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Evaluation of the effectiveness of two irrigation systems in reducing bacterial load in root canals in… Zhang, Duo 2017

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 EVALUATION OF THE EFFECTIVENESS OF TWO IRRIGATION SYSTEMS IN REDUCING BACTERIAL LOAD IN ROOT CANALS IN VITRO BY QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION  by Duo Zhang  D.M.D, Sichuan University, 2015  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)   July 2017 © Duo Zhang, 2017 ii  Abstract  Introduction: Irrigation is regarded as having a key role in one of the main goals of endodontic treatment, the killing and removing bacteria from the infected root canal. Different methods and techniques have been used to deliver irrigating solutions and to facilitate the effectiveness of irrigation. The purpose of this in vitro study was, by using quantitative real-time PCR to determine the effectiveness of two irrigation and cleaning systems in removing multispecies oral biofilms from root canals.   Methods: Fifteen extracted human molars were instrumented to size #15/.02 and then cleaned with the GentleWave System (GW; Sonendo Inc, Laguna Hills, CA). The teeth were autoclaved to provide the same sterile baseline. The molars were filled with mixed plaque suspended in brain-heart infusion broth (BHI; Becton Dickinson, Sparks, MD) and centrifuged to help the bacteria spread all over the root canal system. After two weeks of incubation, samples were divided randomly into two treatment groups and instrumented into size #15/.04 (GW) and #35/.04 (PiezoFlow group) under needle irrigation with sterile water. The teeth were then cleaned either with GentleWave System or ProUltra PiezoFlow Active Ultrasonic System (Dentsply Tulsa, Tulsa, OK) using 3% NaOCl, 8% EDTA and sterile water as irrigants. Root canals were sampled with paper points before and after instrumentation (S1, S2) and after GW or PF cleaning (S3). Quantitative real-time PCR was performed, the presence of microorganism in the samples was determined by using universal bacterial, a genus specific and species-specific primers. Statistical analysis was performed using the Mann-Whitney U test with the significance level set at P < 0.05.   iii  Result: A highly significant bacterial reduction was recorded for both groups (P < 0.001). GW group showed a significantly higher reduction than the PF group of total microbes (P < 0.01), Enterococcus faecalis (P < 0.01) and Streptococcus species (P < 0.05). GW resulted predictably in high reduction of over 99% of the microbes in every canal, whereas in the PF group the reduction of microbes varied between 87% and 99.99%.  Conclusion: While both systems demonstrated effective reduction of intracanal bacteria, a high level of over 99% was more predictably achieved in the GW group. iv  Lay Summary  Apical periodontitis is an infectious oral disease which starts when bacteria invade the root canal inside the tooth. Apical periodontitis can be identified in a radiograph as destruction of bone structures around the root tip of the affected tooth. Healing of the infection requires that bacteria inside the tooth are removed completely or reduced to a very low level. Eradication of bacteria, according to studies and clinical experience is difficult. In this study we examined the ability of a novel equipment specifically designed to clean infected root canals from bacteria for its efficacy and compared it to the most effective system so far. Sensitive, modern techniques were used to measure the effectiveness and compare two the systems. The results showed that the new system, GentleWave, produced cleaner canals with higher predictability than the other system.  v  Preface  The study design was identified by Dr. Duo Zhang under the guidance of Dr. Markus Haapasalo and Dr. Ya Shen. Teeth collection, treatment, and sample taking was carried out by Markus Haapasalo and Prashanthi Vandrangi. DNA extraction and QPCR were performed by Dr. Duo Zhang. Data were collected and analyzed by Dr. Duo Zhang with the assistance of Dr. HsingChi von Bergmann. Thesis was prepared by Dr. Duo Zhang and revised by Dr. Markus Haapasalo and Dr. Ya Shen. Ethics approval was acquired from the University of British Columbia Office of Research Services, Clinical Research Ethics Board (certificate number H15-02793). vi  Table of Contents  Abstract .......................................................................................................................................... ii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables ................................................................................................................................ ix List of Figures .................................................................................................................................x List of Abbreviations ................................................................................................................... xi Acknowledgements .................................................................................................................... xiii Dedication ................................................................................................................................... xiv Chapter 1: Literature Review .......................................................................................................1 1.1 Introduction ..................................................................................................................... 1 1.2 Biofilm ............................................................................................................................ 1 1.3 Apical Periodontitis ........................................................................................................ 6 1.3.1 Location of the Microbes in the Root Canal System .................................................. 7 1.3.2 Distribution of the Microbes in Various Parts of the Root Canal ............................... 9 1.4 Root Canal Treatment ................................................................................................... 10 1.5 Irrigation ....................................................................................................................... 11 1.5.1 Irrigating Solutions ................................................................................................... 12 1.5.2 Irrigation Methods .................................................................................................... 13 1.5.2.1 Conventional Syringe Needle Irrigation (CSNI) .............................................. 13 1.5.2.2 Apical Negative Pressure System (ANP) ......................................................... 13 vii  1.5.2.3 Passive Ultrasonic Irrigation System (PUI) ...................................................... 14 1.5.2.4 Continuous Ultrasonic Irrigation (CUI) ............................................................ 15 1.5.2.5 Laser Activated Irrigation (LAI) and Photon-Induced Photoacoustic Streaming (PIPS) ........................................................................................................................... 15 1.5.2.6 GentleWave System .......................................................................................... 16 1.5.3 Fluid Hydrodynamics................................................................................................ 17 1.6 Methods of Sampling Bacteria from Root Canals ........................................................ 18 1.7 Quantification of the Biofilm Microbes ........................................................................ 19 1.7.1 Culture Based Methods ............................................................................................. 19 1.7.2 qPCR ......................................................................................................................... 19 1.8 Aim of the Study ........................................................................................................... 20 1.9 Hypothesis..................................................................................................................... 21 Chapter 2: Materials and Methods ............................................................................................22 2.1 Tooth Collection and Preparation ................................................................................. 22 2.2 Inoculation .................................................................................................................... 22 2.3 Sampling of the Bacteria from the Canals, First Sample .............................................. 23 2.4 GentleWave Group, Second and Third Sample ............................................................ 23 2.5 Ultrasonic System Group, Second and Third Sample .................................................. 24 2.6 DNA Extraction and Quantitative Real-time PCR Analysis ........................................ 24 2.7 Statistical Analysis ........................................................................................................ 28 Chapter 3: Results........................................................................................................................29 3.1 qPCR Co-efficiency Test and Specificity ..................................................................... 29 3.2 qPCR Results of Bacterial Quantification .................................................................... 