defects to the root canal wall (ie. scratches, ledges, cracks). None was apparent. To ensure randomization over all 420 irrigation tests, a new irrigation group with a different material was tested every 2 to 3 tests. The entire experimental study above using the same irrigation groups and study materials was repeated with the tooth model and thus also the canals placed in a vertical orientation (long axis of canal laying perpendicular to the benchtop; apex downward and crown upward) within the sample holder as shown in Figure 3.15. All pre-treatment material application steps and measurement of length of apical canal filled was performed under the stereo microscope. The tooth was then placed vertically into the holding apparatus. During these vertically oriented   55 experiments, the stereo microscope could not be used due to its physical constraints in angulation. Therefore, while each irrigation group was being performed, it was viewed only on the external monitor which was connected to a SLR camera with macro lens (Nikon D500 camera, Nikon ED 200mm macro lens). Pre- and post-irrigation method photographs of material filled and remaining in the apical canal, respectively, were taken using the camera. Post-irrigation method measurement of length of apical canal remaining with material was performed under the stereo microscope at magnifications described above.    Figure 3.10 Photograph showing GentleWave handpiece with yellow nozzle (size 9) placed into access cavity and pulp chamber of tooth L4.  Note: tooth is placed in a horizontal orientation (long axis parallel to the benchtop) and the photo is taken from above the sample.     56    Figure 3.11 Irrigation needles used in the experimental groups (From top to bottom) NaviTip open-ended needle (30 gauge), ProRinse side-vented needle (30 gauge), ProUltra PiezoFlow needle (25 gauge).    57  Figure 3.12 Syringe needle irrigation setup Distilled water, digitally controlled peristaltic pump (Reglo Digital MS-2/8; Ismatec) set at flow rate of 15 mL/min, and syringe needle (25 mm length, 30 gauge, side-vented, closed-ended ProRinse irrigation needle shown here).  i)   ii)    iii)  Figure 3.13 ProUltra PiezoFlow ultrasonic needle setup i) needle binding at 16 mm, ii) red stopper showing occlusal reference point used was the bottom surface of the hollow space, iii) needle at 15 mm (1 mm coronal to binding).   58   Figure 3.14 ProUltra PiezoFlow Ultrasonic Irrigation overall setup Distilled water, digitally controlled peristaltic pump (Reglo Digital MS-2/8; Ismatec) set at maximum irrigation time of 60 seconds and flow rate of 15 mL/min (not shown), Piezo Pilot ultrasonic unit set at power level 5, and ProUltra PiezoFlow ultrasonic needle (25 gauge).    59  Figure 3.15 Photograph of mandibular molar (L4) placed vertically in sample holder (front view)  3.8 Visual evaluation and measurement of material removal  After the remaining material was visually measured under the stereomicroscope, the percentage of dental and other material removed could be determined at the maximum irrigation time for each respective irrigation group.    The following equation was used to calculate the percentage of material removed: Percentage of material removed = (length filled – length remaining)/length filled x 100%  The percentage of material removed represents a ratio scale numerical variable ranging from 0% to 100%. To analyze and compare the amount of material removed by different irrigation groups at maximum irrigation times, each test was categorically placed into one of three categories based on the percentage of material removed: all removed (100% removed), part removed (1-99% removed), not removed (0% removed). This provided the total number of tests   60 (n) with each material all removed, part removed, and not removed in different irrigation groups. Figure 3.16 illustrates several examples using visual evaluation. It shows different percentages of amalgam ‘dust’ particles removed after irrigation. The percentage removed can then be placed into an amount of material removed category.  To analyze and compare the amount of material removed in different irrigation groups at a standardized 1-minute irrigation time, each test was categorically placed into one of two categories based on the amount of material removed: complete removal, not complete removal. This provided the total number of tests (n) with complete removal and not complete removal of each material in different irrigation groups. Under visual evaluation, if a material was completely removed from the entirety of the tooth (both distal canals and pulp chamber) prior to maximum procedure time, the exact time for complete removal was recorded in seconds.  Using the data collected, the following could also be calculated: - Mean percentage (%) of material removed for each material in different irrigation groups  - Mean time required, in seconds, for complete removal of each material      61 ia)  ib)  iia)  iib)  iiia)  iiib)    62 iva)  ivb)  Figure 3.16 Examples of samples for visual evaluation following irrigation. Photos show amalgam ‘dust’ particles before (ia-iva) and remaining particles (ib-ivb) after irrigation. Subsequently, percentage of material removed could be determined and then placed into an amount of material removed category. All photos taken at 10.4x magnification. ia) Starting length (3 mm) of apical canal filled with material, ib) Remaining length (3 mm) (0% removed, therefore not removed) iia) Starting length (4 mm), iib) Remaining length (3 mm) (25% removed, therefore part removed) iiia) Starting length (4 mm), iiib) Remaining length (0.5 mm) (88% removed, therefore part removed) iva) Starting length (5 mm), ivb) Remaining length (0 mm) (100% removed, therefore all removed)  3.9 Statistical analyses  All statistical tests were performed using the SPSS software version 27 (IBM, Armonk, New York). Data was tested for normality using the Kolmogorov-Smirnov test. Levene’s test was used to assess the equality of variance assumption.  The amount of materials removed in each of the irrigation groups was compared versus the GW system at both maximum recommended irrigation times and at a 1-minute irrigation   63 time. This data was analyzed using the Fisher’s exact test because the conditions for the Chi-Square test were not fulfilled.  Percentage of material removed data was analyzed to determine mean differences among irrigation groups. If data demonstrated a normal distribution (Kolmogorov-Smirnov test p>0.050), one-way analysis of variance (ANOVA) with a Dunnett’s post-hoc test was used to compare each irrigation group to GW (as a comparative group). If data was not normally distributed (Kolmogorov-Smirnov test p<0.050), then the non-parametric equivalent Kruskal-Wallis test was used. Normality assessment showed the percentage of material removed data was approximating a normal distribution. Therefore, one-way analysis of variance (ANOVA) with a Dunnett’s post-hoc test was chosen. To compare if different canal orientations (horizontal vs. vertical) resulted in differences in the mean percentage of material removed for each irrigation group, independent sample t test (normal data) or Mann-Whitney test (non-normal data) was used. As the canal orientation variable was approximating a normal distribution, the comparison was performed using the independent sample t test. The independent sample t test (or non-parametric equivalent) was also used to compare the effect of flow rate on percentage of material removed. As the flow rate variable was approximating a normal distribution, the effect of two irrigation flow rates by the same irrigation method on percentage of material removed was compared using the independent sample t test. The level of statistical significance was set at p<0.050 for all sets of tests.     64  Results  4.1 The amount of different materials removed by each irrigation method  4.1.1 Removal of materials at recommended maximum irrigation times Table 4.1 shows the total number of tests (n) with each material all removed, part removed, and not removed in different irrigation groups at manufacturer recommended maximum irrigation times. Ten canals, five in vertical and five in horizontal position were included in the experiments. The results for horizontal and vertical canal orientation are shown in Tables 4.2 and 4.3, respectively.  When results from both canal orientations were combined (Table 4.1), GW removed all of each material on every irrigation test (10 out of 10), except gold alloy dust (7 out of 10). When comparing each irrigation group to GW, all other irrigation groups removed significantly less glass, titanium, stainless steel and amalgam dust than GW (p<0.050). All other irrigation groups, except for SNI OE (15 mL/min), removed significantly less blue PE microbeads than GW (p<0.050). All other irrigation groups, except for SNI OE (5 mL/min) and SNI OE (15 mL/min), removed significantly less gold alloy dust than GW (p<0.050).          