Dentistry, Faculty of
DSpace
UBCV
Alboloshi, Elaf
Master of Science - MSc
University of British Columbia
Objectives: The objective of this study was to assess the antimicrobial efficacy of Triton, an all-in-one dual-action endodontic irrigation solution used either independently or sequentially after NaOCl, against multispecies biofilms in dentin canals, with and without the smear layer. Additionally, we aimed to compare its effectiveness with traditionally used solutions (NaOCl / EDTA). Methods: We prepared ten infected dentin specimens with a smear layer and ten specimens without a smear layer. Utilizing a previous protocol, we introduced multispecies bacteria into dentinal tubules. Following a 3-week incubation period, the infected dentin blocks underwent exposure to various irrigating solutions, including sterile water, 2% sodium hypochlorite (NaOCl), 2% NaOCl / 17% EDTA, Triton, and 2% NaOCl / Triton. The proportions of live and dead bacteria were analyzed using confocal laser scanning microscopy and viability staining. Results: The Triton groups (Triton and 2% NaOCl / Triton) exhibited significantly higher bacterial killing compared to 2%NaOCl / EDTA, irrespective of the presence of the smear layer. Furthermore, a more pronounced reduction in bacterial load was observed in groups without a smear layer compared to those with a smear layer. Conclusions: The Triton groups (Triton and 2% NaOCl / Triton) demonstrated the most effective bacterial killing, irrespective of the presence of the smear layer. Triton, the all-in-one, dual-action endodontic irrigation solution, whether used independently or in sequence after 2%NaOCl, displayed superior antimicrobial effects against 3-week-old multispecies biofilms in dentin canals compared to traditionally used 2%NaOCl / EDTA irrigation solutions. The combined application of Triton after 2%NaOCl revealed the highest bacterial killing. This study underscores Triton's heightened and synergistic antimicrobial effect when employed as a final rinse following 2%NaOCl.
ANTIMICROBIAL EFFECTIVESNESS OF AN ALL-IN-ONE ENDODONTIC IRRIGATION SOLUTION (TRITON): AN IN VITRO STUDY by Elaf Alboloshi BDS, King Abdulaziz University, 2018 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) May 2024 © Elaf Alboloshi, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Antimicrobial effectiveness of an all-in-one endodontic irrigation solution (Triton): an in vitro study submitted by Elaf Alboloshi in partial fulfilment of the requirements for the degree of Master of Science in Craniofacial Science Examining Committee: Dr. Ya Shen. Professor, Department of Oral Biological & Medical Sciences, UBC Supervisor Dr. Jeffrey Coil. Assistant Professor, Director of the Graduate Endodontic Program, Department of Oral Biological and Medical Sciences, UBC Supervisory Committee Member Dr. Ahmed Hieawy. Clinical Associate Professor, Department of Endodontics, UBC Supervisory Committee Member Dr. Jolanta Aleksejūnienė. Associate Professor, Department of Oral Health Sciences, UBC Additional Examiner iii Abstract Objectives: The objective of this study was to assess the antimicrobial efficacy of Triton, an all-in-one dual-action endodontic irrigation solution used either independently or sequentially after NaOCl, against multispecies biofilms in dentin canals, with and without the smear layer. Additionally, we aimed to compare its effectiveness with traditionally used solutions (NaOCl / EDTA). Methods: We prepared ten infected dentin specimens with a smear layer and ten specimens without a smear layer. Utilizing a previous protocol, we introduced multispecies bacteria into dentinal tubules. Following a 3-week incubation period, the infected dentin blocks underwent exposure to various irrigating solutions, including sterile water, 2% sodium hypochlorite (NaOCl), 2% NaOCl / 17% EDTA, Triton, and 2% NaOCl / Triton. The proportions of live and dead bacteria were analyzed using confocal laser scanning microscopy and viability staining. Results: The Triton groups (Triton and 2% NaOCl / Triton) exhibited significantly higher bacterial killing compared to 2%NaOCl / EDTA, irrespective of the presence of the smear layer. Furthermore, a more pronounced reduction in bacterial load was observed in groups without a smear layer compared to those with a smear layer. Conclusions: The Triton groups (Triton and 2% NaOCl / Triton) demonstrated the most effective bacterial killing, irrespective of the presence of the smear layer. Triton, the all-in-one, dual-action endodontic irrigation solution, whether used independently or in sequence after iv 2%NaOCl, displayed superior antimicrobial effects against 3-week-old multispecies biofilms in dentin canals compared to traditionally used 2%NaOCl / EDTA irrigation solutions. The combined application of Triton after 2%NaOCl revealed the highest bacterial killing. This study underscores Triton's heightened and synergistic antimicrobial effect when employed as a final rinse following 2%NaOCl. v Lay Summary The root canal system has a complex anatomy, and when infected, disinfection of the system is prudent for the success of root canal treatment. Disinfection heavily depends on irrigation with antimicrobial solutions. The solutions used in root canal treatment should possess all the requirements to be able to kill and remove the bacteria. However, to date, no such solution has been shown to be optimal. The use of sodium hypochlorite and EDTA is the most popular irrigation regimen used by the majority of endodontists. Various solutions have been proposed to function as well as or better than this regimen. In this study, the antimicrobial effectiveness of Triton, an all-in one dual-action endodontic irrigation solution was investigated when used alone or in sequence after sodium hypochlorite. vi Preface The project in this thesis ‘‘Antimicrobial effectiveness of an all-in-one endodontic irrigation solution (Triton): an in vitro study.’’ has been approved by the University of British Columbia Clinical Research Ethics Board (Certificate H15-02793). The thesis work, including experiments, data analysis, and thesis writing was performed exclusively by the candidate Elaf Alboloshi. Statistical analysis was done with the guidance and assistance of Dr. Jolanta Aleksejuniene. Dr. Ya Shen, supervisor, contributed to the study design and editing. Committee members, Dr. Ahmed Hieawy and Dr. Jeffrey Coil have contributed to thesis editing. The relative contribution to this research project by Dr. Alboloshi was 90%. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ..................................................................................................................................x List of Figures ............................................................................................................................... xi List of Symbols ............................................................................................................................ xii List of Abbreviations ................................................................................................................. xiii Acknowledgements .................................................................................................................... xiv Dedication .....................................................................................................................................xv Chapter 1: Introduction ................................................................................................................1 1.1 Root canal treatment and biofilm removal ...................................................................... 1 1.2 Biofilms in endodontics .................................................................................................. 2 1.3 Irrigation ......................................................................................................................... 2 1.4 Sodium hypochlorite (NaOCl) ........................................................................................ 4 1.4.1 Mechanism of action of NaOCl .............................................................................. 5 1.4.2 NaOCl concentrations ............................................................................................. 6 1.4.3 Effect of high concentrations of NaOCl on dentin ................................................. 6 1.5 Smear layer and decalcifying solutions .......................................................................... 7 1.6 Ethylenediaminetetraacetic acid (EDTA) ....................................................................... 7 1.6.1 Mechanism of action of EDTA ............................................................................... 8 viii 1.6.2 NaOCl and EDTA ................................................................................................... 8 1.7 Triton ............................................................................................................................... 9 1.8 Dentin block (study model) .......................................................................................... 11 Chapter 2: Aims and Hypotheses ...............................................................................................12 2.1 Aims and objectives ...................................................................................................... 12 2.2 Study hypotheses .......................................................................................................... 12 Chapter 3: Materials and Methods ............................................................................................13 3.1 Sample size calculation ................................................................................................. 13 3.2 Tooth selection and dentin specimen preparation ......................................................... 13 3.3 Dentin specimen infection ............................................................................................ 15 3.4 Dentin disinfection and smear layer production ........................................................... 16 3.5 Groups ........................................................................................................................... 17 3.6 Pre-scan preparation ...................................................................................................... 17 3.7 Confocal laser scanning microscopy (CLSM) examination ......................................... 19 3.8 Data analysis ................................................................................................................. 19 3.8.1 Independent sample t-test ..................................................................................... 19 3.8.2 One-way ANOVA with post-hoc Scheffé ............................................................ 20 Chapter 4: Results ........................................................................................................................21 4.1 Comparison of sensitivity among groups with a smear layer and those without .......... 21 4.2 Comparison between Triton and 2% NaOCl ................................................................ 23 4.3 Comparing the antimicrobial effect of different solutions ............................................ 24 4.4 Confocal imaging of stained bacteria in the dentin block model .................................. 26 Chapter 5: Discussion ..................................................................................................................28 ix 5.1 Study Discussion ........................................................................................................... 28 5.2 Limitations .................................................................................................................... 31 5.3 Future directions ........................................................................................................... 31 Chapter 6: Conclusion .................................................................................................................32 References .....................................................................................................................................33 x List of Tables Table 1.1. Characteristics of an ideal endodontic irrigation solution, described by Basrani & Haapasalo ………………………………………………………………………………………....4 Table 1.2. Ingredients of Triton solution used in this study………………………………..…...10 Table 2.1. Table showing the 10 experimental groups and exposure times………....…......……17 Table 4.1. Mean percentages of dead bacteria in smear layer and no smear layer groups………21 Table 4.2. Proportion of killed bacteria (dead cell volume%) in dentin specimens with and without smear layer after irrigation with different disinfecting solutions………………….........23 Table 4.3. Bacteria in different antimicrobial solutions………………………………………....24 Table 4.4. Comparisons between the proportions of killed bacteria in all groups…………….....25 xi List of Figures Figure 1.1. Triton: the all-in-one dual-action endodontic irrigation solution (Brasseler; Savannah, US) ……………………………………………………………………………………..…….…...9 Figure 2.1. An example of a sectioned sample showing (a) the ground and refined outer surfaces and (b) the inner surface showing the canal space (arrow)………………………………………14 Figure 2.2. Dentin specimen in the filter tube (a) before and (b) after sealing with composite…15 Figure 2.3. Dental specimen after removal from the filter tube and application of varnish….….16 Figure 2.4. Flow chart illustrating the experimental design of this study………………..………18 Figure 4.1. Bacteria killing among different groups of antimicrobial solutions…………...