31 viii  Chapter 4: Discussion ..................................................................................................................36 Chapter 5: Limitations of the Study...........................................................................................41 Chapter 6: Conclusion .................................................................................................................42 Chapter 7: Future Directions ......................................................................................................43 References .....................................................................................................................................44 Appendices ....................................................................................................................................67  ix  List of Tables Table 2.1     Primers Used in Real-time PCR for Bacterial Quantification...................................25 Table 3.1     Level of Total Bacteria in Root Canal Samples of Teeth Taken Before Instrumentation (S1), After Instrumentation (S2) and After GentleWave/PF Ultrasonic System (S3) .............................................................................................31 Table 3.2     Level of Enterococcus faecalis in Root Canal Samples of Teeth Taken Before Instrumentation (S1), After Instrumentation (S2) and After GentleWave/PF Ultrasonic System (S3) .............................................................................................32 Table 3.3     Level of Streptococcus species in Root Canal Samples of Teeth Taken Before Instrumentation (S1), After Instrumentation (S2) and After GentleWave/PF Ultrasonic System (S3) .............................................................................................33  x  List of Figures Figure 3.1   Standard Curve of qPCR Co-efficiency Test.............................................................28 Figure 3.2   Melt Curve of qPCR Assay - qPCR Specificity.........................................................29  xi  List of Abbreviations ANP.....................................................................................................apical negative pressure system ATCC.............................................................................................. American Type Culture Collection BHI.....................................................................................................................brain-heart infusion CFD...................................................................................................... Computational fluid dynamics CFU............................................................................................................counting of colony-forming CHX...................................................................................................................................chlorhexidine CSNI........................................................................................ conventional syringe needle irrigation CUI.......................................................................................................continuous ultrasonic irrigation DNA .................................................................................................................... deoxyribonucleic acid EDTA ................................................................................................... ethylenediaminetetraacetic acid E. faecalis............................................................................................................Enterococcus faecalis EPS...................................................................................................extracellular polymeric substance LAI...................................................................................................................laser activated irrigation Min.............................................................................................................................................minute NaOCl ..............................................................................................................sodium hypochlorite PBS................................................................................................................phosphate buffered saline PIPS...................................................................................... photon-induced photoacoustic streaming PMA.................................................................................................................... propidium monoazide PTED.............................................................................post-treatment endodontic disease PUI................................................................................................passive ultrasonic irrigation system QPCR...................................................................................... quantitative polymerase chain reaction Sec.................................................................................................................................................second xii  UAI...................................................................................................ultrasonically activated irrigation WL..................................................................................................................................working length xiii  Acknowledgements Firstly, I owe my sincerest thankfulness to my supervisor Professor Markus Haapasalo, who is always willing to share his wisdom and expertise to me. His richness of endodontics knowledge, passionate enthusiasm in research and sense of humor motivate me to study and research more in endodontics. I am also grateful to his guidance during the whole project and providing me creative and valuable ideas on every step of my project. What I have achieved would not be possible without his patient guidance.  I also would like to express my sincere gratefulness to Dr. Ya Shen, who is my co-supervisor. Her kind support and professional guidance encourage me to overcome every difficulty in my project.   I am thankful to my committee members Dr. Lari Hakkinen and Dr. Clive R. Roberts for their valuable input to my study design and troubleshooting.   xiv  Dedication To my parents, thank you for your unwavering support and encouragement in my education and life. I can’t achieve this without you.  To my wife, your accompanying and love are always my motivation to pursue our dream.  To Dr. Bo Hu, thank you for your kind care and support on both my education and future career. Thank you for sharing your knowledge and experience.  1  Chapter 1: Literature Review  1.1 Introduction Various factors such as chemical and physical irritants can cause irritation and even necrosis of the pulp, but bacteria and their products entering the pulp are the most common cause for endodontic diseases such as pulpitis and apical periodontitis (Bergenholtz 1974; Bergenholtz 1990). One of the primary goals of the endodontic treatment is to eradicate the microbes in root canal systems (Sjogren et al. 1997; Chugal et al. 2001; Jhajharia et al. 2015). Nowadays, there is still no recognized treatment strategy that can predictably eliminate all bacteria from infected root canals. The successful treatment of endodontic diseases depends on killing and removing as many microbes as possible from root canal system and preventing reinfection. Oral infections, root canal infections included, are biofilm infections. Therefore, successful control and prevention of oral infections depends on success in preventing and controlling oral biofilms.  1.2 Biofilm Traditionally, much of the research in endodontic disinfection has been done with pure cultures of planktonic bacteria, in test tube conditions (Gomes et al. 2001). Many of the available disinfecting agents are quite effective against planktonic microbes and killing may results in just a few seconds, even with low concentration of the disinfecting agent. These results were in stark contrast with the clinical reality, where it seemed particularly difficult to eradicate the microbes from the necrotic, infected root canal (Nair et al. 1990; Nair et al. 1999). The realization that endodontic microbes are organized as biofilms around the root canal system, has opened a new approach to the understanding of endodontic microbiology and disinfection in clinical 2  endodontics. Biofilm is a complex bacterial community in which cells attach to each other and are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS) (Costerton et al. 1994; Costerton et al. 1999; Socransky and Haffajee 2002; Stoodley et al. 2002). EPS is a matrix material consisting of nucleic acid, polysaccharides, proteins and salt. Some of the bacteria have the ability to rapidly build a self-aggregating collection of a large number of bacterial cells embedded in EPS which helps them escape the host defense system (Nair et al. 2008). Because of the three-dimensional structure, the residing bacteria in a biofilm community have a metabolic cooperation between resident cells of different species. Thus, the bacteria in a biofilm have high resistance against the host's immune system and disinfecting agents (Gilbert et al. 1997; Costerton et al. 1999; Chavez de Paz et al. 2007). In the oral cavity, there are three basic steps involving biofilm formation: Pellicle formation, bacterial colonization and biofilm maturation (Gilbert et al. 1993; Jenkinson and Lappin-Scott 2001; Katsikogianni and Missirlis 2004). Oral biofilms are involved in the development of many oral diseases such as caries and periodontal diseases.   Bacterial cells, a fluid medium, and a solid surface are the three components involved in biofilm formation. The process of bacterial biofilm formation begins when a cell attaches to a surface coated with an organic conditioning polymeric matrix or “conditioning film” (Grenier and Mayrand 1986). In the initial phase, the contact between the cell and conditioning film is initially weak. Then the sticky exopolymers secreted by the cells help the bacterial cells to adhere to the surface irreversible (Handley et al. 1985). In the next stage, the development of a biofilm occurs as a result of adherent cells replicating and by additional cells adhering to the biofilm (Jenkinson 3  and Lappin-Scott 2001). During the biofilm development and maturation process, some cells will eventually detach from the biofilm.    