65 MATERIAL REMOVED (combined orientations n = 10) Material Material Removal (at max irrigation times) GW              SNI OE SNI OE SNI SV SNI SV PF US PF US (5 mL/min) (15 mL/min) (5 mL/min) (15 mL/min) (0 mL/min) (15 mL/min)       Gr 1 Gr 2 Gr 3 Gr 4 Gr 5 Gr 6 Gr 7 1. Blue Polyethylene All removed                                                        Part removed                                                      Not removed                    10                         0               0 2                       8                        0 8                       2                       0 0                         10                         0 0                        10                        0 0                        0                       10 0                          7                         3   P value as compared to GW#  <0.001 0.237 <0.001 <0.001 <0.001 <0.001 2. Glass All removed                                                        Part removed                                                      Not removed                    10                         0               0 0                       10                         0 2                       8                      0 0                         10                         0 0                        10                        0 0                        0                       10 0                         9                         1   P value as compared to GW#  <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 3. Titanium Metal All removed                                                        Part removed                                                      Not removed                    10                         0               0 0                       10                         0 1                       9                      0 0                         10                         0 0                        10                        0 0                        0                       10 0                        10                         0   P value as compared to GW#  <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 4. Stainless Steel Metal All removed                                                        Part removed                                                      Not removed                    10                         0               0 1                       9                        0 4                       6                       0 0                         10                         0 0                        10                        0 0                        1                       9 0                         9                         1   P value as compared to GW#  <0.001 0.005 <0.001 <0.001 <0.001 <0.001 5. Gold Alloy ‘Dust’ All removed                                                        Part removed                                                      Not removed                    7                         3                0 5                         5                        0 6                       4                       0 0                         10                         0 0                         9                        1 0                       0                       10 0                         9                         1   P value as compared to GW#  0.650 1.000 0.003 0.003 <0.001 0.003 6. Amalgam Filling ‘Dust’ All removed                                                        Part removed                                                      Not removed                    10                         0               0 0                       10                         0 0                       10                       0 0                          9                          1 0                       10                        0 0                        0                       10 0                        10                         0   P value as compared to GW#  <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 #Fisher's exact test.     66 Table 4.1 Number of tests (n) with material all removed, part removed, and not removed in different irrigation groups (Gr) at maximum irrigation times. Combined horizontal and vertical canal orientations. Note 1: Maximum irrigation times for each irrigation group removal test: GW (7 minutes), SNI (2 minutes), PF US (1 minute). Note 2: all removed = 100%, part removed = 1-99%, not removed = 0%. Note 3: GW (GentleWave), SNI OE (Syringe Needle Irrigation open-ended), SNI SV (Syringe Needle Irrigation side-vented), PF US (PiezoFlow Ultrasonic). Note 4: Number in brackets indicates flow rate.  67 In horizontal canal orientation (Table 4.2), all other irrigation groups removed significantly less glass, titanium, and amalgam dust than GW (p<0.050). All other irrigation groups, except for SNI OE (15 mL/min), removed significantly less blue PE microbeads and stainless steel than GW (p<0.050). Only PF US (0 mL/min) removed significantly less gold alloy dust than GW (p<0.050). 68 MATERIAL REMOVED (horizontal orientation n = 5) Material Material Removal (at max irrigation times) GW              SNI OE SNI OE SNI SV SNI SV PF US PF US (5 mL/min) (15 mL/min) (5 mL/min) (15 mL/min) (0 mL/min) (15 mL/min)       Gr 1 Gr 2 Gr 3 Gr 4 Gr 5 Gr 6 Gr 7 1. Blue Polyethylene All removed                                                        Part removed                                                      Not removed                    5                     0                         0 0                         5                         0 4                         1                         0 0                        5                        0 0                         5                         0 0                         0                         5 0                          2                          3   P value as compared to GW#  0.008 1.000 0.008 0.008 0.008 0.008 2. Glass All removed                                                        Part removed                                                      Not removed                    5                     0                         0 0                         5                         0 0                         5                        0 0                        5                        0 0                         5                         0 0                         0                         5 0                           4                          1   P value as compared to GW#  0.008 0.008 0.008 0.008 0.008 0.008 3. Titanium Metal All removed                                                        Part removed                                                      Not removed                    5                     0                         0 0                         5                         0 1                         4                        0 0                        5                        0 0                         5                         0 0                         0                         5 0                          5                          0   P value as compared to GW#  0.008 0.048 0.008 0.008 0.008 0.008 4. Stainless Steel Metal All removed                                                        Part removed                                                      Not removed                    5                     0                         0 1                         4                         0 4                         1                        0 0                        5                        0 0                         5                         0 0                         1                         4 0                          5                          0   P value as compared to GW#  0.048 1.000 0.008 0.008 0.008 0.008 5. Gold Alloy ‘Dust’ All removed                                                        Part removed                                                      Not removed                    2                      3                          0 0                          5                          0 1                         4                       0 0                        5                        0 0                           4                          1 0                         0                         5 0                           4                          1   P value as compared to GW#  0.444 1.000 0.444 0.444 0.008 0.444 6. Amalgam Filling ‘Dust’ All removed                                                        Part removed                                                      Not removed                    5                     0                         0 0                          5                          0 0                          5                        0 0                         4                        1 0                         5                         0 0                         0                         5 0                          5                          0   P value as compared to GW#  0.008 0.008 0.008 0.008 0.008 0.008 #Fisher's exact test.    69 Table 4.2 Number of tests (n) with material all removed, part removed, and not removed in different irrigation groups (Gr) at maximum irrigation times. Canal oriented horizontally.    70 In vertical canal orientation (Table 4.3), all other irrigation groups removed significantly less titanium, stainless steel and amalgam dust than GW (p<0.050). All other irrigation groups, except for SNI OE (5 mL/min) and SNI OE (15 mL/min), removed significantly less blue PE microbeads and gold alloy dust than GW (p<0.050). All other irrigation groups, except for SNI OE (15 mL/min), removed significantly less glass than GW (p<0.050). 