…….22 Figure 4.2. Confocal imaging (CLSM) showing viability-stained biofilms confirming biofilm penetration into dentinal tubules (green) and the antimicrobial effects (red) of all solutions used………………………………………………………………………………………………27 xii List of Symbols α Alpha β Beta °C Degree Celsius µ Micro p P-value xiii List of Abbreviations AAE American Association of Endodontics ANOVA Analysis of variance BHI Brain heart infusion CFU Colony forming unit CLSM Confocal laser scanning microscopy EDTA Ethylenediaminetetraacetic acid EPS Extracellular polymeric substance NaOCl Sodium hypochlorite OD Optical density rpm Revolutions per minute Sd Standard deviation SEM Scanning electron microscopy Triton all-in-one, dual-action endodontic irrigation solution xiv Acknowledgements I would like to convey my enduring gratitude to my supervisor, Dr. Shen, for without your mentorship and guidance, this study would not have existed. Also, I would also like to extend my gratitude to Dr. Ahmed Hieawy and Dr. Jeffrey Coil for their input and thoughts for this study and throughout the program. I would also like to thank Dr. Jeffrey Coil for giving me the opportunity of joining this program. I also owe special thanks to my colleagues, members of Dr. Shen’s lab; Dr. Alaa Zaghwan, Dr. Mohamed Amhmed, and Dr. Abdullah AlMegbel for their guidance and assistance in the lab. I thank all of UBC graduate endodontic instructors for their time and effort spent on teaching and guiding us. Finally, I thank Lois Bermudez, Francisco Briseno H, and my co-residents for providing a healthy and friendly atmosphere and making this journey easier for me. xv Dedication To my lovely family; my devoted parents and my siblings. I am forever grateful for their limitless love, support, and encouragement. A special dedication to the source of support in my life, my backbone, my mother, Fatma Wali. My gratitude is unconditional and will forever be insufficient. Lastly, I thank my grandmother and grandfather; Zainab Wali and Abdulrahim Wali, the pillars of our family, a source of kindness and affection in my life. . 1 Chapter 1: Introduction 1.1 Root canal treatment and biofilm removal Root canal treatment is a procedure done to treat or prevent apical periodontitis. Apical periodontitis is defined as “inflammation and destruction of apical periodontium that is of pulpal origin, radiographically might or might not be associated with an apical radiolucent area.” (1). In order for inflammation to occur, bacteria in the canal system needs to reach the apical area (2). It has also been proven that bacteria and their by-products are the main cause of peri-radicular infections (2, 3). Root canal treatment consists of a triad which includes biomechanical instrumentation, 3-dimensional cleaning, and filling the root canal system.(4) The aim of the biomechanical instrumentation is to remove the infected tissues and dentin from the canal system; this allows and creates adequate space for irrigation. Irrigation is the chemical part of root canal treatment. The aim of irrigation is to reduce and kill the number of bacteria present in the canal system. Mechanical instrumentation alone is insufficient for removing and cleaning the pulpal space and reaching all the canal irregularities. Biofilms have been found in isthmuses, lateral and accessory canals, and ramifications invading deep into the dentinal tubules (5). Moreover, it has been quantified that up to 55% of the root surfaces remain untouched during instrumentation (6). Chemical cleaning is needed as an adjunct to mechanical instrumentation to kill bacteria, remove the biofilms, flush debris out, and remove the smear layer from all the irregularities of the canal system. Therefore, it has been stated that both chemical and mechanical cleaning is essential and important to successfully achieve the goal of root canal treatment (7). 2 1.2 Biofilms in endodontics When a tooth becomes necrotic due to infection, it becomes an ideal environment for microbes to survive and grow (8). Bacteria in the canal form biofilms in which they are densely layered (up to 300 layers) and attached together on the tooth surface (9). A biofilm is defined as a group of microorganisms that are stable, stationary, attached to each other on a surface, and produce a matrix that surrounds them (extracellular polymeric substance; EPS) (10). This matrix provides stability, adhesion, and nutrition to the biofilm and protects the biofilm from antimicrobial agents and the host cells (11). It has been proven that biofilms take up to 10 days to mature (12). Despite this, in terms of structural development, it has also been shown that it takes 3 weeks for dental biofilms to mature (13). The more mature the biofilm gets, the more resistant it becomes (13). Bacteria in biofilms are up to 1000-fold more resistant to antimicrobials than planktonic bacteria (14). There are several possible proposed mechanisms by which biofilms resist antimicrobials. Firstly, the EPS plays an important role in resistance by creating a barrier that delays antimicrobial penetration into the biofilm (15). In addition, microbes located in the deeper layers of the biofilm exert several characteristics in order to develop tolerance to antimicrobials, and the biofilm has a communication system involving a mechanism known as quorum sensing. Lastly, the biofilm produces persister cells that remain dormant and resist antimicrobials (16). 1.3 Irrigation Irrigation in endodontics is defined by the American Association of Endodontics (AAE) as “washing by a stream of fluid; intracanal irrigation facilitates physical removal of materials from the canal and introduction of chemicals for antimicrobial activity, demineralization, tissue dissolution, bleaching, deodorizing and hemorrhage control.” (1). Several irrigation solutions 3 have been proposed for use in disinfecting the root canals during treatment. The characteristics of an ideal irrigation solution have been described by Basrani & Haapasalo in 2012 (17). The solution must be biocompatible, nontoxic, not caustic or irritating to peri-radicular tissues, non-carcinogenic, and does not cause anaphylaxis, but have the ability to kill bacteria and fungi. The irrigation solution should not adversely affect the tooth structure, but it should have the ability to penetrate deep into the dentinal tubules. Table 1.1 further describes all the characteristics of an ideal endodontic irrigation solution. An optimal irrigation solution should have all the positive properties and none of the negative ones. The irrigation solution should also be able reach the infected areas that hasn’t been instrumented in teeth with complex canal anatomy. None of the currently available solutions are optimal. However, a combination of products in the correct sequence contributes to a successful treatment outcome. Due to their characteristics, the most commonly used irrigation solutions in endodontics are sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid (EDTA). 