In endodontic infection, microorganisms penetrate into the pulpal space and spread further apically along the root canal. Biofilm formation in root canals is initiated after the invasion of the pulp chamber by oral organisms and pulp tissue breakdown (Mohammadi et al. 2013). Nutritional and ecologictal status of the root canal has an impact on the progression of the infection. Development of an anaerobic environment and depletion of nutrition create a tough ecological niche for the surviving microorganisms. During root canal treatment, the anatomical and geometrical complexities in the root canal system shelter the biofilms from the mechanical cleaning procedures. A recent study indicated  that the dead bacteria can provide the surviving bacteria a large amount of microbial biomass as a nutritional source for growth (Herrero et al. 2017). The presence of dead bacteria may have resulted in an outgrowth of several pathogenic species in complex multi-species biofilms. Additionally, upon contact with dead oral bacteria, virulence genes of P. intermedia and P. gingivalis were up-regulated. This resulted in a more pronounced epithelial cytotoxicity (Herrero et al. 2017).   In vitro/ex vivo models on extracted teeth Optimally, the most natural environment to do research on the effectiveness of disinfecting agents in endodontics would be natural teeth “in vivo” with apical periodontitis. However, because of numerous reasons, both ethical, practical and cost related, this is rarely possible. In addition, the wide variation between different teeth and different infections adds to the need of large patient material due to the confounding factors. The use of extracted teeth or prepared blocks of root 4  dentin in vitro and ex vivo has been a widely used approach in studies of the efficacy of endodontic disinfectants is an effort to better simulate the in vivo conditions and the in vivo reality of the root canal. Typically, just a single species, E. faecalis, or polymicrobial plaque collected from an infected root canal infection or from interdental space is incubated in the root canal (or partial root canal) for 1 – 30 days (Dunavant et al. 2006; Kishen et al. 2006; Shen et al. 2009; Williamson et al. 2009; de Paz et al. 2010; Shen et al. 2011; Stojicic et al. 2013). After the biofilm growth, different treatment protocols are performed, with an emphasis on various irrigation parameters, and microbiological samples e.g. for culturing are collected (Dunavant et al. 2006; Kishen et al. 2006; Williamson et al. 2009; Liu et al. 2010). Although these studies have provided much useful information, the dentin block model and other ex vivo models also have weaknesses and shortcomings. In many of these studies, the magnitude of biofilm growth and presence was not confirmed with any microscopic method. There are also great variations regarding the time of incubation of bacteria and how often nutrients were refreshed (Siqueira et al. 1997; Kishen et al. 2006; Bhuva et al. 2010; Liu et al. 2010). When the incubation is started, the microbes are mostly in a planktonic state, even exponential growth phase is possible. Such bacteria are extremely sensitive to disinfecting agents when compared to bacteria in established biofilms. Portenier et al. (2005) reported that bacteria in the starvation phase were 1,000 times more resistant to disinfecting agents than bacteria during the exponential or even stationary growth phase. In addition to the difference between planktonic bacteria in different growth phase, microbes in biofilms of different age and maturation level can demonstrate considerable differences in their susceptibility to antimicrobial agents (Shen et al. 2011; Stojicic et al. 2013).   5  Mono- and multi-species biofilms  Research of in vivo, natural biofilms in their own environment if difficult. Therefore, efforts have been made to develop in vitro biofilm models which could closely mimic the key features of biofilms in the necrotic root canal of a tooth with apical periodontitis, e.g. In oral infections, the biofilms are almost always multispecies biofilms. In persistent endodontic infections, cultural studies have indicated that in some cases a monospecies E. faecalis infection may be involved (Peciuliene et al. 2000). Over the past years, endodontic research on biofilms has focused on both single-species (Du et al. 2013) and multi-species biofilm models (Shen et al. 2009; Arias-Moliz et al. 2010). In one study, the ability of four root canal bacteria to establish a multi-species biofilm community was studied with special focus on various structural and ecological aspects of the biofilm (de Paz 2012). The species included were Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus gordonii, and E. faecalis, all originally isolated from infected root canals. The four species were able to develop stabile in vitro biofilms with a relatively low proportion of dead cells. However, the overall time for biofilm growth examined was relatively short in this and many other experiments.   Several new biofilm models have been reported by a group from the University of British Columbia in recent years (Shen et al. 2009; Shen et al. 2010; Ma et al. 2011; Shen et al. 2011). These in vitro models include mono- and multispecies biofilms grown on collagen coated hydroxyapatite discs, dentin discs and in microscopic dentin canals  (Shen et al. 2009; Shen et al. 2010; Ma et al. 2011; Shen et al. 2011). E. faecalis has been the most commonly used species in the monospecies biofilms, whereas the multispecies biofilms have been based on supra- and subgingival plaque, thus containing tens of different species, both facultative and anaerobic 6  bacteria. Anaerobic growth conditions and limited availability of nutrients (added only once per week) has been chosen as a strategy to invite the biofilm microbes to depend more on interbacterial interactions than continuous supply of nutrients from an external source. As a result, spirochetes are regularly detected in these biofilms even after extended periods of incubation for several weeks, something which has not been described earlier in literature for in vitro biofilms (Shen et al. 2009). In dentin canal biofilm model by the same group, centrifugation has been used as a method to force large numbers of bacteria into the dentinal tubules (Ma et al. 2011). Pure cultures of E. faecalis and mixed plaque have both been used in the dentin canal model with centrifugation. The method has made it possible to produce high numbers of parallel specimens with similar type of biofilm inside dentin, for the study of the effect of endodontic disinfecting agents in deeper layer of dentin (Wang et al. 2012; Du et al. 2014; Wang et al. 2014; Du et al. 2015; Yang et al. 2016).   1.3 Apical Periodontitis Apical periodontitis is an inflammatory lesion around the apex of a tooth root caused by biofilm-related infection in the necrotic root canal (Moller et al. 1981; Lee et al. 2016). The bacteria in teeth with apical periodontitis are mainly obligate anaerobic bacteria, which often constitute as much as 90% of the microbial flora (Wittgow and Sabiston 1975). The microbiota is a polymicrobial mix with approximately equal proportions of Gram-positive and Gram-negative species, dominated by obligately anaerobic bacteria (Figdor and Sundqvist 2007). Within the numerous species, Enterococcus faecalis and Streptococcus species are among those often isolated from infected root canals of human teeth (Marton and Kiss 2000; Wang et al. 2015; Tatikonda et al. 2017). Enterococcus faecalis is the most frequent species present in post-treatment disease and may play a significant role in persistent periapical infections following root canal treatment 7  (Sundqvist et al. 1998; Peciuliene et al. 2001; Pinheiro et al. 2003; Rocas et al. 2004; Siqueira and Rocas 2004; Skucaite et al. 2010). Streptococcus mutans is considered to be the microbial species most strongly associated with enamel caries and initiation of tooth surface biofilm formation (Nicolas and Lavoie 2011).   It has been previously estimated that about 500 species of bacteria inhabit the human oral cavity (Wilson et al. 1997). However, currently more than 1,000 bacterial species belonging to 13 phylotypes have already been identified in the oral microbiota and oral cavity (Wade 2013). Many of these species can invade the root canal system and thus are potential endodontic pathogens. Culture-based approaches have been widely used to identify microbes in infected root canals. The average number of isolated species per canal was reported from 2.4-6.8 (Aderhold et al. 1981; Brook et al. 1991; Sundqvist 1992; Brauner and Conrads 1995). Studies using molecular biological methods have shown that more than 200 different microbial species can be found in infected root canals, more than 20 species per canal can be detected (Rolph et al. 2001; Vianna et al. 2005). Over 900 bacterial species have been found within the root canal system by using barcoded multiplex pyrosequencing, a high-throughput multiplexed 16S rRNA gene barcoded pyrosequencing approach (Santos et al. 2011). Of these species, 486 were found in acute endodontic infections, 265 in chronic infections, and 165 in both cases (Santos et al. 2011).   1.3.1 Location of the Microbes in the Root Canal System The root dentin is hard tissue with countless ca. 2 μm wide dentin canals traveling from the pulp to the root surface cement. Several studies have shown that bacteria can and will penetrate the 8  dentin canals under favorable conditions Bacterial presence in dentinal tubules has been demonstrated in most teeth with necrotic canal and apical periodontitis.   