71 #Fisher's exact test. MATERIAL REMOVED (vertical orientation n = 5) Material Material Removal (at max irrigation times) GW              SNI OE SNI OE SNI SV SNI SV PF US PF US (5 mL/min) (15 mL/min) (5 mL/min) (15 mL/min) (0 mL/min) (15 mL/min)       Gr 1 Gr 2 Gr 3 Gr 4 Gr 5 Gr 6 Gr 7 1. Blue Polyethylene All removed                                                        Part removed                                                      Not removed                    5                         0                         0 2                         3                         0 4                       1                       0 0                         5                         0 0                        5                        0 0                        0                        5 0                          5                          0   P value as compared to GW#  0.167 1.000 0.008 0.008 0.008 0.008 2. Glass All removed                                                        Part removed                                                      Not removed                    5                         0                         0 0                          5                          0 2                       3                       0 0                         5                         0 0                        5                        0 0                        0                        5 0                          5                          0   P value as compared to GW#  0.008 0.167 0.008 0.008 0.008 0.008 3. Titanium  All removed                                                        Part removed                                                      Not removed                    5                         0                         0 0                          5                          0 0                       5                       0 0                          5                          0 0                        5                        0 0                       0                       5 0                          5                          0   P value as compared to GW#  0.008 0.008 0.008 0.008 0.008 0.008 4. Stainless Steel  All removed                                                        Part removed                                                      Not removed                    5                         0                         0 0                          5                          0 0                       5                       0 0                          5                          0 0                        5                        0 0                       0                       5 0                           4                          1   P value as compared to GW#  0.008 0.008 0.008 0.008 0.008 0.008 5. Gold Alloy Dust All removed                                                        Part removed                                                      Not removed                    5                         0                         0 5                         0                         0 5                       0                       0 0                          5                          0 0                        5                        0 0                       0                       5 0                          5                          0   P value as compared to GW#  1.000 1.000 0.008 0.008 0.008 0.008 6. Amalgam Filling Dust All removed                                                        Part removed                                                      Not removed                    5                         0                         0 0                          5                          0 0                       5                       0 0                          5                          0 0                        5                        0 0                       0                       5 0                          5                          0   P value as compared to GW#  0.008 0.008 0.008 0.008 0.008 0.008  72 Table 4.3 Number of tests (n) with material all removed, part removed, and not removed in different irrigation groups (Gr) at maximum irrigation times. Canal oriented vertically.           73 4.1.2 Removal of materials at 1-minute of irrigation The recommended irrigation times by the manufacturers for each equipment were different. Therefore, a standard time of 1-minute was also used to compare the effectiveness of the different irrigation methods. Table 4.4 shows the total number of tests with complete removal of each material in different irrigation groups at a standardized 1-minute irrigation time. Results from the two canal orientations are combined. After 1-minute, GW overall was still appearing to be the most effective. GW was able to remove all materials completely, except stainless steel metal and gold alloy dust, in less than 1 minute. In addition, there did not appear to be a gradual, continuous increase of material removed between 1 and 2 minutes of SNI.  All other irrigation groups removed significantly less glass, titanium, and amalgam dust than GW (p<0.050) (Table 4.4). All other irrigation groups, except for SNI OE (15 mL/min), removed significantly less blue PE microbeads and stainless steel than GW (p<0.050). All other irrigation groups, except for SNI OE (5 mL/min) and SNI OE (15 mL/min), removed significantly less gold alloy dust than GW (p<0.050). 74 Table 4.4 Number of tests (n) with complete material removal (Yes) and not complete material removal (No) at a standardized 1-minute irrigation time in different irrigation groups (Gr). Combined horizontal and vertical canal orientations.  Note 1: complete removal indicates all material removed (100% removed), not complete removal indicates not all material removed from canal and pulp chamber (1-99% removed). Note 2: GW (GentleWave), SNI OE (Syringe Needle Irrigation open-ended), SNI SV (Syringe Needle Irrigation side-vented), PF US (PiezoFlow Ultrasonic). Note 3: Number in brackets indicates flow rate.MATERIAL REMOVED (combined orientations n = 10) Material Complete Material Removal  (at 1-min) GW              SNI OE SNI OE SNI SV SNI SV PF US PF US (5 mL/min) (15 mL/min) (5 mL/min) (15 mL/min) (0 mL/min) (15 mL/min)       Gr 1 Gr 2 Gr 3 Gr 4 Gr 5 Gr 6 Gr 7 1. Blue Polyethylene Yes                                                                                                            No                    100 2 8 8 2 0 10 0 10 0 10 0 10   P value as compared to GW#  <0.001 0.474 <0.001 <0.001 <0.001 <0.001 2. Glass Yes                                                                                                            No                    100 0 10 2 8 0 10 0 10 0 10 0 10   P value as compared to GW#  <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 3. Titanium Metal Yes                                                                                                            No                    100 0 10 1 9 0 10 0 10 0 10 0 10   P value as compared to GW#  <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 4. Stainless Steel Metal Yes                                                                                                            No                    82 1 9 4 6 0 10 0 10 0 10 0 10   P value as compared to GW#  0.006 0.170 <0.001 <0.001 <0.001 <0.001 5. Gold Alloy ‘Dust’ Yes                                                                                                            No                    55 4 6 5 5 0 10 0 10 0 10 0 10   P value as compared to GW#  1.000 1.000 0.033 0.033 0.033 0.033 6. Amalgam Filling ‘Dust’ Yes                                                                                                            No                    100 0 10 0 10 0 10 0 10 0 10 0 10   P value as compared to GW#  <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 #Fisher's exact test.     75 4.2 The percentage of different materials removed by each irrigation method The mean percentages (%) of different materials removed in each irrigation group at its maximum irrigation time is shown as both canal orientations combined (Table 4.5), horizontal canal orientation (Table 4.6) and vertical canal orientation (Table 4.7). The overall mean percentage removed of all six materials combined in each irrigation group is shown in Table 4.5 (first row). The ability of GW to remove the six materials tested ranged from 67-100% with a mean (sd) removal (for all six materials combined) of 98.6% (6.3). Mean percentage removal for other irrigation groups ranged from 0% to 86% (Table 4.5 – first row).  When results from both canal orientations and all six materials were combined, one-way ANOVA showed significant results (p<0.001) (Table 4.5 – second row). There is a significant difference in the ability of different irrigation methods to remove dental and other materials from the apical part of a root canal. All other irrigation groups removed significantly less materials than GW (p<0.050) (Table 4.5 – second row). Figure 4.1 illustrates how to interpret a box and whisker plot. The inter-quartile range (IQR) represents a range whereby the middle half (50%) of the data set is included. The vertical length of the IQR can be used to indicate variation. Variation is a measure of how far the data is spread out. The median represents the middle of the dataset whereby 50% of the data is less than this value. The upper and lower ends of the whiskers represent maximum and minimum values, respectively, excluding outliers. An outlier is a data point located outside the whiskers. It is an observation that is numerically distant from the rest of the data. It can be indicated by a small circle “o” which represents a possible outlier. It can also be indicated by a “*” which represents a most probable outlier.  