4 Table 1.1 Characteristics of an ideal endodontic irrigation solution, described by Basrani & Haapasalo (17). Effective germicide and fungicide Non-irritating to the periapical tissues Stable in solution Prolonged antimicrobial effect and a sustained antibacterial effect after use Active in the presence of blood, serum, and protein derivatives of tissue Able to completely remove the smear layer Low surface tension Able to disinfect the dentin/dentinal tubules Does not interfere with repair of periapical tissues Does not stain tooth structure Inactivation in a culture medium Does not induce a cell-mediated immune response. Is non-antigenic, non-toxic, and non-carcinogenic to tissue cells surrounding the teeth Has no adverse effects on the physical properties of exposed dentin Has no adverse effect on the sealing ability of filling materials Easy to use/apply Inexpensive 1.4 Sodium hypochlorite (NaOCl) The AAE describes NaOCl as being a “clear, pale, greenish-yellow, strongly alkaline liquid with a strong chlorine odor; has a solvent action on organic tissue and is a potent antimicrobial agent; used for irrigating root canals either in full strength or diluted solutions” (1). It was first 5 introduced into endodontics by Coolidge in 1920 (18) and is one of the most commonly used irrigation solutions in endodontic treatments. It is the irrigation solution of choice due to its low cost, strong antimicrobial effect, and ability to dissolve pulpal tissue and the organic part of dentin (19). Nevertheless, NaOCl is highly toxic, corrosive, and does not remove the inorganic part of the smear layer. NaOCl also has a minimal killing effect on Enterococcus faecalis (17). NaOCl also gets buffered and the pH becomes neutral when it contacts dentinal debris, this lowers the bacterial killing ability of NaOCl (20). Although, this drawback is overcome by using EDTA or increasing the concentration of NaOCl. Another drawback is its relatively high surface tension, which prevents its penetration into the dentinal tubules or its flow into the isthmuses and irregularities of the canal system (21); this is overcome by using it in combination with other chelators (22) or using it in sequence with EDTA(23). 1.4.1 Mechanism of action of NaOCl NaOCl, when in contact with pulpal tissues and microbes, reacts via three main chemical reactions: saponification, neutralization, and chloramination (24). The organic tissue dissolution property of NaOCl is demonstrated by the first reaction, saponification (24). Saponification also decreases the surface tension of the solution (24). It occurs by degrading fatty acids resulting in a soap (fatty acid salts) and glycerol (alcohol) (24). The neutralization reaction occurs when NaOCl contacts amino acids, resulting in water, salt, and the uptake of hydroxyl ions; this leads to a pH reduction (24). Chloramination occurs when hypochlorous acid contacts organic tissues, releasing chlorine, which reacts with a protein amino group, forming chloramines (24). The chloramines interfere with the cellular metabolism of the microbes, causing irreversible 6 inactivation by degrading amino acids and causing hydrolysis (24). Thus, infected and necrotic tissues are dissolved and removed, enabling the solution to reach deeper into the canals (17). 1.4.2 NaOCl concentrations Currently, there is no shared agreement on the best concentration of NaOCl to be used for irrigation. However, clinicians usually lean towards using higher concentrations to achieve better disinfection (25). There have been studies showing the antimicrobial effect of different concentrations of NaOCl, including one that has shown no difference in the antimicrobial effect of low (0.5%) vs. higher (5%) concentrations (26). Nonetheless, higher tissue dissolution was found when higher concentrations were used (27). On the other hand, there have been studies showing that higher concentrations (5.25%–6%) demonstrated significantly greater antimicrobial effects than lower concentrations (2%–2.5%) (28, 29). 1.4.3 Effect of high concentrations of NaOCl on dentin Despite all the studies that show the greater antimicrobial effect of higher concentrations of NaOCl, there have been studies that show the detrimental effect of its use on tooth structure. A study done on bovine teeth has shown that 6% NaOCl decreases dentin microhardness more than 2% NaOCl (30). Another study found that 3% and 5% NaOCl significantly decreases both the flexural strength and modulus of elasticity of human dentin (31). It has been proven that the higher the concentration of NaOCl, the more adverse the effect on the mechanical properties of dentin (32). Not only are the elastic modulus and flexural strength affected by the higher concentration, but its toxicity also increases (33). All these studies were done in vitro and have not been tested clinically, but it does raise the question of whether this increases the risk of 7 vertical root fracture. Some additional methods to improve the clinical effectiveness and killing rates of NaOCl are the use of higher volumes of lower concentration NaOCl, increasing the temperature, activation of the solutions, and using surface active agents (27, 34, 35). 1.5 Smear layer and decalcifying solutions A smear layer is formed during root canal instrumentation. It consists of organic and inorganic components which includes pulpal tissues, dentin debris, bacteria, bacterial toxins, bacterial enzymes, and bacterial byproducts (36). There has been speculation that this layer retains microorganisms (36, 37), occludes the dentinal tubules during instrumentation which impedes the removal of the bacteria and debris (38), and prevents the adhesion of sealers and obturation materials (37, 39, 40). During irrigation with NaOCl, the organic component of the smear layer is removed, leaving the inorganic component. Decalcifying agents or chelators are known to be used to remove the inorganic component (37). Several decalcifying solutions are used in endodontics, including citric acid and EDTA (17), with EDTA being one of the most commonly used decalcifying solutions among endodontists. 1.6 Ethylenediaminetetraacetic acid (EDTA) Ethylenediaminetetraacetic acid (EDTA) is a polycarboxylic amino acid. It was first described in 1935 by Ferdinand Munz and introduced into endodontics in 1957 by Nygaard-Østby (41). It is a colourless, water-soluble chelating solution that is usually used at a concentration of 17%. The AAE defined EDTA as “an odorless, white, crystalline solid whose various salts are soluble in water; disodium salt of ethylenediaminetetraacetic acid in a buffered aqueous solution is used as 8 a chelating agent in root canal preparation; used to remove calcium, demineralize and soften dentin, and remove the smear layer” (1). 1.6.1 Mechanism of action of EDTA EDTA works by binding to the calcium ions in the inorganic part of the smear layer and forming complexes that are removed with irrigation (41). This opens up the dentinal tubules and increases the permeability of the antimicrobial solutions into the deeper parts of the dentinal tubules (38, 42). Once formed, these complexes are stable, and when all ions have been bound, no further complexes are formed; this explains the self-limiting characteristic of EDTA (43). EDTA is biocompatible with tissues and has limited antimicrobial effects when used alone (23). However, it causes cell wall damage and disrupts the membrane in Gram-negative bacteria by binding to metal ions (Ca2+ and Mg2+) on the cell envelope, rendering it permeable to the antimicrobial solutions (44). When the cation (Ca2+ and Mg2+) concentration decreases, the water solubility of the EPS of the biofilm increases causing biofilm disruption (45). 