Ørstavik & Haapasalo (1990) investigated the effect of endodontic antibacterial agents in dentin blocks prepared from bovine dentin. The authors culture bacteria in the root canal from where some bacteria penetrated into dentin canals. E. faecalis penetrated into the dentin canals more effectively than some other species. Other studies have reported bacterial penetration of 200 μm or more into dentinal tubules (Haapasalo and Orstavik 1987; Love and Jenkinson 2002) Lateral canals, which are small ramifications from the main root canal may also harbor bacteria/bacterial biofilm causing lateral lesions. Histological studies have shown that lateral canals are difficult or impossible to completely clean by presently available methods (Ricucci and Siqueira 2010). Therefore, bacteria in lateral canals can cause post-treatment endodontic disease (PTED).  The presence of a biofilm with complex structure has been reported occupying the isthmus of a tooth, which had been treated 10 years earlier and then re-treated two years from the original treatment. The persisting infection eventually lead to extraction of the tooth (Carr et al. 2009). The study emphasizes the importance of obtaining as complete cleanliness of the whole root canal system as possible.   In addition to established biofilms, instrumented root canals contain smear layer unless chemically removed after the instrumentation. Smear layers in teeth with apical lesion are likely to contain microorganisms and their antigens. Therefore, smear layer and the infective material embedded 9  into it should be removed during the irrigation (McComb and Smith 1975; Mader et al. 1984; Czonstkowsky et al. 1990; Sen et al. 1995).  1.3.2 Distribution of the Microbes in Various Parts of the Root Canal Root canal microbiota is extremely complex, close to 1000 different species have been reported in different studies (Siqueira and Rocas 2009; Santos et al. 2011; Wade 2013). While all of these bacteria and other microorganisms have found their ecological niche in the necrotic root canal, there are also great differences between the ecological requirements between different species. Availability of oxygen and type of nutrients are among the main factors affecting the growth of microorganisms in the root canal. Interbacterial relationships also play a big role in the ability of specific species to establish themselves in the various parts of the root canal, as many species are dependent on metabolic end products produced by some other species.    Özok et al. (2012) examined bacterial ecology of different parts of the necrotic root canal and found that the apical root canal favors selection of a more anaerobe microbiota than the coronal canal. Often the coronal canal is richer in nutrients from the oral cavity. As a result, fast growing and carbohydrate utilizing species take over the coronal canal flora (Kassen et al. 2000, Pham et al. 2009). In the apical canal limitation of nutrients may prevent overgrowth by a single or just a few species, and the survival of many species is in fact dependent on survival of other species as well, which seems to favor diverse anaerobic microbiota (Kreft 2004).  10  1.4 Root Canal Treatment Root canal treatment, after diagnosis and treatment decision, consists of mechanical instrumentation, irrigation, intracanal medicaments between appointments, root canal filling, and coronal restoration (Eckerbom et al. 1989; Siren et al. 1997; Sjogren et al. 1997; Haapasalo and Endal 2003). A variety of different factors will affect the prognosis of the treatment. These include the initial size of the periapical lesion, complexity of the root canal anatomy, type and location of the flora, and different types of mechanical and/or biological complications during the treatment (Nair et al. 1990). Sjogren et al. (1997) reported that complete elimination (as shown by negative culture result) of bacteria prior to root filling was essential to high success rate of the root canal treatment. Complete periapical healing was detected in 94% of cases with negative culture before root filling whereas in cases with positive culture (growth of bacteria) before the filling the success rate was only 68%.    Mechanical instrumentation and irrigation both play key roles in the eradication of microbes. Instrumentation removes necrotic tissue and microorganisms from the root canal, and irrigation with chemical agents optimally completes the removal of tissue and killing of microorganisms. Due to the anatomic irregularities of the root canal including isthmuses, fins and apical deltas, many areas  in the root canal system are still untouched during the instrumentation (Wu and Wesselink 2001; Ricucci and Bergenholtz 2003). Therefore, irrigation has a great responsibility to reach those areas of the root canal wall that are beyond the reach of instrumentation (Haapasalo et al. 2014).  11  The procedures of conservative endodontics, mechanical, chemical and biological, only affect tissue and microbes which are within the confines of the root canal system. In some cases, bacteria have been shown to reside outside the root canal, on the root surface or as independent colonies in the periapical lesion as biofilm islands (Nair et al. 1999). In these situations, conservative root canal treatment cannot bring healing of the lesion without completing the treatment with an endodontic microsurgical procedure, e.g. apicoectomy, where ca. 3 mm of the root tip is cut off and a retrograde cavity of a minimum of 3 mm is filled with a retrograde filling material (Frenkel and Stellmach 1973; Kim and Kratchman 2006; Tsesis et al. 2006).   1.5 Irrigation The goal of irrigation is not only to dissolve pulp tissue and to remove bacteria from the root canal but also to remove the smear layer and dentin debris created during instrumentation (Baugh and Wallace 2005; Haapasalo et al. 2005). Studies have reported that the efficacy of irrigation can determine the residual number of bacteria in root canal system and have a great influence on the short and long term prognosis (Haapasalo et al. 2014; Plotino et al. 2016). A successful treatment outcome is contributed by a combination of optimal irrigants with chemical agents and an effective irrigation method. A large number of studies have focused on development of new irrigant solutions (Kovac and Kovac 2011; Singla et al. 2011; Mohammadi 2015; Goncalves et al. 2016). However, over several decades, Sodium hypochlorite (NaOCl) and EDTA are still the most important and popular irrigants in root canal treatment (Siqueira et al. 2000; Dunavant et al. 2006; Haapasalo et al. 2014). On the other hand, numerous innovative irrigation techniques have been developed.  12  1.5.1 Irrigating Solutions NaOCl is the most important irrigants in root canal treatment (Siqueira et al. 2000; Dunavant et al. 2006). It effectively kills microbes and dissolves organic matter in the root canals (Cobankara et al. 2010; Paque et al. 2012). NaOCl can ionize into sodium (Na+) and ClO- in water. ClO- and hypochlorous acid (HClO) both have strong antimicrobial effect, HClO being stronger than ClO-. (Gomes et al. 2001; Zehnder 2006). The tissue dissolving effect of NaOCl is dependent on concentration, volume and contact time. In addition, increasing the temperature, flow, and surface tension of NaOCl improve its effectiveness (Stojicic et al. 2010). NaOCl is used in concentrations between 0.5 ‑6 %, recently even 8% solution has been adopted by many endodontic specialists.   Ethylenediaminetetraacetic acid (EDTA) is another important irrigant, used to finalize the removal of the smear layer. EDTA is used in concentration of 17%, 15% or 8%. It only affects the inorganic part of dentine and smear layer, therefore it is used as final rinse after NaOCl (Goldman et al. 1982; Yamada et al. 1983). If mixed or used alternatively, EDTA weakens the effect of NaOCl.   Chlorhexidine (CHX) is used by many dentists as a final solution after EDTA particularly in cases of persistent root canal infections (Russell and Day 1993). Since CHX has no effect of dissolving organic or inorganic tissue, it cannot replace NaOCl and be used as the only irrigant. Its antibacterial effectiveness is weaker than that of 5% NaOCl, close to 2% NaOCl (Ma et al. 2011; Wang et al. 2012).   13  1.5.2 Irrigation Methods  1.5.2.1 Conventional Syringe Needle Irrigation (CSNI) CSNI is the classical method of irrigation in root canal treatment. It can be effective in cleaning the main root canal when used properly (Kahn et al. 1995; van der Sluis et al. 2006). One recent study reported that a flow rate of 4 mL/min can achieve the optimal effect of irrigant exchange and yet cause a relative low apical pressure, which is important in reducing the risk of extrusion (Park et al. 2013). The use of side-vented needles can provide safer irrigation than open-ended needles, which may cause higher apical pressure at the foramen (Shen et al. 2009). Depending on canal anatomy and size, it is often difficult for the needle to reach the apical canal. Therefore, apical vapor lock can form and further prevent the solutions from reaching to the apical area of the root canal (Boutsioukis et al. 2014). Different modifications such as side vented or flexible needles have been introduced to improve the effectiveness and safety of syringe-needle irrigation. CSNI is not as effective as some other methods using additional energy in cleaning the complex root canal system (Siu and Baumgartner 2010).  1.5.2.2 Apical Negative Pressure System (ANP) ANP has been introduced to simultaneously deliver the irrigant to and suction from the canal in order to obtain a good and safe flow of irrigant into the entire root canal system. EndoVac (KerrEndo, Orange County, CA, USA) is a commercial realization of the ANP principle. It delivers a constant flow of irrigant from a Master Delivery Tip, and at the same time remove debris and irrigant solution at the apical third by MacroCannula or MicroCannula using suction. Negative pressure irrigation contributes to improved safety as compared to positive pressure, and it has been proven to be significantly safer than CSNI (Malentacca et al. 2012). Also, when compared to 14  ultrasonic irrigation methods, teeth treated by ANP have shown less debris extrusion (Tambe et al. 2013). As for efficacy, some early studies have shown that the ANP can effectively clean the apical part of the root since it has been shown to allow the irrigants to safely reach the most apical canal, help overcome apical vapor lock, and prevent extrusion of irrigant and debris into the periapical tissue (Siu and Baumgartner 2010; Tambe et al. 2013). However, some recent studies reported ANP was only slightly better than CSNI in debridement efficacy, smear layer removal, and dentinal tubule penetration (Malentacca et al. 2017; Turkel et al. 2017).  