The percentage of material removed in each irrigation group is illustrated in Figure 4.2 using a box and whisker plot. It visualizes how GW removed almost all material completely,  76 whereas PF US (0 mL/min) removed almost no material. However, both GW and PF US (0 mL/min) did have some outliers. In addition, it shows SNI OE (5 mL/min) and SNI SV (15 mL/min) had the largest variance of values.  When results from both canal orientations were combined (Table 4.5), all other irrigation groups, except for SNI OE (5 mL/min) and SNI OE (15 mL/min), removed significantly less blue PE microbeads than GW (p<0.050). These results are illustrated in Figure 4.3. It also visualizes that GW removed all blue PE microbeads completely and SNI OE (15 mL/min) removed almost all blue PE completely except for a couple outliers, while PF US (0 mL/min) removed no blue PE microbeads.  In addition, the most variation was observed in the PF US (15 ml/min) group. All other irrigation groups, except for SNI OE (15 mL/min), removed significantly less glass than GW (p<0.050) as illustrated in Figure 4.4. It visualizes that GW removed all glass completely, while PF US (0 mL/min) removed no glass. In addition, it shows SNI OE (5 mL/min) had minimal to no variation in its values. All other irrigation groups removed significantly less titanium than GW (p<0.050) as illustrated in Figure 4.5. It visualizes that GW removed all titanium completely, while PF US (0 mL/min) removed no titanium. All other irrigation groups, except for SNI OE (15 mL/min), removed significantly less stainless steel than GW (p<0.050) as illustrated in Figure 4.6. It visualizes that GW removed all stainless steel completely, while PF US (0 mL/min) removed almost no stainless steel. In addition, the most variation was observed in the SNI OE (5 mL/min) group. PF US (15 mL/min) had no variation in its values and a few outliers. All other irrigation groups, except for SNI OE (5 mL/min) and SNI OE (15 mL/min), removed significantly less gold alloy dust than GW (p<0.050) as illustrated in Figure 4.7. It visualizes that PF US (0 mL/min) removed no gold alloy dust. The most variation was observed in the SNI OE (5 mL/min) group. All other irrigation  77 groups, except for SNI OE (15 mL/min), removed significantly less amalgam dust than GW (p<0.050) as illustrated in Figure 4.8. It visualizes that GW removed all amalgam dust completely, while PF US (0 mL/min) removed no amalgam dust. SNI OE (5 mL/min) had the most variation in its values.   78 #One-way analysis of variance (ANOVA) with a Dunnett's post-hoc test.   MATERIAL REMOVED (combined orientations) Material GW SNI OE SNI OE SNI SV SNI SV PF US PF US (5 mL/min) (15 mL/min) (5 mL/min) (15 mL/min) (0 mL/min) (15 mL/min) Gr 1 Gr 2 Gr 3 Gr 4 Gr 5 Gr 6 Gr 7  Mean + sd (%) Mean + sd (%) Mean + sd (%) Mean + sd (%) Mean + sd (%) Mean + sd (%) Mean + sd (%) All materials combined (n = 60) 98.6 + 6.3 74.6 + 20.9 86.0 + 16.7 42.0 + 17.1 46.4 + 16.5 0.2 + 1.4 45.5 + 18.7 P value# Gr 2 vs. GW (<0.001); Gr 3 vs. GW (<0.001); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) Blue Polyethylene (n = 10) 100.0 + 0.0 82.3 + 16.4 93.2 + 17.7 48.7 + 15.3 48.9 + 15.8 0.0 + 0.0 34.8 + 25.0 P value# Gr 2 vs. GW (0.062); Gr 3 vs. GW (0.833); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) Glass (n = 10) 100.0 + 0.0 77.4 + 21.8 83.3 + 14.7 50.7 + 16.1 56.5 + 14.6 0.0 + 0.0 45.4 + 20.6 P value# Gr 2 vs. GW (0.007); Gr 3 vs. GW (0.073); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) Titanium Metal (n = 10) 100.0 + 0.0 73.0 + 15.4 81.1 + 15.3 50.2 + 14.7 55.7 + 11.2 0.0 + 0.0 43.7 + 9.0 P value# Gr 2 vs. GW (<0.001); Gr 3 vs. GW (0.002); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) Stainless Steel Metal (n = 10) 100.0 + 0.0 65.5 + 21.7 84.1 + 19.5 36.3 + 14.5 37.0 + 15.2 1.1 + 3.5 44.7 + 18.6 P value# Gr 2 vs. GW (<0.001); Gr 3 vs. GW (0.106); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) Gold Alloy ‘Dust’ (n = 10) 91.7 + 14.1 76.5 + 28.5 86.3 + 24.0 32.8 + 15.7 42.3 + 18.8 0.0 + 0.0 47.5 + 17.4 P value# Gr 2 vs. GW (0.292); Gr 3 vs. GW (0.971); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) Amalgam Filling ‘Dust’ (n = 10) 100.0 + 0.0 73.1 + 20.9 88.1 + 3.7 33.3 + 18.3 37.8 + 13.8 0.0 + 0.0 57.1 + 15.2 P value# Gr 2 vs. GW (<0.001); Gr 3 vs. GW (0.193); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001)  79 Table 4.5 Mean percentage (%) of materials removed with standard deviations at maximum irrigation times in different irrigation groups (Gr). Both horizontal and vertical canal orientations are combined. Note 1: Maximum irrigation times for each irrigation group removal test: GW (7 minutes), SNI (2 minutes), PF US (1 minute). Note 2: GW (GentleWave), SNI OE (Syringe Needle Irrigation open-ended), SNI SV (Syringe Needle Irrigation side-vented), PF US (PiezoFlow Ultrasonic). Note 3: Number in brackets indicates flow rate.   80  Figure 4.1 Interpretation guide for box and whisker plot (courtesy of Dr Jolanta Aleksejuniene)   81 Figure 4.2 Percentage of material removed in irrigation groups (all materials and canal orientations combined; 60 tests per group) Figure 4.3 Percentage of blue PE removed in irrigation groups (combined canal orientations; 10 tests per group)  Figure 4.4 Percentage of glass removed in irrigation groups (combined canal orientations; 10 tests per group)   82  Figure 4.5 Percentage of titanium metal removed in irrigation groups (combined canal orientations; 10 tests per group)   Figure 4.6 Percentage of stainless steel metal removed in irrigation groups (combined canal orientations; 10 tests per group)  83  Figure 4.7 Percentage of gold alloy ‘dust’ removed in irrigation groups (combined canal orientations; 10 tests per group)   Figure 4.8 Percentage of amalgam filling ‘dust’ removed in irrigation groups (combined canal orientations; 10 tests per group)  84 In horizontal canal orientation (Table 4.6), all other irrigation groups, except for SNI OE (5 mL/min) and SNI OE (15 mL/min), removed significantly less blue PE microbeads, gold alloy dust, and amalgam dust than GW (p<0.050). All other irrigation groups, except for SNI OE (5 mL/min), removed significantly less glass than GW (p<0.050). All other irrigation groups, except for SNI OE (15 mL/min), removed significantly less titanium and stainless steel than GW (p<0.050).  In vertical canal orientation (Table 4.7), all other irrigation groups removed significantly less titanium than GW (p<0.050). All other irrigation groups, except for SNI OE (5 mL/min) and SNI OE (15 mL/min), removed significantly less blue PE microbeads, gold alloy dust, and amalgam dust than GW (p<0.050). All other irrigation groups, except for SNI OE (15 mL/min), removed significantly less glass and stainless steel than GW (p<0.050).  After performing an irrigation test in some of the irrigation groups, it was also occasionally observed that some of the material had “risen” from the apical root canal, but not completely removed from the entirety of the tooth model. Although no specific calculations were done on how often this was observed, it appeared that the “risen” material had relocated into the coronal aspect of the distal canals or into the pulp chamber.     85  Table 4.6 Mean percentage (%) of materials removed with standard deviations at maximum irrigation times in different irrigation groups (Gr). Canal oriented horizontally.  MATERIAL REMOVED (horizontal orientation n = 5) Material GW SNI OE SNI OE SNI SV SNI SV PF US PF US (5 mL/min) (15 mL/min) (5 mL/min) (15 mL/min) (0 mL/min) (15 mL/min) Gr 1 Gr 2 Gr 3 Gr 4 Gr 5 Gr 6 Gr 7  Mean + sd (%) Mean + sd (%) Mean + sd (%) Mean + sd (%) Mean + sd (%) Mean + sd (%) Mean + sd (%) Blue Polyethylene 100.0 + 0.0 86.4 + 4.7 97.6 + 5.4 40.2 + 13.6 39.8 + 16.2 0.0 + 0.0 16.4 + 22.5 P value# Gr 2 vs. GW (0.308); Gr 3 vs. GW (0.999); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) Glass 100.0 + 0.0 85.0 + 7.2 74.8 + 14.3 60.0 + 16.2 62.0 + 11.2 0.0 + 0.0 34.2 + 24.3 P value# Gr 2 vs. GW (0.314); Gr 3 vs. GW (0.027); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) Titanium Metal 100.0 + 0.0 70.0 + 19.5 81.6 + 19.0 57.4 + 17.4 54.6 + 15.6 0.0 + 0.0 40.8 + 2.3 P value# Gr 2 vs. GW (0.008); Gr 3 vs. GW (0.169); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) Stainless Steel Metal 100.0 + 0.0 65.8 + 26.8 90.0 + 22.4 37.8 + 18.8 38.6 + 15.8 2.2 + 4.9 49.4 + 14.9 P value# Gr 2 vs. GW (0.019); Gr 3 vs. GW (0.864); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) Gold Alloy ‘Dust’ 83.4 + 16.5 53.0 + 21.0 72.6 + 28.8 32.4 + 10.6 29.0 + 16.9 0.0 + 0.0 40.2 + 22.9 P value# Gr 2 vs. GW (0.073); Gr 3 vs. GW (0.870); Gr 4 vs. GW (0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (0.006) Amalgam Filling ‘Dust’ 100.0 + 0.0 70.4 + 26.6 85.8 + 3.8 29.4 + 25.5 31.2 + 15.7 0.0 + 0.0 59.4 + 22.1 P value# Gr 2 vs. GW (0.054); Gr 3 vs. GW (0.623); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (0.005) #One-way analysis of variance (ANOVA) with a Dunnett's post-hoc test.   86 MATERIAL REMOVED (vertical orientation n = 5) Material GW SNI OE SNI OE SNI SV SNI SV PF US PF US (5 mL/min) (15 mL/min) (5 mL/min) (15 mL/min) (0 mL/min) (15 mL/min) Gr 1 Gr 2 Gr 3 Gr 4 Gr 5 Gr 6 Gr 7  Mean + sd (%) Mean + sd (%) Mean + sd (%) Mean + sd (%) Mean + sd (%) Mean + sd (%) Mean + sd (%) Blue Polyethylene 100.0 + 0.0 78.2 + 23.3 88.8 + 25.0 57.2 + 12.6 58.0 + 9.7 0.0 + 0.0 53.2 + 7.2 P value# Gr 2 vs. GW (0.107); Gr 3 vs. GW (0.674); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) Glass 100.0 + 0.0 69.8 + 29.5 91.8 + 10.2 41.4 + 10.2 51.0 + 16.6 0.0 + 0.0 56.6 + 7.1 P value# Gr 2 vs. GW (0.011); Gr 3 vs. GW (0.867); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) Titanium Metal 100.0 + 0.0 76.0 + 11.4 80.6 + 12.8 43.0 + 7.2 56.8 + 5.9 0.0 + 0.0 46.6 + 12.6 P value# Gr 2 vs. GW (0.001); Gr 3 vs. GW (0.008); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) Stainless Steel Metal 100.0 + 0.0 65.2 + 18.4 78.2 + 16.4 34.8 + 10.6 35.4 + 16.3 0.0 + 0.0 40.0 + 22.4 P value# Gr 2 vs. GW (0.004); Gr 3 vs. GW (0.109); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) Gold Alloy ‘Dust’ 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 33.2 + 21.0 55.6 + 8.3 0.0 + 0.0 54.8 + 4.4 P value# Gr 2 vs. GW (1.000); Gr 3 vs. GW (1.000); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) Amalgam Filling ‘Dust’ 100.0 + 0.0 75.8 + 16.0 90.4 + 1.5 37.2 + 8.0 44.4 + 8.6 0.0 + 0.0 54.8 + 4.4 P value# Gr 2 vs. GW (<0.001); Gr 3 vs. GW (0.231); Gr 4 vs. GW (<0.001);  Gr 5 vs. GW (<0.001); Gr 6 vs. GW (<0.001); Gr 7 vs. GW (<0.001) #One-way analysis of variance (ANOVA) with a Dunnett's post-hoc test.   