1.6.2 NaOCl and EDTA It has been proven that the sequential use of NaOCl/EDTA effectively removes the smear layer (46). Also, it have proven that the tissue dissolving capacity and the amount of chlorine from NaOCl is reduced and inhibited by EDTA when both solutions are combined and mixed (47). However, EDTA, when mixed with NaOCl, retains its ability to chelate Ca2+ (47). This finding led to the clinical suggestion advising against mixing both solutions together (17). It is known that NaOCl removes the organic part of the smear layer and the collagen part of dentin, while EDTA removes the inorganic part and hydroxyapatite. It was shown that this combined effect, in 9 the form of 5.25% NaOCl/17% EDTA, increased the dentin strain and changed the viscoelastic properties of dentin (48). It has been recommended that irrigation with NaOCl throughout the instrumentation phase and finishing with a final flush of 17% EDTA followed by 5.25% NaOCl is effective in removing the smear layer (49). The alternating action between NaOCl and EDTA causes dissolution of dentin around the dentinal tubules, where NaOCl dissolves the organic part of dentin and EDTA demineralizes the organic part (46). To minimize any erosive effects, it was recommended that EDTA application to the root canal system should not exceed 1 minute (50). Another study showed that the application of 5.25% NaOCl following EDTA irrigation causes extensive erosion of root canal dentin and should be avoided (51). Moreover, this erosion may be a contributing factor to vertical root fracture (52). 1.7 Triton Triton is an all-in-one dual-action endodontic irrigation solution introduced by Brasseler (Savannah, US) in 2021 (see Figure 1.1) that comes in a two-part solution where the bottle has two separate components (Parts A and B). It has an auto-mix cap that mixes both parts upon drawing out the solution. Part A is comprised of surfactants, chelators, stabilizers, pH modifiers, and water. Part B is comprised of 8% NaOCl, water, and a pH modifier. According to the manufacturer, when both parts are combined, the resultant mixture is a 4% NaOCl solution with other components that are patent pending and have not been disclosed (Table 1.2). These components consist of 14 chelators and surfactants. Furthermore, the components do not buffer the NaOCl to a point that it becomes ineffective in organic tissue dissolution. Triton differs from other irrigation solutions by using a single irrigation throughout the procedure. This shortens the chairside time and precludes the need for using several solutions and syringes. 10 Figure 1.1. Triton: the all-in-one, dual-action endodontic irrigation solution (Brasseler; Savannah, US). The manufacturer claims that this solution is the first to allow removal and dissolution of both the organic and inorganic tissues. The non-NaOCl components of this solution allow for the use of a lower concentration of NaOCl (4%), which removes the smear layer and dissolves the organic tissue in a more effective manner compared to the use of NaOCl and EDTA. This solution follows the concept of continuous chelation. This concept was introduced to combine the NaOCl benefits with chelating solutions to remove inorganic parts of the smear layer and to disinfect and dissolve the organic part (22). A previous study has shown that this concept results in removal of the smear layer in a more homogenous manner than the sequential use of NaOCl and EDTA (53). Despite the potential benefits, limited studies are available regarding Triton, its antimicrobial and antibiofilm effect, and its efficiency in smear layer removal. 11 Table 1.2. Ingredients of Triton solution used for this study. Ingredient Name Percentage (%) Part A 1,2,4-Butanetricarboxylic acid, 2-phosphono ≤ 15% Citric acid ≤ 2% Sodium dodecylbenzenesulfonate ≤ 2% Alcohols, C9-11, ethoxylated, liquids ≤ 2% Polyethylene glycol 4-(tert-octylphenyl) ether, liquid ≤ 2% Sodium lauryl sulfate ≤ 2% 2-Ethylhexyl sodium sulfate ≤ 2% Sodium cumenesulphonate ≤ 2% Sodium hydroxide <1% Part B Sodium hypochlorite ≤ 10% Sodium hydroxide ≤ 2% 1.8 Dentin block (study model) The aim of creating a model using dentin is to mimic infections in teeth. When bacteria enter the dentin, they invade deep into the dentinal tubules, by which enables them to evade the antimicrobial effects of irrigation. Several models using dentin have been developed for research purposes to examine antimicrobial effectiveness and dentin disinfection (54). However, the number of infected dentinal tubules and the depth of invasion were uneven and a constant challenge. In 2011, a new model was introduced that involved centrifugation to obtain a deep, evenly distributed infection (55). That model is used in this study. 12 Chapter 2: Aims and Hypotheses 2.1 Aims and objectives The aims of this in vitro study were as follows: 1. Explore the antimicrobial effectiveness of Triton, an all-in-one, dual-action endodontic irrigation solution, when used independently against multispecies biofilms in dentin canals, both with and without a smear layer. 2. Assess the antimicrobial impact of Triton, an all-in-one, dual-action endodontic irrigation solution, when employed sequentially after NaOCl against multispecies biofilms in dentin canals, considering the presence or absence of a smear layer. 3. Contrast the antimicrobial efficacy of Triton, an all-in-one, dual-action endodontic irrigation solution, with traditionally used solutions (NaOCl / EDTA) in addressing multispecies biofilms in dentin canals. 2.2 Study hypotheses The null hypotheses of this study were as follows: 1. Triton exhibits minimal antimicrobial effects on infected dentin canals. Limited improvement with Triton in sequence after 2% NaOCl 2. The sequential use of Triton after 2% NaOCl does not result in significantly enhanced antimicrobial effects. 3. There are no discernible differences in the antibacterial efficacy between Triton and the traditionally employed solutions (2% NaOCl / EDTA). 13 Chapter 3: Materials and Methods 3.1 Sample size calculation We calculated the necessary minimal sample size employing an online sample size calculator (https://www.stat.ubc.ca/~rollin/stats/ssize/n2.html). This calculation used the power of β = 0.90, an α = 0.05 significance level, and an effect size of 3.3 and showed that we need a minimum of 2 samples per experimental group. 