1.5.2.3 Passive Ultrasonic Irrigation System (PUI) PUI is utilized after instrumentation and without the intent to enlarge, instrument, or impact the walls of the root canal. Unfortunately, however, even though the intent is not to make contact the root canal walls, this will occur due to the oscillating motion of the ultrasonic instrument tip. Because of this, the phrase ultrasonically activated irrigation (UAI) was recently suggested instead of passive ultrasonic instrumentation (Boutsioukis et al. 2013).  The effectiveness of PUI/UAI to remove tissue and debris has been studied. PUI was reported to remove debris significantly better than CSNI (Tanomaru-Filho et al. 2015; Topcuoglu et al. 2015). De Greporio et al. (2010) compared the penetration of NaOCl into the simulated working length and lateral canals and found that PUI enhanced the NaOCl penetration into the lateral canals more than ANP. A large number of studies have reported a significant reduction in the number of bacteria following the use of PUI when compared with CSNI (Huque et al. 1998; Sjogren et al. 1998; Spoleti et al. 2003; Townsend and Maki 2009; Cachovan et al. 2013). Culturing and counting the colony forming units (CFU) was used as the method of quantification.  15  1.5.2.4 Continuous Ultrasonic Irrigation (CUI) Although PUI was reported to improve the cleanliness of root canal system, the lack of irrigant replenishment is an issue. CUI was developed to resolve this issue. In CUI, the needle simultaneously activates and replenishes the irrigant in the canals. The high-power stream generated has a promising cleaning effect on the rough surfaces and otherwise difficult to reach areas within the root canal system. Castelo-Baz et al. (Castelo-Baz et al. 2016) reported that CUI with the Piezoflow™ system (Dentsply Tulsa, Tulsa, OK, USA) was more effective than PUI/UAI in getting irrigant into lateral canals. The authors found that using CUI as the final rinse can significantly increase the penetration of irrigant into the simulated lateral canals and apical third of curved roots. CUI has been reported to have a significant higher efficacy in removing pulp tissue and debris from root canal system than PUI and CSNI (Jiang et al. 2012; Neelakantan et al. 2016). Malentacca et al. (Malentacca et al. 2012) compared the efficacy and safety of CUI, PUI, ANP and CSNI in vitro study and found CUI was the most effective system while ANP was the safest, but only by a slight margin compared with CUI, which was reported to be the best to reconcile efficacy and safety among these four irrigation systems.  1.5.2.5 Laser Activated Irrigation (LAI) and Photon-Induced Photoacoustic Streaming (PIPS) LIA is a method which uses laser to “activate” the liquid. The liquid vaporizes and expands to form bubbles. When the laser pulse ceases, bubbles begin to shrink and result in acoustic waves which aid in cleaning the root canal by shearing debris off the walls (Matsumoto et al. 2011).  PIPS (Fidelis, Fotona, Ljubljana, Slovenia) is a special form of LIA that works indirectly and without the thermal effect by activating irrigants. It creates a strong photoacoustic shockwave that streams 16  irrigants throughout the root canal system. Unlike other conventional laser applications, the PIPS tip is not placed inside the canal system but in the pulp chamber. Peters et al. (2011) compared the disinfection and disruption of biofilm within the root canal in the apical third and found PIPS generated more negative samples and removed biofilm better than PUI, but was not able to completely remove the biofilm. LIA / PIPS have also been reported to be more effective in debris removal than PUI (de Groot et al. 2009; Arslan et al. 2014). One recent study on irrigation safety reported that there was no extrusion of root canal irrigants during the use of LIA / PIPS (Peeters and Mooduto 2013).  1.5.2.6 GentleWave System GentleWave System (Sonendo Inc, Laguna Hills, CA, USA) is a novel type of endodontic device developed for root canal cleaning and disinfection (Haapasalo et al. 2014; Ma et al. 2015; Molina et al. 2015; Wohlgemuth et al. 2015; Charara et al. 2016). It uses high-speed fluid dynamics to deliver the irrigants into the root canal system without requiring the tip of the instrument to enter the root canals. Instrumentation can therefore be minimized, which can positively contribute to maintaining strong tooth structure. A few studies have reported its superior soft-tissue dissolution compared to ultrasound and excellent cleaning efficacy in the root canal system (Haapasalo et al. 2014; Ma et al. 2015; Molina et al. 2015). An in vitro study of tissue dissolution by GW and several other irrigation systems showed eight (8) times faster soft tissue dissolution by GW than by any other system studied (Haapasalo et al. 2014). GW, when used with sterile water only dissolved tissue comparable to PUI with 2% NaOCl, giving basis for speculation that the high speed irrigant flow hitting the end plate of the nozzle of the GW cleaning instrument creates a strong cavitation effect. Further, all irrigants in the GW system are degassed in real time just before entering the 17  pulp chamber and root canal system, which might explain the spread of the cavitation effect much farther away from the instrument tip than with “conventional” ultrasound used with solutions which are not degassed (Haapasalo et al. 2014).   Two clinical studies have reported a high level of success for cases treated by GentleWave System (Sigurdsson et al. 2016; Sigurdsson et al. 2016). So far, there are no studies of the effectiveness of the GentleWave system to remove bacteria from the root canals.  1.5.3 Fluid Hydrodynamics Irrigation dynamics is important (Moser and Heuer 1982) for the effectiveness of irrigation. This depends on the mechanism(s) of action of the irrigant and on how the irrigant can be brought in contact with the microorganisms and tissue debris (Chow 1983). Smear layer and biofilm are removed by chemical action and physical shear stress by the irrigant on the canal wall. Wall shear stress is difficult to measure. It depends on the flow velocity gradient in closest contact with the wall. Computational fluid dynamics (CFD) is a branch of research focusing on the various phenomena in irrigant flow at different environments. CFD studies have evaluated the effect of root canal taper and apical preparation size on irrigant flow inside a root canal during final irrigation (Boutsioukis et al. 2010a; Boutsioukis et al. 2010b). Higher taper particularly in the apical canal improves irrigant replacement and wall shear stress and reduces apical pressure and extrusion of the irrigant. An irrigant in a non- or minimally tapered root canal with a large apical preparation also showed better irrigant replacement and wall shear stress than in canals with a small preparation size. Irrigant velocity is very important for the overall effectiveness of irrigation (Fariniuk et al. 2003). Previous studies have showed that the irrigant in the canal is only reaching 18  1-3 mm beyond the tip of the needle in simulated curved canals. In order to obtain total replacement of an irrigating solution in the root canal, the irrigation needle should be placed at close proximity of the apical foramen (Park et al. 2013). However, this also increases the risk for irrigant extrusion, which in particular with NaOCl should be avoided at all cost. In recent years studies using CFD models to examine irrigant flow have shed light to many poorly understood areas in root canal irrigation and helped to increase the evaluation of risk for irrigant extrusion (Boutsioukis et al. 2010; Shen et al. 2010; Snjaric et al. 2012).   1.6 Methods of Sampling Bacteria from Root Canals In the process of studying the composition of the root canal microbiota and in quantifying the endodontic microbes, the major challenge is adequate and representative sampling of the complete root canal system. Sampling of the microbes has been done with paper points, endodontic files, or by aspirating the sample fluid in the root canal (Berber et al. 2006; Harrison et al. 2010; Huffaker et al. 2010). The root canal anatomy makes it impossible for a paper point or file to touch all areas of the root canal system (Sathorn et al. 2007). These sampling methods are best suited for planktonic bacteria and those only loosely attached to biofilm. With regard to biofilms, sampling with paper points is unlikely to effectively detach and collect bacteria from them. Further, these sampling methods only address areas where instrumentation has created access. Many areas are simply left untouched by the paper point or file. To increase the yield, agitation of the sample fluid by sonic or ultrasonic equipment is recommended (Harrison et al. 2010; Huffaker et al. 2010; Grundling et al. 2011). However, even then the impact on biofilm is uncertain. In some studies, the whole dentin block or root which has been cut off the crown after extraction, has been frozen and pulverized to maximize the numbers of bacteria for the culture (Kho and Baumgartner 2006; 19  Miller and Baumgartner 2010). A study that compared the efficacy of pulverization and sterile paper point for sampling root canals concluded that the pulverization was more effective than paper point sampling. However, pulverization is an invasive and destructive method which may bring contamination e.g. from the root surface.    