Table 4.7 Mean percentage (%) of materials removed with standard deviations at maximum irrigation times in different irrigation groups (Gr). Canal oriented vertically. 87 4.3  The percentage of materials removed based on canal orientation  When the mean percentage removed in all irrigation groups and for all materials were combined, the mean percentage (sd) of material removed at maximum irrigation times from the apical root canal in the horizontal orientation was 54.1% (34.8) and in the vertical orientation was 58.3% (33.7) (Table 4.8). The mean percentage of material removed from canals oriented horizontally was not significantly different than from the canals oriented vertically (p=0.208) (Table 4.8). This is illustrated in Figure 4.9. In addition, both canal orientations had similar minimum and maximum values for percentage of material removed with the lowest being no material removed (0%) and the highest being all material removed (100%), respectively.  Canal Orientation Mean % of material removed P value#      Horizontal  54.1 + 34.8 0.208      Vertical  58.3 + 33.7      #Independent sample t test.   Table 4.8 Mean percentage (%) of materials removed compared between horizontal versus vertical canal orientation (all irrigation groups and materials combined; 210 tests per canal orientation). Note: All irrigation groups performed with a respective maximum irrigation time: GW (7 minutes), SNI (2 minutes), PF US (1 minute).   88  Figure 4.9 Percentage of material removed by canal orientation (all irrigation groups and materials combined; 210 tests per canal orientation)  When the results from all materials were combined, for each irrigation group the mean percentage of material removed from the apical root canal in the horizontal orientation and in the vertical orientation is shown in Table 4.9. This is illustrated in Figure 4.10. All irrigation groups, except for PF US (15 mL/min), showed no significant difference in the percentage of material removed from the canal oriented horizontally versus the canal oriented vertically (p>0.050) (Table 4.9). PF US (15 mL/min) removed in average by 10.9% significantly more of the material from vertically oriented canals than from horizontally oriented canals (p<0.050). Figure 4.10 shows PF US (15 mL/min) had much more variation in values in a horizontal rather than vertical canal orientation. In addition, Figure 4.10 visualizes how GW removed almost all material in both orientations, while PF US (0 mL/min) removed essentially no material in both orientations.    89 Irrigation Group Canal Orientation  % of material removed (mean + sd) P values#     GW Horizontal (n = 30) 97.2 + 8.8 0.095     Vertical (n = 30) 100.0 + 0.0     SNI OE (5 mL/min) Horizontal (n = 30) 71.8 + 21.3 0.293     Vertical (n = 30) 77.5 + 20.5     SNI OE (15 mL/min) Horizontal (n = 30) 83.7 + 18.5 0.295     Vertical (n = 30) 88.3 + 14.7     SNI SV (5 mL/min) Horizontal (n = 30) 42.9 + 20.0 0.699     Vertical (n = 30) 41.1 + 13.9     SNI SV (15 mL/min) Horizontal (n = 30) 42.5 + 18.5 0.071     Vertical (n = 30) 50.2 + 13.4     PF US (0 mL/min) Horizontal (n = 30) 0.4 + 2.0 0.326     Vertical (n = 30) 0.0 + 0.0     PF US (15 mL/min) Horizontal (n = 30) 40.1 + 22.4 0.023     Vertical (n = 30) 51.0 + 12.1     #Independent sample t test.   Table 4.9 Mean percentage (%) of materials removed compared between horizontal versus vertical canal orientation. For each irrigation group while combining all materials (n = 30).  Note: All irrigation groups performed with a respective maximum irrigation time: GW (7 minutes), SNI (2 minutes), PF US (1 minute).   90  Figure 4.10 Percentage of material removed by canal orientation (horizontal or vertical) for each irrigation group at a respective maximum irrigation time (all materials combined; 30 tests per group)  4.4 The percentage of materials removed based on flow rate Tables 4.10, 4.11, and 4.12 compare the mean percentage of materials removed by different irrigation methods at maximum irrigation times based on differences in flow rate.  When the results for each material were combined, the mean percentage (sd) of material removed from the apical root canal by SNI OE at a flow rate of 5 mL/min was 74.6% (20.9) and at a flow rate of 15 mL/min was 86.0% (16.7) (Table 4.10). These percentages of material removed are illustrated in Figure 4.11. It also visualizes that SNI OE (5 mL/min) had much more variation in values than SNI OE (15 mL/min). However, although SNI OE (15 mL/min) had much less variation, it did have a few outliers. Table 4.10 shows SNI OE (15 mL/min) removed in average by 11.4% a significantly greater mean percentage of all materials combined than SNI OE (5 mL/min) (p=0.001). Looking at each material individually, no significant differences were  91 found. SNI OE (15 mL/min) did not remove a significantly greater mean percentage of any material individually than SNI OE (5 mL/min) (p>0.050) (Table 4.10). These results are illustrated in Figure 4.12. In addition, Figure 4.12 visualizes that the most variation was observed for gold alloy dust and stainless steel metal removal both using the SNI OE (5 mL/min) group.   Materials Irrigation Method  (flow rate) % of material removed (mean + sd) P values#     All materials combined SNI OE (5 mL/min)  (n = 60) 74.6 + 20.9 0.001     SNI OE (15 mL/min) (n = 60) 86.0 + 16.7     Blue Polyethylene SNI OE (5 mL/min)  (n = 10) 82.3 + 16.4 0.171     SNI OE (15 mL/min)  (n = 10) 93.2 + 17.7     Glass SNI OE (5 mL/min)  (n = 10) 77.4 + 21.8 0.487     SNI OE (15 mL/min)  (n = 10) 83.3 + 14.7     Titanium Metal SNI OE (5 mL/min)  (n = 10) 73.0 + 15.4 0.253     SNI OE (15 mL/min)  (n = 10) 81.1 + 15.3     Stainless Steel Metal SNI OE (5 mL/min)  (n = 10) 65.5 + 21.7 0.059     SNI OE (15 mL/min)  (n = 10) 84.1 + 19.5     Gold Alloy ‘Dust’ SNI OE (5 mL/min)  (n = 10) 76.5 + 28.5 0.416     SNI OE (15 mL/min)  (n = 10) 86.3 + 24.0     Amalgam Filling ‘Dust’ SNI OE (5 mL/min)  (n = 10) 73.1 + 20.9 0.050     SNI OE (15 mL/min) (n = 10) 88.1 + 3.7     #Independent sample t test.   Table 4.10 Mean percentage (%) of materials removed by syringe needle irrigation, open-ended (SNI OE) based on differences in flow rate (5 mL/min versus 15 mL/min). Maximum irrigation time of 2 minutes.    92  Figure 4.11 Percentage of material removed by syringe needle irrigation, open-ended (SNI OE) at flow rates of 5 mL/min and 15 mL/min (all materials combined; 60 tests per group)    93 Figure 4.12 Percentage of material removed by syringe needle irrigation, open-ended (SNI OE) at flow rates of 5 mL/min and 15 mL/min for each material.  When the results for each material were combined, the mean percentage (sd) of material removed from the apical root canal using SNI SV at a flow rate of 5 mL/min was 42.0% (17.1) and using SNI SV at a flow rate of 15 mL/min was 46.4% (16.5) (Table 4.11). These percentages of material removed are illustrated in Figure 4.13. Both flow rates had similar variation in values (Figure 4.13). In addition, Table 4.11 shows SNI SV (15 mL/min) did not remove a significantly greater mean percentage of all materials combined than SNI SV (5 mL/min) (p=0.157). Looking at each material individually, SNI SV (15 mL/min) did not remove a significantly greater mean percentage of any material individually than SNI SV (5 mL/min) (p>0.050) (Table 4.11). These results are illustrated in Figure 4.14. The only material that appeared to have any outliers at both flow rates was blue PE.             94 Materials Irrigation Method  (flow rate) % of material removed (mean + sd) P values#     All materials combined SNI SV (5 mL/min) (n = 60) 42.0 + 17.1 0.157     SNI SV (15 mL/min) (n = 60) 46.4 + 16.5      Blue Polyethylene SNI SV (5 mL/min) (n = 10) 48.7 + 15.3 0.977     SNI SV (15 mL/min) (n = 10) 48.9 + 15.8     Glass SNI SV (5 mL/min) (n = 10) 50.7 + 16.1 0.410     SNI SV (15 mL/min) (n = 10) 56.5 + 14.6     Titanium Metal SNI SV (5 mL/min) (n = 10) 50.2 + 14.7 0.358     SNI SV (15 mL/min) (n = 10) 55.7 + 11.2     Stainless Steel Metal SNI SV (5 mL/min) (n = 10) 36.3 + 14.5 0.917     SNI SV (15 mL/min) (n = 10) 37.0 + 15.2     Gold Alloy ‘Dust’ SNI SV (5 mL/min) (n = 10) 32.8 + 15.7 0.236     SNI SV (15 mL/min) (n = 10) 42.3 + 18.8     Amalgam Filling ‘Dust’ SNI SV (5 mL/min) (n = 10) 33.3 + 18.3 0.543     SNI SV (15 mL/min) (n = 10) 37.8 + 13.8     #Independent sample t test.    