3.2 Tooth selection and dentin specimen preparation Approval from the University of British Colombia Clinical Research Ethics Committee Review Board (H15-02793) was obtained. Twenty extracted caries-free and single-rooted human teeth were selected for this study. Teeth were placed in a glass jar, soaked in a 0.05% NaOCl solution, and stored at room temperature (20°C). Twenty root dentin block specimens were prepared following a protocol described by Ma et al. (55). The teeth were horizontally cut 1 mm below the cemento-enamel junction to a length of 4 mm using a 0.6 mm thick diamond saw (Isomet 5000; Buehler Ltd, Lake Bluff, IL) at 1000 rpm. The canal space inside the blocks were enlarged at 300 rpm using a Gates Glidden size#6 drill (Tulsa Dentsply, Tulsa, OK). This is equivalent to a diameter of 1.5 mm. A groove was made in the middle of the specimen using a low-speed handpiece with a small round bur. The blocks were then fractured with a blade and a hammer resulting in two semicylindrical halves. Using 600-grit silicon carbide paper (Carbine; Buehler Ltd, Lake Bluff, IL), the outer surfaces were ground. This step was done to standardize the thickness and remove the cementum to expose the dentinal tubules. A filter tube with a 0.45-mm pore size (Pall Corporation, Ann Arbor, MI) was used to hold the dentin specimens. The 14 specimens needed further preparations to be able to fit in the filter tubes. As such, they were further shaped and refined using a water-cooled low-speed handpiece with a fine carbide bur (Tulsa Dentsply) at 300 rpm (Figure 2.1). Figure 2.1. An example of a sectioned sample showing (a) the ground and refined outer surfaces and (b) the inner surface showing the canal space (arrow). After refinement and before placement in the filter tube, the specimens were immersed in 5.25% NaOCl (Clorox Company of Canada Ltd, Brampton, ON, Canada) for 4 minutes and 6% citric acid (pH 4.0 Sigma-Aldrich, 18 St Louis, Mo) for 4 minutes subsequently while placed in an ultrasonic bath (Sankei Giken Industry Co Ltd, MIE, Tokyo, Japan). They were then rinsed with sterile water for 1 minute. This step was done to remove the smear layer from the specimens. Twenty dentin specimens were placed in 20 filter tubes with the inner surface showing the canal facing up. All gaps around the specimens were sealed using A2 shade flowable composite (Kerr Co, Orange, CA) and light-cured for 20 seconds (Figure 2.2). a b 15 Figure 2.2. Dentin specimen in the filter tube (a) before and (b) after sealing with composite. 3.3 Dentin specimen infection From a healthy adult donor, multispecies bacteria from both supragingival and subgingival plaque were collected, suspended in brain heart infusion (BHI) broth (Becton- Dickinson, Sparks, MD), and grown overnight in an incubator. The cell density of the suspension was standardized to OD405= 0.1 nm above the stock BHI optical density (OD) and to 3 x 106 colony-forming units (CFU)/mL. This was done by measuring the bacterial suspension’s OD value using a microplate reader (Model 3350; Bio-Rad Laboratories, Richmond, CA). The final solution volume needed to complete the infection process was calculated using the OD dilution formula from an online calculator. For each filter tube (n=20), 500 µL of bacterial suspension in BHI broth was added and centrifuged for 5 minutes at 5000× rpm (twice), 6000× rpm (twice), 8000× rpm (twice), and 10000× rpm (twice) in sequence. The bacterial suspension in BHI broth (500 µL) was changed between each cycle for each filter tube. Centrifugation was done to allow the bacteria to enter the dentinal tubules. a b 16 After centrifuging, 500 µL of sterile BHI broth was added to each tube. The tubes were then incubated under anaerobic conditions (AnaeroGen, OXOID, Hampshire, UK) at 37°C for 3 weeks with sterile BHI being refreshed weekly for each tube. This step was done to facilitate bacterial recovery and to allow for biofilm growth on the specimen. 3.4 Dentin disinfection and smear layer production After 3 weeks, the infected dental specimens were removed from the filter tubes, the composite was removed, and the specimens were rinsed with sterile water for 1 minute and dried with air. Nail varnish was applied on the outer surface of the specimen (Figure 2.3). This was done to seal the dentinal tubules from the cemental side. For the smear layer groups, a smear layer was created on the root canal side of the dentin specimen using a medium-grit cylinder flat-end bur with a diameter of 1.4mm, and 1.4mm tip length. Figure 2.3. Dental specimen after removal from the filter tube and application of varnish. 17 3.5 Groups Twenty dentin specimens were included in this study. They were randomly divided into 10 experimental groups based on the irrigation solution used and then subdivided based on the presence or absence of a smear layer (Table 2.1). Table 2.1. Table showing the 10 experimental groups and exposure times. Experimental groups Solutions No smear layer (N=10) Smear Layer (N=10) Exposure time (minutes) Exposure time (minutes) Water (control) 3 3 2% NaOCl 3 3 2% NaOCl / 17% EDTA 3+1 3+1 Triton 3 3 2% NaOCl / Triton 3+1 3+1 3.6 Pre-scan preparation In a 96-well plate, and with the use of a pipette, a droplet of 50 µL of each solution was placed on the root canal side of the dentin specimen for the assigned number of minutes according to Figure 2.4. The specimen was then washed in sterile water for 1 minute. To expose the dentinal tubules and make them visible for confocal laser scanning microscopy (CLSM) examination, the specimens were longitudinally fractured and stained with the fluorescent (LIVE/DEAD BacLight Bacterial Viability kit L-7012, Molecular probe, Eugene, OR, USA). The stain kit contains two components: SYTO 9 and propidium iodide. According to the manufacturer instructions, both 18 components should be mixed in a ratio of 1:1, placed on the specimen for 10 minutes, and then rinsed with phosphate buffered saline for 1 minute. The SYTO 9 stains bacteria with an intact cell membrane a fluorescent green colour and the propidium iodide stains bacteria with a damaged cell membrane a red fluorescent colour. This is viewed by using CLSM (Nikon Eclipse C1. Nikon Canada, Mississauga, ON, Canada). Figure 2.4 Flow chart illustrating the experimental design of this study. Fluorescent LIVE/DEAD BacLight Bacterial Viability staining Confocal Laser Scanning Microscopic (CLSM) Examination Reconstructing the confocal scan slices into a 3D model using Imaris 7.2 softwareWater(control)Water(control)2%NaOCl2%NaOCl2% NaOCl / 17% EDTA2% NaOCl / 17% EDTA2% NaOCl / Triton2% NaOCl / TritonTritonTritonSingle rooted human teethDentin blocks formationDentin blocks infection with multispecies biofilm for 3 weeksSmear layer groupN= 10No smear layer groupN= 10Statistical analysis using the SPSS version 29.0 software 19 3.7 Confocal laser scanning microscopy (CLSM) examination The specimens were examined using CLSM imaging (Nikon Eclipse C1; Nikon Mississauga, ON, Canada). After locating the border of the root canal and the fractured dentin surface, areas were randomly selected from the specimen and scanned, 20 slices per scan were obtained, each slice had a 0.5 μm step size. Next, the Live/Dead bacteria ratios were analyzed. This was done by reconstructing the 20 slices/scan into a 3D model using Imaris 7.2 software (Bitplane Inc., St Paul, MN). The volume ratio of the red fluorescence to the combined red and green fluorescence represented the dead bacteria for each group. 