1.7 Quantification of the Biofilm Microbes  1.7.1 Culture Based Methods Culture-based methods have traditionally been used to identify and quantify bacteria in different infections (Chu et al. 2005; Martinho et al. 2015). Counting of colony-forming units (CFUs) on selective or nonselective agar culture plates is a technique which has been used to quantify microorganisms for more than a century. However, some bacteria are difficult to grow because they have specific requirements for nutrients and around one-third of oral bacteria cannot be cultured using conventional methods (Conrads et al. 1997; Wade et al. 2016). Furthermore, low sensitivity and long time required are also limitations for the conventional methods (Siqueira and Rocas 2005).   1.7.2 qPCR To overcome the limitations of culture-based detection methods and analyses, molecular methods have been applied to detect and quantify microbes. Quantitative Real-time Polymerase Chain Reaction (qPCR) is a technique of molecular biology based on the polymerase chain reaction (PCR). It can monitor the amplification of DNA products not just at its end as in conventional PCR, but in real-time during the run. As early as 2001, real-time qPCR was used to 20  detect and quantify the number of bacteria in oral plaque samples (Sakamoto et al. 2001). qPCR is increasingly used in microbiological investigations because of its high sensitivity and efficiency in both in vitro and in vivo studies to quantify specific species or e.g. the whole bacterial load in polymicrobial infections (Fouad et al. 2002; Kim et al. 2013; Antunes et al. 2015; Rocas et al. 2015; Rodrigues et al. 2015). qPCR can detect DNA already from 10 copies, thus being much more sensitive than culture method, which can only quantify culturable organisms when they are present in high enough numbers (Araujo et al. 2015).  Studies which have compared the qPCR and conventional culture method with their abilities to quantify bacteria concluded that qPCR offered significant advantages regarding speed and sensitivity of detection (Boutaga et al. 2003; Sedgley et al. 2006).   Some recent studies reported DNA had a strong binding affinity to dentin and hydroxyapatite (Brundin et al. 2013; Brundin et al. 2014). The resultant dentin-bound DNA preserved DNA against natural decomposition and protected DNA against nuclease activity in bacterial culture. Excreted extracellular DNA is a natural part of biofilm development and growth (Whitchurch et al. 2002; Martins et al. 2010; Barnes et al. 2012). The contact of extracellular DNA with dentin in infected root canal may well increase the amount of preserved bacterial DNA. The preserved DNA can be recovered when DNA samples are taken from the root canals.   1.8 Aim of the Study The aim of this study was to compare the in vitro effectiveness of removal of bacterial load from infected root canals, as measured by qPCR, by GentleWave System and an Ultrasonic System. Total bacterial load, as well as the amount of E. faecalis and Streptococcus species were 21  determined before instrumentation, after instrumentation and after the two different energy intensive treatments.  1.9 Hypothesis The null hypothesis is: There is no significant difference between GentleWave System and ProUltra PiezoFlow Active Ultrasonic System in their effectiveness of removing bacteria from infected root canals in vitro.   22  Chapter 2: Materials and Methods 2.1  Tooth Collection and Preparation Ethics permission was obtained from the University of British Columbia Office of Research Services, Clinical Research Ethics Board (certificate number H15-02793). Sample size was calculated based on the pilot study with power of 0.95 using statistical software (G*Power 3.1.9.2, Heinrich Heine, University of Dusseldorf). Fifteen extracted human molars were collected and stored in PBS until use. Any teeth with decay or fractures below the cementoenamel junction, internal or external resorption, open apices, or previous root canal therapy were excluded.   Endodontic access was achieved as per standard practice, and patency was confirmed utilizing a #10 K-file. Working length (WL) was defined as 1 mm from the radiographic apex. The teeth were instrumented to #15/.02 and then cleaned with the GentleWave System to provide the same baseline for all the teeth. Samples were submerged in 10 mL of PBS and autoclaved for sterility at 121°C for 25 minutes. The apices of the roots of all the teeth were sealed using hot glue.  2.2 Inoculation The supragingival and subgingival plaque was collected from interdental spaces of molar teeth of an adult volunteer using sterile wooden sticks. The plaque was suspended in BHI and incubated anaerobically at 37°C for two days. Individual molars were filled with approximately 120 µL of mixed plaque in BHI suspension. Specimens were centrifuged at 3500 g for 5 minutes. The process was repeated 3 times with a fresh solution of bacteria during each centrifugation. All the teeth were incubated in BHI broth for 2 weeks at 37°C in air. 23  2.3 Sampling of the Bacteria from the Canals, First Sample After two weeks of incubation, samples were divided randomly into two treatment groups. The initial sample (S1) was taken before treatment. Teeth were taken out from the BHI broth by using sterile tweezers, the outer surface of the teeth was first carefully wiped with CaviWipes (Metrex Research, Orange, CA, U.S.A) to clean and disinfect the teeth surface. Pulp chamber was carefully dried with sterile cotton pellets without affecting the canals. The samples were taken by sterile paper points (#15, Diadent Group International, Seoul, Korea) inserted into each root canal without touching the wall of the pulp chamber. Several paper points were used until the fluid in the root canal was soaked and the root canal appeared dry. Samples from each canal were collected into separate, sterile 1 mL Eppendorf tubes filled with PBS and frozen at -20°C until used.   2.4 GentleWave Group, Second and Third Sample Seven teeth were allocated into GentleWave group. A #10 (or smaller) hand K-file was inserted into the canals to measure the WL (apical foramen minus 1 mm). Pulp chamber was filled with sterile water and the canals were instrumented with #15 hand K-file (Dentsply Tulsa, Tulsa, OK, U.S.A) to WL followed by Vortex Blue™ Rotary #15/.04 files at 350 rpm. One mL of sterile water was used to irrigate the canals with a 5mL syringe and a 30G side vented needle after the hand and rotary file. The irrigation needle was placed as deep as possible without binding, but not closer than 1 mm from the WL. A #10 hand K-file was employed to verify that WL could be reached after finished instrumentation. Pulp chamber was dried as described above and a second bacteriological sample (S2) was taken from the canals with sterile paper points as above and frozen. The teeth were then treated using the GentleWave System, according to the following 24  protocol: 3 min irrigation using 3% NaOCl, sterile water for 30 sec, 8% EDTA for 2 min. Final irrigation was with sterile water for 15 sec. The pulp chamber was dried as above, and the third sample (S3) was taken from each canal and frozen for later analysis.  2.5 Ultrasonic System Group, Second and Third Sample Eight teeth were divided into Ultrasonic System group. A #10 (or smaller) hand K-file was used to measure WL. Pulp chamber was filled with sterile water and the canals were instrumented with #15 and #20 hand K-file to WL. Vortex Blue Rotary 04 taper files were then used at 350rpm in the following order: #15, #20, #25, #30 and #35, all to WL. One mL of sterile water was used to irrigate the canals between each file. After instrumentation, a second sample (S2) was taken as described above. Final irrigation was performed with the ProUltra PiezoFlow Active Ultrasonic System as follows: 3% NaOCl 1 min per canal, sterile water 10 sec per canal, 8% EDTA 1 min per canal and sterile water 10 sec per canal. After the ultrasonic treatment, the pulp chamber was dried and the final sample (S3) was taken as described above.  2.6 DNA Extraction and Quantitative Real-time PCR Analysis The frozen samples were thawed to room temperature, and DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA) following the protocol recommended by the manufacturer. The final volume of DNA solution of each sample was 150 L and was taken into account during calculation.   The total bacterial load was quantified by using 16S ribosomal RNA gene–targeted qPCR. The levels of Enterococcus faecalis and Streptococcus species (genus specific primer) were evaluated 25  by using specific primers for E. faecalis and Streptococcus species. Table 1 shows the sequences and annealing temperatures of each primer. The qPCR was performed with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) on a StepOnePlus Real-Time PCR Systems (Applied Biosystems, Foster City, CA). The total reaction volume was 20 L. Each reaction included 10 L of Power SYBR Green PCR Master Mix, 6 L of sterile distilled water, 1 L of each 10 M primers and 2 L DNA template. The cycling conditions for universal bacteria and Streptococcus species contained 10 min at 95°C followed by 40 repeats of 95°C for 1 min, annealing for 1 min (temperatures shown in Table 2.1), and 72°C for 1 min. The temperature setting for E. faecalis was 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. Triplicate measurements were done for all samples. Each measurement included triplicate negative controls which contained all reactants but no template DNA. Melt curve analysis was performed after amplification to confirm the specificity of the amplified reaction. The melt curve was detected from 60°C to 95°C. Fluorescence measurements were taken continuously at every 1% increase in temperature. StepOne Software v2.3 (Applied Biosystems) was used to acquire and analyze the data.  