Table 4.11 Mean percentage (%) of materials removed by syringe needle irrigation, side-vented (SNI SV) based on differences in flow rate (5 mL/min versus 15 mL/min). Maximum irrigation time of 2 minutes.   95  Figure 4.13 Percentage of material removed by syringe needle irrigation, side-vented (SNI SV) at flow rates of 5 mL/min and 15 mL/min (all materials combined; 60 tests per group)  Figure 4.14 Percentage of material removed by syringe needle irrigation, side-vented (SNI SV) at flow rates of 5 mL/min and 15 mL/min for each material.  96 When the results for each material were combined, the mean percentage (sd) of material removed from the apical root canal using PF US at a flow rate of 0 mL/min was 0.2% (1.4) and using PF US at a flow rate of 15 mL/min was 45.5% (18.7) (Table 4.12). These percentages of material removed are illustrated in Figure 4.15. PF US (0 mL/min) appears to have almost no variation in its values whereas PF US (15 mL/min) appears to have variation and a few outliers. In addition, Table 4.12 shows PF US (15 mL/min) removed in average by 45.4% a significantly greater mean percentage of all materials combined than PF US (0 mL/min) (p<0.001). Looking at each material individually, PF US (15 mL/min) removed a significantly greater mean percentage of all materials individually than PF US (0 mL/min) (p<0.050) (Table 4.12). These results are illustrated in Figure 4.16. In addition, Figure 4.16 visualizes that the most variation was observed for removal of blue PE microbeads using the PF US (15 mL/min) group.   Materials Irrigation Method  (flow rate) % of material removed (mean + sd) P values#     All materials combined PF US (0 mL/min)  (n = 60) 0.2 + 1.4 <0.001     PF US (15 mL/min) (n = 60) 45.5 + 18.7     Blue Polyethylene PF US (0 mL/min) 0.0 + 0.0 0.002     PF US (15 mL/min) 34.8 + 25.0     Glass PF US (0 mL/min) 0.0 + 0.0 <0.001     PF US (15 mL/min) 45.4 + 20.6     Titanium Metal PF US (0 mL/min) 0.0 + 0.0 <0.001     PF US (15 mL/min) 43.7 + 9.0     Stainless Steel Metal PF US (0 mL/min) 1.1 + 3.5 <0.001     PF US (15 mL/min) 44.7 + 18.6     Gold Alloy ‘Dust’ PF US (0 mL/min) 0.0 + 0.0 <0.001     PF US (15 mL/min) 47.5 + 17.4     Amalgam Filling ‘Dust’ PF US (0 mL/min) 0.0 + 0.0 <0.001     PF US (15 mL/min) 57.1 + 15.2     #Independent sample t test.    97 Table 4.12 Mean percentage (%) of materials removed by PiezoFlow Ultrasonic (PF US) based on differences in flow rate (0 mL/min versus 15 mL/min). Maximum irrigation time of 1 minute.   Figure 4.15 Percentage of material removed by PiezoFlow Ultrasonic (PF US) at flow rates of 0 mL/min and 15 mL/min (all materials combined; 60 tests per group)   98  Figure 4.16 Percentage of material removed by PiezoFlow Ultrasonic (PF US) at flow rates of 0 mL/min and 15 mL/min for each material. 99 4.5 The time required for complete removal of materials  Table 4.13 shows the mean times required in an irrigation group to completely remove each material when results from both canal orientations were combined. The results for horizontal and vertical canal orientation are shown in Tables 4.14 and 4.15, respectively. The mean time for complete removal was calculated by averaging the times from the tests with complete removal. Not complete removal indicates material remaining within the tooth model at the maximum allotted time for each irrigation group removal test. If material was not completely removed, no mean time for complete removal could be calculated. As shown earlier in Table 4.1, GW completely removed all materials on every irrigation test (ten out of ten tests), except gold alloy dust (seven out of ten tests). Table 4.13 (second column from the left) shows the mean time required for complete removal of each material by GW. The mean times ranged from 4 seconds to 66 seconds. These results for the time GW required for complete removal of each material is illustrated in Figure 4.17. It visualizes that there was a large variance of time observed for complete removal of gold alloy dust by GW. However, within each of the other five materials, the time required for complete removal showed only little variation. In addition, Figure 4.17 shows all materials, except gold alloy dust, had similar times compared to one another required for complete removal by GW. All materials, except gold alloy dust, were removed equally fast using GW.  All other irrigation groups either had a combination of complete and not complete removal of materials, or only not complete removal of materials at their respective maximum irrigation times. Tables 4.13, 4.14, and 4.15 shows the results for combined orientations, horizontal orientation, and vertical orientation, respectively.    100 MATERIAL REMOVED (combined orientations n = 10) Material GW SNI OE SNI OE SNI SV SNI SV PF US PF US (5 mL/min) (15 mL/min) (5 mL/min) (15 mL/min) (0 mL/min) (15 mL/min) Gr 1 Gr 2 Gr 3 Gr 4 Gr 5 Gr 6 Gr 7  Mean + sd (s) Mean + sd (s) Mean + sd (s) Mean + sd (s) Mean + sd (s) Mean + sd (s) Mean + sd (s) Blue Polyethylene 4.0 + 3.3                                    Completely removed (n = 10) 46.5 + 16.3                         Completelyremoved (n = 2)           Not completely removed (n = 8) 28.5 + 4.3                      Completely removed (n = 8)       Not completely removed (n = 2) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Glass 4.0 + 1.6                            Completely removed (n = 10) Not completely removed (n = 10) 14.0 + 12.7                   Completely removed (n = 2)           Not completely removed (n = 8) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Titanium Metal 4.9 + 4.3                            Completely removed (n = 10) Not completely removed (n = 10) 29.0 + 0.0                                Completely removed (n = 1)        Not completely removed (n = 9) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Stainless Steel Metal 33.9 + 65.8                        Completely removed (n = 10) 26.0 + 0.0                        Completely removed (n = 1)        Not completely removed (n = 9) 23.0 + 1.8                      Completely removed (n = 4)        Not completely removed (n = 6) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Gold Alloy ‘Dust’ 66.4 + 127.9                     Completely removed (n = 7)        Not completely removed (n = 3) 44.4 + 13.5                   Completely removed (n = 5)        Not completely removed (n = 5) 40.3 + 44.8                    Completely removed (n = 6)        Not completely removed (n = 4) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Amalgam Filling ‘Dust’ 7.5 + 11.1                          Completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10)    101 Table 4.13 Mean time required, in seconds (s), for complete removal of materials with standard deviations. Combined horizontal and vertical canal orientations. Note 1: Maximum time for each irrigation group removal test: GW (7 minutes), SNI (2 minutes), PF US (1 minute). Note 2: completely removed = 100%, not completely removed = 1-99%. Note 3: 'n' indicates number of tests performed. Note 4: GW (GentleWave), SNI OE (Syringe Needle Irrigation open-ended), SNI SV (Syringe Needle Irrigation side-vented), PF US (PiezoFlow Ultrasonic).    102  Table 4.14 Mean time required, in seconds (s), for complete removal of materials with standard deviations. Canal oriented horizontally.  MATERIAL REMOVED (horizontal orientation n = 5) Material GW SNI OE SNI OE SNI SV SNI SV PF US PF US (5 mL/min) (15 mL/min) (5 mL/min) (15 mL/min) (0 mL/min) (15 mL/min) Gr 1 Gr 2 Gr 3 Gr 4 Gr 5 Gr 6 Gr 7  Mean + sd (s) Mean + sd (s) Mean + sd (s) Mean + sd (s) Mean + sd (s) Mean + sd (s) Mean + sd (s) Blue Polyethylene 2.8 + 1.3                        Completely removed (n = 5) Not completely removed (n = 5) 31.8 + 3.3                      Completely removed (n = 4)        Not completely removed (n = 1) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Glass 3.4 + 1.7                       Completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Titanium Metal 5.2 + 5.5                       Completely removed (n = 5) Not completely removed (n = 5) 29.0 + 0.0                            Completely removed (n = 1)        Not completely removed (n = 4) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Stainless Steel Metal 5.0 + 5.0                       Completely removed (n = 5) 26.0 + 0.0                             Completely removed (n = 1)        Not completely removed (n = 4) 23.0 + 1.8                     Completely removed (n = 4)        Not completely removed (n = 1) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Gold Alloy ‘Dust’ 4.0 + 1.4                       Completely removed (n = 2)        Not completely removed (n = 3) Not completely removed (n = 5) 127.0 + 0.0                   Completely removed (n = 1)        Not completely removed (n = 4) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Amalgam Filling ‘Dust’ 10.6 + 15.9                   Completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10) Not completely removed (n = 10)  103 MATERIAL REMOVED (vertical orientation n = 5) Material GW SNI OE SNI OE SNI SV SNI SV PF US PF US (5 mL/min) (15 mL/min) (5 mL/min) (15 mL/min) (0 mL/min) (15 mL/min) Gr 1 Gr 2 Gr 3 Gr 4 Gr 5 Gr 6 Gr 7  Mean + sd (s) Mean + sd (s) Mean + sd (s) Mean + sd (s) Mean + sd (s) Mean + sd (s) Mean + sd (s) Blue Polyethylene 5.2 + 4.3                       Completely removed (n = 5) 46.5 + 16.3                   Completely removed (n = 2)        Not completely removed (n = 3) 25.3 + 2.1                     Completely removed (n = 4)        Not completely removed (n = 1) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Glass 4.6 + 1.5                       Completely removed (n = 5) Not completely removed (n = 5) 14.0 + 12.7                   Completely removed (n = 2)        Not completely removed (n = 3) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Titanium Metal 4.6 + 3.1                       Completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Stainless Steel Metal 62.8 + 87.4                   Completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Gold Alloy ‘Dust’ 91.4 + 147.7                 Completely removed (n = 5) 44.4 + 13.5                   Completely removed (n = 5) 23.0 + 16.0                   Completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Amalgam Filling ‘Dust’ 4.4 + 1.7                       Completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5) Not completely removed (n = 5)  Table 4.15 Mean time required, in seconds (s), for complete removal of materials with standard deviations. Canal oriented vertically.  