3.8 Data analysis Statistical analysis was performed using SPSS version 29.0 software (SPSS Inc, Chicago, IL) with a threshold for significance set at p<0.050. The dead cell volume (quantitative) was the study’s outcome (dependent variable). Means and standard deviations (Sd) of the proportions of dead bacteria cell volume were compared among the study groups. The predictors tested were the type of antimicrobial solution (nominal variable) and the presence of a smear layer (dichotomous variable; yes vs. no). 3.8.1 Independent sample t-test Independent sample t-tests compared the mean of the dead cell volume of the groups with the smear layer (data from five groups combined) to the mean of the groups without the smear layer (data from five groups combined). 20 3.8.2 One-way ANOVA with post-hoc Scheffé One-way ANOVA with post-hoc Scheffé adjustments (control: water) compared the means of the dead cell volume among the five groups of antimicrobial solutions. 21 Chapter 4: Results This study examined the effectiveness of the Triton irrigation solution in killing bacteria in dentin canals and compared it to traditionally used NaOCl in the presence or absence of a smear layer. The antimicrobial effect of Triton, when used as a final rinse after 2% NaOCl, was also compared to the traditionally used EDTA. In addition, evidence of bacterial penetration into the specimens was viewed using CLSM. 4.1 Comparison of sensitivity among groups with a smear layer and those without There was a statistical significance in the percentage of dead bacteria between all groups (with and without a smear layer). The percentage of dead bacteria was an average of 4.8% higher in groups with no smear layer, compared to the groups with a smear layer (p=0.032) (Table 4.1). Table 4.1. Mean percentages of dead bacteria in smear layer and no smear layer groups. *Independent sample t-test. Percentage of dead bacteria in dentin specimens (%) No smear layer mean ±sd Smear layer mean±sd Significance* 37.7±16.4 32.9±14.8 .032 22 Figure 4.1. Percentage of dead bacteria (%) among different groups of antimicrobial solutions. In comparison to water (control group), all the antimicrobial solutions were effective at killing bacteria, and the mean percentage of dead bacteria that were killed by the antimicrobial solutions ranged from 9.5% to 52.8% (see Figure 4.1). More bacteria were killed in the groups without a smear layer (up to 52.8%) compared to groups with a smear layer (up to 46.7%). The control group had the smallest proportion of dead bacteria (9.5%- 10.5%) (Table 4.2). Percentaage of dead bacteria (%)Water (control)2% NaOCl 2% NaOCl/ 17% EDTA2% NaOCl/ TritonTritonNo smear layerSmear layer23 4.2 Comparison between Triton and 2%NaOCl In the 2% NaOCl group, the mean percentage of killed bacteria was 32.6% in groups with no smear layer, a significant mean difference compared to the mean of the groups with a smear layer (28.8%) (p<.001). In the Triton only group, 49% of bacteria were killed in groups with no smear layer and 46.8% were killed in groups with a smear layer; this mean difference was not significant (p=.267). The mean percentage of killed bacteria in the 2%NaOCl / EDTA group with smear layer was (33.2%) and (43.5%) in groups with no smear layer with significant difference (p<.001). (Table 4.2) Overall, the highest proportion of dead bacteria found in the 2% NaOCl / Triton groups with no smear layer (52.8%) with no significant difference than the same group with smear layer. (46.5%) (p=.036). Table 4.2 Proportion of killed bacteria (dead cell volume %) in dentin specimens with and without a smear layer after irrigation with different disinfecting solutions. Groups No smear layer mean±sd Smear layer mean±sd Significance* Water (control) 10.5±2.4 9.5±4.0 .339 2% NaOCl 32.6±4.2 28.8±2.1 <.001 2% NaOCl / 17% EDTA 43.5±5.2 33.2±4.9 <.001 Triton 49.0±6.6 46.8±5.6 .267 2% NaOCl / Triton 52.8±10.1 46.5±8.4 .036 *Independent sample t- test 24 4.3 Comparing the antimicrobial effect of different solutions A one-way analysis of variance (ANOVA) compared the means of the dead cell volume (dependent variable) among the groups exposed to different antimicrobial solutions. The mean percentage of killed bacteria in the 2% NaOCl / Triton group was 11.6% higher than the one in the 2% NaOCl / 17% EDTA group (p=0.001). However, the mean of the killed bacterial in the 2% NaOCl / Triton group was only 2% higher than the one observed for the Triton only group. (p=0.828) (see Tables 4.3 and 4.4). Table 4.3 Proportion of killed bacteria in different antimicrobial solutions. Proportion of killed bacteria in infected dentin canals (%) Water (control) mean±sd 2% NaOCl mean±sd 2% NaOCl / 17% EDTA mean±sd Triton mean±sd 2% NaOCl / Triton mean±sd Significance* 10.0±3.3 31.0±3.8 38.4±7.2 48.0±6.2 50.0±9.7 <.001 * One-way ANOVA. 25 Table 4.4 Comparisons between the proportions of killed bacteria in all groups. Groups Irrigation solution Bacteria killing mean±sd Significance* Water (control) 2% NaOCl -20.7±1.4 <.001 2% NaOCl / 17% EDTA -28.3±1.4 <.001 Triton -37.8±1.4 <.001 2% NaOCl / Triton -39.6±1.4 <.001 2% NaOCl Water (control) 20.7±1.4 <.001 2% NaOCl / 17% EDTA -7.7±1.4 <.001 Triton -17.2±1.4 <.001 2% NaOCl / Triton -19.0±1.4 <.001 2% NaOCl / 17% EDTA Control (water) 28.3±1.4 <.001 2% NaOCl 7.6±1.4 <.001 Triton -9.5±1.4 <.001 2% NaOCl / Triton -11.3±1.4 <.001 Triton Control (water) 38.0±1.4 <.001 2% NaOCl 17.2±1.4 <.001 2% NaOCl / 17% EDTA 9.5±1.4 <.001 2% NaOCl / Triton -1.8±1.4 .828 2% NaOCl / Triton Control (water) 39.6±1.4 <.001 2% NaOCl 19.0±1.4 <.001 2% NaOCl / 17% EDTA 11.3±1.4 <.001 Triton 1.8±1.4 .828 * One-way ANOVA with post-hoc Scheffé test. 26 4.4 Confocal imaging of stained bacteria in the dentin block model Following staining with LIVE/DEAD stain, CLSM images were obtained showing the killing of biofilm microbes (Figure 4.2). Viable bacteria are stained a fluorescent green colour and dead bacteria are fluorescent red. The rows show the antimicrobial solution groups (water, 2% NaOCl, 2% NaOCl / 17% EDTA, Triton, 2% NaOCl / Triton)) indicating the killed bacteria when the smear layer is either removed or not (columns). 