Standard curves were constructed by 10-fold diluted DNA extracted from E. faecalis ATCC 29212 and Streptococcus mutans ATCC 25175. The concentration of the pure extracted DNA was quantified using GeneQuant™ pro RNA/DNA Calculator (GE Healthcare, Little Chalfont, UK). Genome copy levels were calculated using the formula m = n(1 mole/6 × 1023 [bp])(660 [g]/ mole) = n(1.096× 10-21 [g/bp]) (Antunes et al. 2015), where m is the genomic mass of a single cell and n is the genome size. Streptococcus mutans ATCC 25175 was also used for total 26  bacteria quantification because it contained 5 copies of 16S rRNA gene and 5 is the approximate average number of 16S rRNA genes which most oral bacteria have (Rocas et al. 2015). 27   TABLE 2.1 Primers Used in Real-time PCR for Bacterial Quantification   * Another primer for E. faecalis previously used in (Rocas and Siqueira 2012) was tried in pilot experiments, but the results showed variation and indicated of less than optimal performance. Therefore, a new primer sequence was chosen based on the analysis of Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and preliminary testing. Target Primer sequence Annealing temp (°C) Reference Universal 16S rRNA gene 5'-CAD ACT CCT ACG GGA GGC-3’ 59 (Dorn-In et al. 2015) 5'-ATC CTG TTT GMT MCC CVC RC-3’ Enterococcus faecalis* 5'-CAA ACT GTT GGC ATT CCA CAA-3’ 60  5'-TGG ATT TCC TTT CCA GTC ACT TC-3’ Streptococcus species 5'-AGA GTT TGA TYM TGG CTC AG-3’ 58 (Rocas and Siqueira 2012) 5'-TTA GCC GTC CCT TTC TGG T-3’ 28   2.7 Statistical Analysis Statistical analysis was performed with SPSS 16.0 software (SPSS Inc, Chicago, IL). The bacterial reduction from S1 to S2 as well as S1 to S3 were compared between two systems for total bacteria, E. faecalis and Streptococcus species, respectively by using Mann-Whitney U test. The significance level was set at P < 0.05.   29  Chapter 3: Results 3.1 qPCR Co-efficiency Test and Specificity  A total of 43 canals were included in this study (17 canals for Gentlewave group and 26 canals for PF ultrasonic group). Three samples (S1, S2, S3) were taken from each canal. There were 129 samples evaluated by qPCR. All runs were taken in triplicate for samples and standards. Standard curve of qPCR Co-efficiency test was used to evaluate the amplification efficiency of each run (Figure 3.1). Purified DNA from standard strains was diluted from 105 copies/L to 10 copies/L to construct the standard curve. The Co-efficiency was calculated based on the slope of the standard curve by using the formula: Efficiency =10(-1/slope)-1. In this study, the average Co-efficiencies for Universal, E. faecalis and Streptococcus species were 94.67%, 98.65% and 92.59%, respectively. Co-efficiency for each run was within the normal range of the Co-efficiency for all qPCR assays (90%-110%).   The regression coefficient was calculated from the regression line in the standard curve. The R2 value indicated the closeness of fit between the standard curve regression line and the individual Ct data points from the standard reaction. A value of 1 indicated a perfect fit between the regression line and the data points. In this study, the average R2 values of all runs for Universal, E. faecalis and Streptococcus species were 0.992, 0.999, and 0.996, respectively, which illustrated a high correlation between the bacterial copy counts and Ct values (Figure 3.1).  30   Figure 3.1. Standard Curve of qPCR Co-efficiency Test. The average Co-efficiencies for Universal, E. faecalis and Streptococcus species were 94.67%, 98.65% and 92.59%. R2 values of all runs for Universal, E. faecalis and Streptococcus species were 0.992, 0.999, and 0.996.  Melt curve of each qPCR assay was used to confirm the specificity of the primers. In this study, melt curve with a single peak and no amplification in negative control were detected in every qPCR assay, which indicated a high specificity of the primers (Figure 3.2).    31   Figure 3.2. Melt Curve of qPCR Assay - qPCR Specificity. Melt curve with a single peak and no amplification in negative control was detected in every qPCR assay, which indicated a high specificity of the primers.  3.2 qPCR Results of Bacterial Quantification Both treatment groups presented a high reduction of bacterial numbers from S1 to S3, as calculated from the number of DNA copies. The reduction by GentleWave System was greater than by the Ultrasonic System in all three bacterial groups (Table 3.1-3.3). In GentleWave System group, a mean number of 4.28×107 total bacterial cell equivalents per canal were detected in S1 samples and significantly decreased in S3 to a mean of 5.58×104 cells per canal (P 32  < 0.001); the mean reduction in total bacterial counts was 99.85% with the range from 99.27% to 99.99%. In Ultrasonic System group, the reduction in the mean number of bacteria from S1 to S3 was from 1.84×107 to 5.13×104 cells per canal, or 97.96%, with individual canals ranging from 85.77% to 99.99%. The difference between GentleWave System and the Ultrasonic System was statistically significant (P = 0.004).   E. faecalis was observed at a mean value of 6.28×105 before instrumentation and 1.35×103 after treatment in GentleWave System group. The mean reduction (S1 to S3) was 99.73% by GentleWave and 97.18% by the Ultrasound (P = 0.008). Table 3.2 shows the mean numbers of E. faecalis per canal and the mean reduction percentages for the two treatment groups.  Slightly smaller but still statistically significant difference between the two cleaning methods was measured in the Streptococcus group (genus specific primer), 99.61% for GentleWave and 98.53% for the Ultrasonic System (P = 0.028) (Table 4).  There was no significant difference in the decrease of the microbes between the two groups after mechanical instrumentation only (S1 to S2) (P > 0.05) (Tables 3.1-3.3). 33  TABLE 3.1 Level of Total Bacteria in Root Canal Samples of Teeth Taken Before Instrumentation (S1), After Instrumentation (S2) and After GentleWave / PF Ultrasonic System (S3) (Mean ± Standard error). The difference of reduction% from S1 to S3 between GentleWave System and the Ultrasonic System was statistically significant (P = 0.004)    Groups N (Canals) Mean (copies/canal) ± SE Reduction% S1 to S2 Reduction% S1 to S3 S1 S2 S3            Mean ± SE Mean ± SE        Range GentleWave  17 4.14×107±9.26×106 8.58×106±4.23×106 5.48×104±1.57×104 85.55%±3.16% 99.85%±0.04% 99.27% - 99.99% PF Ultrasonic  26 1.85×107±4.41×106 4.17×106±1.54×106 5.97×104±1.90×104 81.70%±2.20% 97.96%±0.71% 85.77% - 99.99% 34  TABLE 3.2 Level of Enterococcus faecalis in Root Canal Samples of Teeth Taken Before Instrumentation (S1), After Instrumentation (S2) and After GentleWave / PF Ultrasonic System (S3) (Mean ± Standard error). The difference of reduction% from S1 to S3 between GentleWave System and the Ultrasonic System was statistically significant (P = 0.008)    Groups N (Canals) Mean (copies/canal) ± SE Reduction % S1 to S2 Reduction% S1 to S3 S1 S2 S3 Mean ± SE Mean ± SE Range GentleWave 17 6.28×105±1.70×105 1.56×105±3.60×104 1.35×103±3.30×102 73.73%±4.69% 99.73%±0.08% 99.19% - 99.99% PF Ultrasonic 26 3.39×105±1.11×105 8.63×104±1.72×104 1.79×103±5.50×102 66.59%±4.85% 97.18%±0.83% 84.21% - 99.99% 35  TABLE 3.3 Level of Streptococcus species in Root Canal Samples of Teeth Taken Before Instrumentation (S1), After Instrumentation (S2) and After GentleWave / PF Ultrasonic System (S3) (Mean ± Standard error). The difference of reduction% from S1 to S3 between GentleWave System and the Ultrasonic System was statistically significant (P = 0.028)    Groups N (Canals) Mean (copies/canal) ± SE Reduction % S1 to S2 Reduction% S1 to S3 S1 S2 S3 Mean ± SE Mean ± SE Range GentleWave  17 3.10×104±1.36×104 4.74×103±9.71×102 3.15×101±9.46×100 65.63%±5.40% 99.61%±0.21% 96.31% - 99.99% PF Ultrasonic 26 1.02×104±2.95×103 2.68×103±6.39×102 4.05×101±8.86×100 64.69%±5.13% 98.53%±0.58% 86.02% - 99.99% 36  Chapter 4: Discussion  Treatment with both GentleWave multisonic and ProUltra PiezoFlow ultrasonic systems greatly reduced the bacterial load in the canals. The average reduction of total bacteria, streptococci and E. faecalis was 99.61 – 99.85 % in the GW group and 97.18 - 98.53 % in the ultrasound group. There were no differences in bacterial reduction between canals from teeth with two, three or four canals (data not shown). The change in bacterial counts in the three measured bacterial groups (total bacteria, E. faecalis, streptococci) from S1 (start) to S2 (after mechanical instrumentation), before cleaning using the two energy-driven systems was not significantly different between the two groups (Table 3.1- 3.3).  When the results are evaluated based on the magnitude of reduction in bacterial DNA, the differences between the average reductions in each group may seem relatively small, although statistically significant. However, from the point of view of elimination of the endodontic infection and healing of the periapical lesion, it is the number of residual bacteria that is likely to be more important rather than the mere amount of reduction in numbers. In canals cleaned with the GW system, an average of 0.15% of the original flora was still found. In PF treated canals the average residual flora was 2.04% of the original flora. When these numbers are compared between the two groups, it shows that more than ten times as many bacteria are expected to be found in PF cleaned than in GW cleaned canals.   So far, no treatment strategy, cleaning method or antibacterial agent and medicament have been able to predictably provide sterile canals at the end of the chemomechanical treatment, before the root canals are filled. While the results of this study cannot be directly compared to earlier studies, 37  particularly those where bacterial culture has been used to quantitate the number of bacteria, the results do indicate that the GW cleaning is moving the bar closer to ideal (no bacteria) than previously obtainable. Recent, short term healing results one year after the root canal treatment using  GW cleaning showed very high success rates (Sigurdsson et al. 2016), which might be explained at least partly by the excellent results of the present qPCR study of bacterial elimination. However, it is important to emphasize that more long term in vivo clinical studies are needed on healing of apical periodontitis after GW treatment. Also, the results of the present study must be confirmed by others, both by ex vivo and in vivo studies.   