104  Figure 4.17 Time required, in seconds (s), for complete removal of each material using GentleWave. Both canal orientations combined.   105  Discussion  5.1 Study importance To the best of our knowledge, the present study is the first to evaluate and compare the ability of different irrigation methods to remove pieces of dental and other materials from the apical aspect of a root canal. The different irrigation methods tested in this study included syringe needle irrigation, ProUltra PiezoFlow ultrasonic irrigation, and the multisonic GW system. These were chosen to compare traditional irrigation approaches (SNI) with different designs and flow parameters to newer irrigation methods (ultrasonics and GW). Previous studies with some of these types of irrigation systems have evaluated their ability to remove pulpal calcifications (Chen et al., 2020), separated instruments (Wohlgemuth et al,, 2015), and calcium hydroxide paste (Ma et al., 2015). This study was designed to simulate actual clinical conditions whereby materials, typically from coronal restorations, can passively fall into the apical root canal during access cavity preparation. Finding a novel method for removal has significance as these materials can compromise the outcome of endodontic treatment if not removed. Furthermore, traditional methods have shown to be time-consuming and result in complications such as excess removal of root dentin and potential material extrusion (Friedman et al., 1990; Hülsmann, 1993; Pai et al., 2013; Al Ghamdi, 2017).  This in vitro study used a transparent, 3D printed, micro-CT scan-based resin mandibular molar tooth model to assess material removal from the apical root canal. This model was chosen for multiple reasons: i) to allow direct visualization of irrigant flow dynamics, ii) to visually measure the amount of material removed, and iii) to document the exact time required for complete removal of a material. In addition, unlike using extracted teeth, root canal morphology  106 between all groups could be standardized (Reymus et al., 2019). However, it should be noted that a 3D printed tooth model does present some limitations. Of primary importance is that this tooth model less closely approximated the in vivo clinical situation than using extracted teeth. In artificial, manufactured tooth models, the physical and chemical characteristics of the synthetic root canal wall do not resemble those of extracted teeth (Reymus et al., 2019). Furthermore, all experiments were performed on the same canal of only one mandibular tooth model. Firstly, the canals length and overall tooth anatomy is not representative of all teeth. Secondly, although only distilled water was being used for all irrigation tests, it is possible that the physical characteristics of the tooth model could have changed over all the tests performed. It is uncertain how much of an effect these differences in characteristics would have had on the results, but it must be considered when evaluating the data. Considering that the purpose of this study was to primarily assess the effect of flow dynamics on material removal and not surface chemistry, these limitations do not compromise the experimental model and investigated aims of the study.  The apices of both roots of the tooth model were sealed with glue to create a closed environment that would be representative of the clinical setting. This has importance as Tay et al. (2010) showed that there was a significant difference in the apical irrigation efficacy of open and closed systems using SNI SV. In the tooth model chosen, the apical diameter of the distal canal was 0.25 mm. In an ex vivo study by Bronnec et al. (2010) who evaluated irrigant penetration, it was found irrigating solution flows more apically with increased taper of the apical root canal. This is due to there being more space for deeper needle penetration and backflow of irrigating solution in a coronal direction. Future studies with smaller or larger canal tapers may result in different results for removal of materials using the irrigation systems above.   107 Six materials were passively placed into the apical part of the distal root canal. Microsphere beads were chosen as they could have a standardized shape and size, as well as beads of different density comparable to dentin could be chosen. Beads of different density have clinical significance. Originally, before endodontic treatment starts, the only "particles" that often are in the canal space are pulp stones, some loose, some attached (Johnson & Bevelander, 1956). Chen et al. (2020) recently found the GentleWave system has the ability to remove pulpal calcifications, which have the same density as dentin, from uninstrumented root canals. Therefore, it was important to test heavier and more dense materials such as titanium and stainless steel. Dental materials were chosen to provide more realism to the study as these materials could fall into the root canal during access cavity preparation. In addition, in an in vivo situation, the materials would be of different sizes and shapes. Thus, the benefit of an in vitro study and use of a standardized model is that such confounding factors can be controlled.     A visual measurement of the apical length of each material before and after different irrigation methods was used to determine the amount and percentage of material removed and the time for complete removal. As this was performed by only one primary examiner (the main author A.K.), there is the possibility of subjectivity, operator fatigue and bias. To prevent this, a photo was taken after placement of each material and again after each irrigation method. Videos of randomly chosen irrigation tests were also taken. These photos and videos were used at a later date by the primary examiner as reference to confirm all previous measurements. In addition, a second examiner not associated with the study (A.C.), was trained with measuring the parameters above. The second examiner randomly selected photos and videos to confirm the values obtained by the primary examiner and no differences were noted.   108 5.2 Discussion of major findings  First hypothesis  The first hypothesis was that the different irrigation methods compared to the GW system do not differ in their ability to remove materials from the apical part of a root canal. This hypothesis has not been examined in previous studies. The effectiveness of the different irrigation methods at manufacturer recommended maximum irrigation times and at a standardized one minute irrigation time was compared to the GW system. Based on the results of the study, the null hypothesis could be rejected because GW was the most effective at maximum and one minute irrigation times. Combining all materials, all other irrigation methods removed a significantly less mean percentage than GentleWave. Surprisingly, looking at each material individually, SNI OE (5 mL/min) and/or SNI OE (15 mL/min) occasionally did not remove a significantly different mean percentage than GW. If the sample size had been larger, one can speculate that significant differences may have been observed.   It was not surprising to see the SNI OE needle to be more effective at removing materials than the SNI SV needle. Park et al. (2013) showed that the flow through 30 gauge OE needles induces significantly more apical pressure and more apical clearance of dye than 30 gauge SV needles.   In this study, the PF US (15 mL/min) was less effective at removing all materials than SNI OE (15 mL/min). The PiezoFlow system provides ultrasonic energy and subsequently results in acoustic microstreaming of the irrigation fluid (Ahmad et al., 1987a). It uses a 25 gauge open-ended needle and the manufacturer recommends it should not be taken deeper than 75% of WL or placed 1mm coronal to binding. Therefore, in the distal canal of this tooth model, it could only be placed at 15 mm (1 mm coronal to binding). The OE needle was inserted to a  109 maximum depth of 24 mm. Therefore, it appears that when using the same needle design with the same irrigant flow rate, the removal of material is possibly more dependent upon needle insertion depth rather than ultrasonic activation. That being said, OE needles and CFUI should be used with caution as studies have shown both can lead to complications during treatment such as apical extrusion of irrigant (Mitchell & Baumgartner, 2011). It is also important to consider that ultrasonically activated tips have the potential to cause iatrogenic damage, such as ledges, transportations and perforations, to the root canal wall (Caron et al., 2010).  All other irrigation groups were less effective in completely removing each material, except gold alloy dust, than GW. If GW was able to completely remove a material, this usually occurred in less than one minute. Therefore, between each irrigation test with any irrigation method, GW was used in this study to remove any remaining material within the apical canal. GW was not able to completely remove gold alloy dust from the tooth model in three tests at maximum irrigation time. Interestingly, the gold alloy dust particles moved in a rapid and random fashion within the energized irrigant flow created by GW. It would be of interest to know if this rapid movement would help scape off biofilms and tissue remnants from the apical canal walls. In some cases of partial removal of a material by an irrigation method, it was observed that the material had ‘risen’ from the apical root canal to the coronal root canal or pulp chamber. This appeared to occur using all irrigation groups, except GW, and most commonly in the PF US (0 mL/min – no flow) group. Perhaps this was seen only in the PF US (0 mL/min – no flow) group due to its cavitation and acoustic microstreaming effect on irrigation fluid (Ahmad et al., 1987a). It is questionable whether this relocation is clinically significant.     110 Second hypothesis  The second hypothesis of the present study was that the canal orientation does not influence the ability of different irrigation methods to remove materials from the apical part of a root canal. The effect of canal orientation on material removal was assessed using independent sample t test. When the results from all materials and all irrigation groups were combined, the null hypothesis could not be rejected because no significant differences in the removal of materials between canal orientations were found. This finding is of great clinical importance as it shows gravity does not play a part in irrigant flow efficacy to remove materials from the apical root canal. However, it should be noted that when looking at each irrigation group individually, PF US at a flow rate of 15 mL/min was the only irrigation group to show a significant difference between canal orientations. It removed significantly more material when the canal was oriented vertically rather than horizontally. To the best of our knowledge, there are no published studies on the relationship between gravity and effectiveness of irrigation methods.   Third hypothesis  The third hypothesis studied was that the success of removing particles of materials does not depend upon the type of the material. This hypothesis was not statistically analyzed; however, some trends can be seen from the results. In general, each irrigation group individually seemed to remove a similar amount of each material. Therefore, the results indicate that perhaps the type, size and density of a material is not an important factor in removal from the apical root canal. For GW specifically, gold alloy dust was the only material that it could not completely remove on every attempt. Interestingly, all three attempts GW could not completely remove the gold alloy dust occurred when the canal was in a horizontal orientation. It would be interesting to  111 explore further why the gold alloy dust particles behaved in such a manner. Perhaps it was due to their flake-like shape or small particle size. In addition, compared to the other materials, the mean time for complete removal of gold alloy dust using GW had the largest variation. The larger standard deviation indicates it is unpredictable how much time it will take to remove gold alloy dust completely from the root canal using GW.   Fourth hypothesis The fourth hypothesis studied was that flow rate is not associated with a larger percentage of material removed. This hypothesis could be partially rejected for SNI OE. SNI OE (15mL/min) removed significantly more material from the apical root canal than SNI OE (5 mL/min) when all materials were combined, but not for each material individually. This hypothesis could not be rejected for SNI SV. SNI SV (15 mL/min) did not remove significantly more material from the apical root canal than SNI SV (5 mL/min) for both when all materials were combined and for each material individually. The results above for each material individually for SNI OE and SNI SV were not surprising. Park et al. (2013) showed irrigant does not progress further than 2 to 3 mm apical to both needle tip designs and there is no further increase in the extent of irrigation from the needle tip at flow rates beyond 4 mL/min. Therefore, if an OE needle design is to be used for material removal, then a flow rate of 5 mL/min is adequate for effectiveness, while providing improved safety. In addition, this hypothesis could be rejected for PF US. PF US (15 mL/min) removed significantly more material from the apical root canal than PF US (0 mL/min) for both when all materials were combined and for each material individually. This was expected because without a continuous irrigant flow or intermittent flushing, the irrigation solution only undergoes  112 cavitation and microstreaming (Ahmad et al., 1987a). Jiang et al. (2012) found that without the replenishment of fresh irrigant solution, there is minimal removal and coronal displacement of debris. The minimal coronal movement of materials could be visualized under the stereo microscope during PF US (0 mL/min) irrigation.   5.3 Novelty of the study   The present study suggests that some irrigation methods can be effectively used to remove materials of different size and density that have accidently fallen into the apical root canal. More specifically, the GW system showed it can completely clean root canals of most materials in less than 1-minute irrigation time. This is of clinical importance because it allows conservation of root dentin and time savings to the dental provider.   5.4 Limitations of the study As previously mentioned, some limitations of this in vitro study include using a transparent resin tooth model and using microsphere beads as materials. A third limitation is the larger than ideal standard deviations seen for the mean percentage of materials removed and mean time for complete removal of materials. A fourth limitation of this study is the smaller than ideal sample size. As discussed earlier, an online sample size calculator was used to select the sample size based on anticipated means and standard deviations from a pilot study. However, the results of the study showed higher standard deviations than those anticipated, therefore the selected sample size was smaller than actually required. Placing the results into the online sample size calculator (https://www.stat.ubc.ca/~rollin/stats/ssize/n2.html accessed on October  113 29, 2020) showed a minimum of 16 irrigations (tests) per group would be necessary to reach statistical significance.   5.5 Future directions  In the future, the study should be performed ex vivo on an extracted tooth or on a chemically cleared extracted tooth to account for any physical, chemical, or anatomical differences seen in a natural tooth. In addition, multiple different extracted teeth should be tested to account for variations in root canal anatomy, size, taper, curvature and length. Other dental materials of different size and density that could potentially fall into the apical root canal should also be tested. These may include particles of composite resin, porcelain, zirconium metal, glass ionomer cements, and metal posts. Also, further modifications in the size and shape of the gold alloy dust particles should be performed to determine if they behave the same or differently. Other irrigation methods, such as EndoVac (Kerr dental, Orange, CA), IrriSafe ultrasonic file irrigation (Acteon, Gustave Eiffel, France) or EndoActivator (Endo Inventions LLC, Santa Barbara, CA), could also be added as a comparison. These systems may provide the advantage of increased dispersion of irrigant flow apically, while in a safe and effective manner. Other clinically relevant flow rates could also be assessed for effectiveness to determine if there is an optimal flow rate required for complete material removal. Lastly, a bigger sample size should be used for each irrigation group to account for the large standard deviations that were observed in the current study.      114  Conclusions  Under the limitations of this in vitro study: 1. The GentleWave system was more effective, at 7-minute maximum and 1-minute irrigation times, in removing materials from the apical root canal than the other irrigation methods.  2. The orientation of the canal, horizontally or vertically, did not affect the ability of the irrigation methods to remove the materials.  3. Higher irrigant flow rates using open-ended syringe needle and PiezoFlow ultrasonic were more effective than lower flow rates in removing materials.    115 Bibliography  Abou-Rass, M., & Piccinino, M. V. (1982). 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