While the overall performance of each cleaning system, i.e. the average reduction of microbiota in the root canal system is important, there is another factor which is at least equally important. The range of reduction of total bacterial counts in the individual canals in the GW group was much smaller (99.27% - 99.99%) than in the PF group (85.77% - 99.99%). The result indicates that GW much more predictably achieved a high level of removal of microbes from the root canal system.  There is a growing consensus in endodontics that while complete sterility may not be achievable, there is a threshold value for the number of residual microbes, below which healing will occur. It is also possible or even likely that this value is different in individual cases. Based on this kind of hypothesis one can speculate that the high predictability of bacterial reduction by the GW system (very low range and standard deviation), increases the probability that the strong effect brings the residual flora below the threshold value, more often that e.g. with the PF system. It is clear that it is difficult or impossible to make a study designed to directly determine the threshold value of bacteria for predictable healing. However, future in vivo studies of healing combined with analysis 38  of the residual microflora with sensitive molecular methods, may give indirect information on this topic.   One of the main benefits of in vitro studies is that it is usually possible to standardize the experimental conditions fairly well for different groups and thereby minimize the effect of confounding factors. In the present study, this was not possible to the same extent as in many other in vitro studies because of the different type of action of the systems as well as recommendations for use by the manufacturers (Zhu et al. 2013; Al Shahrani et al. 2014). The PF system is supposed to be used after conventional size instrumentation (#35/.04 in this study), which allows the tip to be placed freely in the coronal third of the canal (Castelo-Baz et al. 2016). To secure maximal efficacy, the tip must avoid contact with canal walls. The GW system, on the other hand, is designed to work on minimally or even uninstrumented canals, as instead of being inserted into the canal the tip of the instrument is placed in the pulp chamber, just above the chamber floor throughout the treatment. For this reason, the canal dimensions between the groups or within the GW group were not standardized. Another key difference is that PF is used in each canal separately, whereas GW circulates the irrigant simultaneously in all root canals. In other words, PF system provides active ultrasonic cleaning in one canal while the other canals at the same time are more passively exposed to the irrigant. With GW, all canals are actively targeted all the time. The experimental design in the present study was made to conform with the recommended use of the systems rather than testing the ultimate cleaning power of the two in identical conditions, i.e. same canal size and same active time of cleaning per canal facilitated by the multisonic and ultrasonic energy. Therefore, conclusions from the results must be drawn with particular caution. The NaOCl irrigation per tooth was limited to 3 min with GW and 3 or 4 min with PF. If the active ultrasonic 39  (PF) irrigation with NaOCl had been 3 min per canal, the total NaOCl irrigation per a molar in the present study would have been 9 or 12 minutes, depending on the number of canals (3 or 4), instead of the 3 or 4 minutes now used. Nine to twelve-minute ultrasonic irrigation is not realistic in any clinical situation, in addition, would also come the time for water and EDTA irrigation, multiplied by the number of canals. Corresponding studies with GW and other cleaning and treatment methods in the future are likely to set the focus in different ways, which may result in different experimental designs.  Sampling was done with sterile paper points from the root canals. It is generally accepted that paper point sampling is not the most effective way of collecting all microbes from the root canal system (Antunes et al. 2015). Cutting off the roots and pulverizing them for DNA extraction is likely to yield more bacteria in the samples (Tran et al. 2013). However, when whole roots are sampled, microbes and even residual DNA from root surface, contaminated e.g. during extraction, handling and incubating the root canal microbiota would increase the risk for an error. The exterior apical third of the roots in the present study was sealed with hot glue to prevent the transfer of microbes between the root surface and the root canal system. Another point worth noting is the different volume of the canals in the two groups. One can argue that from a larger canal more bacteria can be picked than from a narrow canal. It is also possible that contrary to the previous claim removal of more dentin by instruments in the PF group mechanically removed the bacteria-rich zones around the main canal, which was the sampling area, and improved the effectiveness of the PF system due to better flow of the irrigants. Nevertheless, the canal dimensions in both groups in samples S2 and S3 remained constant throughout the experiment, and the changes (reduction) in bacterial counts were calculated within each group. 40  Previous histological studies have indicated that both PF and GW effectively clean hard to reach areas in the root canal system from organic matter (Molina et al. 2015). A micro-CT study with molar canals filled with calcium hydroxide (CH) showed that GW was the only system studied which completely removed the CH paste even from lateral canals and isthmus areas in teeth instrumented to conventional preparation sizes (Ma et al. 2015). PUI was one of the methods in this and other studies that was not able to remove all calcium hydroxide. So far, PF has not been examined in a corresponding setting. The present study showed that GW removed bacteria at a high level of predictability in minimally (#15/.04) instrumented molar canals. The reduction in the amount of total bacterial DNA was in every case at least 99.27%. This is an important finding as effective disinfection is one of the key requirements for minimal instrumentation in endodontics to claim sound scientific basis. Preservation of root dentin can be supposed to contribute to maintaining stronger root and tooth structure than previously possible with conventional instrumentation sizes. Other studies are needed to address technologies and quality of root fillings in minimally instrumented root canals, another key requirement.   41  Chapter 5: Limitations of the Study In the present study, sampling was done by paper points, which was considered to be the most suitable method for this study. However, paper point sampling may not optimally reflect the microbiological status in the whole root canal system, especially in the isthmuses and lateral canals. A previous micro-CT study has shown that GW was the only system tested which completely removed a calcium hydroxide paste even from lateral canals and isthmus areas in all molar teeth examined (Ma et al. 2015). It is therefore logical to speculate that GW has the potential to eliminate the microbes in areas unreachable for paper points in root canal system, and bring residual bacteria from the peripheral areas to the main canal, where they may be sampled with the paper points.   The binding affinity of DNA to dentin is another potential concern. Since the present study employed a quantitative qPCR method, the DNA preserved earlier in the root dentin could be recovered when the samples were taken from the root canals. Therefore, this is a factor that should be taken into consideration when designing sample taking procedures for qPCR analysis. Sampling with paper points can minimize the amount of extracellular DNA from dentin as the points cannot penetrate into dentin, whereas dentin/root pulverizing method would include all dentin bound DNA in the sample. Variation in canal anatomy and different length of the roots may also cause differences in the sample volume. However, S1, S2 and S3 of one tooth were taken within the same canal each and compared to each other. Thus, reduction in DNA/bacterial counts were calculated each time within one tooth.  42  Chapter 6: Conclusion In conclusion, within the limitation of this ex vivo qPCR study, both PiezoFlow ultrasonic and GentleWave systems effectively reduced the level of bacteria in the root canals of molar teeth. GentleWave System showed more predictably a constantly higher level (> 99%) of bacterial reduction than the Ultrasonic System. Additional in vivo studies are also needed for further investigations into the antibacterial effectiveness of the PiezoFlow Ultrasonic and the GentleWave Systems in the treatment of endodontic infections. 43  Chapter 7: Future Directions qPCR is a sensitive and rapid technique to quantify microbes. However, its main limitation is its inability to discriminate between live and dead cells. A method using propidium monoazide (PMA) together with qPCR has been developed to overcome this problem, with reportedly promising results for different bacterial species in different types of samples (Alvarez et al. 2013). In the present study, the effectiveness of removing bacteria (live and dead) was evaluated. A future direction may be using qPCR to detect and compare the ability of different techniques / irrigants in removing and killing microbes.  GW is designed to work on minimally or even uninstrumented canals. 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