27 Figure 4.2. Confocal imaging (CLSM) showing viability-stained biofilms confirming biofilm penetration into dentinal tubules (green) and the antimicrobial effects (red) of all solutions used. No Smear Layer Smear Layer Triton Control Water 2% NaOCl 2% NaOCl / EDTA 2% NaOCl / Triton 28 Chapter 5: Discussion 5.1 Study Discussion The main goal of root canal treatment is to eliminate the bacteria infiltrating the canal system and prevent recontamination and reinfection after treatment. In order to effectively clean the canals, the antimicrobial used should have all the characteristics needed to kill and eliminate microbes in the canal system (23). The presence of biofilms in the ramifications of the canals is one of the contributing factors to the difficulty of achieving such a goal (56). NaOCl and EDTA are the most popular irrigation solutions used in endodontic therapy. Despite the advantages of NaOCl, it does not completely remove the smear layer (17). Therefore, irrigation with EDTA is used as a final irrigation step to remove inorganic part of the smear layer (23). Brasseler, USA proposed an irrigation solution (Triton) as a single dual action solution that removes the smear layer and provides better antimicrobial effects than the multiple solutions used. In this in vitro study, a multispecies biofilm model was used. Using multispecies biofilm models to test antimicrobial effects has been used several times previously (13, 57). The method includes collecting a sample from the subgingival and supragingival plaque of a donor patient, which has the advantage of being easy and guarantees bacteria collection (58). It has also been proven that microorganisms in endodontic lesions are similar to the ones found in plaque and carious lesions (59). The dentin infection model used in this study was proposed in 2011. This model has the advantage of gaining uniform and deep infection in the dental tubules of the specimens by 29 adding a centrifugation step (55). The centrifugation step introduces standardization and allows for comparisons between specimens. In addition, creating the dentin blocks from human dentin simulates the same clinical conditions as found in a tooth in a patient’s mouth (55, 60). In this study, we found that the groups with Triton (Triton and 2% NaOCl / Triton) had the highest significant bacterial killing when compared to 2% NaOCl / EDTA, despite whether the smear layer was present or not. Also, a larger bacterial load reduction was more evident in groups with no smear layer compared to the groups with a smear layer. Nevertheless, Triton and 2% NaOCl / Triton have been proven to have effective antimicrobial activities. 2% NaOCl was used in this study as the main irrigation solution, the concentration is low due to the evidence presented on the adverse effects that high NaOCl concentrations have on dentin (30-33). Our aim was to attain a solution that provided the same or higher bacterial killing as is obtained with 6% NaOCl. Our study found that using 2% NaOCl / Triton kills (50.0% ± 9.7%) of the bacteria, which is significantly higher than 2% NaOCl / 17% EDTA solution (38.4% ±7.2%) (p=0.001). This shows that Triton has a better antimicrobial effect when used as a final rinse in sequence after 2% NaOCl. This finding also highlights the fact that Triton outperformed 2% NaOCl / EDTA. This may make it convenient for clinicians to add Triton to their routine endodontic therapy without being overly affected by its cost. The findings in this study confirm that sequentially combining the use of NaOCl with Triton boosts the antimicrobial effect and killing of bacteria. Triton stands out as an all-in-one irrigation solution, streamlining the procedure by combining multiple components into a single solution, reducing chairside time, and eliminating the need for multiple solutions and syringes. 30 Overall, among all the solutions included in this study, a higher bacterial load was killed in the groups with no smear layer (37.7% ± 16.4%) compared to the groups with a smear layer (32.9% ±14.8%) (p=0.032). The presence of a smear layer directly affects the effectiveness of the solution, higher antimicrobial effects are found when the smear layer is absent. Triton is composed of two parts that are automatically mixed upon withdrawal from the bottle, giving a 4% NaOCl solution. One part is 8% NaOCl and the other part is mainly surfactants and citric acid. Citric acid increases the antibiofilm effectiveness of NaOCl and allows deeper penetration and better removal of the smear layer (61). Sodium hydroxide is also a component of Triton and is a pH modifier that maintains NaOCl at a high pH, resulting in elevated antimicrobial effects (62). The evidence pertaining to Triton is scarce and has yet to be fully investigated, making direct comparisons unfeasible. A recent study tested Triton on extracted, instrumented, and irrigated teeth under scanning electron microscopy (SEM). It compared the smear and debris layer removal of Triton to 6% NaOCl / 17% EDTA. They found that Triton was significantly more effective in removing debris from the middle and incisal third but not the coronal third of the tooth (p<0.05). They also found that Triton removed more smear layer from the apical third of the tooth than from the rest of the tooth ( p<0.05 ) (63). Another study compared Triton to NaOCl and NaOCl / EDTA using methodology similar to what was used in this study. They tested the antimicrobial effectiveness of Triton and its ability to remove the smear layer from both multispecies and E. faecalis biofilms. They found results 31 similar to this study, where Triton showed the highest bacterial killing among all groups (64). This further confirms the positive antimicrobial effect of Triton. However, Triton was tested as the only irrigation solution using different exposure times. In this present study, the combined effect of using 2% NaOCl and Triton in sequence was investigated and this group showed the highest bacterial killing. This finding underscores the potential Triton possesses as an effective antimicrobial solution for endodontic therapy. 5.2 Limitations This study has succeeded in proving the antimicrobial effectiveness of Triton and has proven its potential as an alternative irrigation solution. Nevertheless, all studies have limitations that should be mentioned to shed light on areas where future research can be improved upon. This study was done on an in vitro model. However, to simulate and replicate an exact clinical situation, ex vivo models should be employed. In addition, to further investigate and confirm the smear layer removal and antibiofilm effect of Triton, SEM, molecular tests, and polymerase chain reaction assays should be considered. 5.3 Future directions Further studies on Triton are prudent. Several potential areas for future studies include the antimicrobial effect of Triton against E.faecalis biofilm, the long-term antimicrobial effect of Triton, its effect on dentin structure, and whether other treatment factors such as activation and exposure time could impact the effectiveness of Triton. Also, the biocompatibility and effect of Triton on sealer adhesion to dentin should be studied. 32 Chapter 6: Conclusion In summary, the findings from this study provide valuable insights: 1. The Triton groups (Triton and 2% NaOCl / Triton) consistently demonstrated the most effective bacterial eradication, irrespective of the presence of a smear layer. 2. The all-in-one, dual-action endodontic irrigation solution, whether employed independently or sequentially after 2% NaOCl, exhibited superior antimicrobial efficacy against 3-week-old multispecies biofilms in dentin canals compared to 2% NaOCl / EDTA irrigation solution. Particularly, the sequential use of Triton after 2% NaOCl showcased the highest level of bacterial elimination. This study underscores Triton's heightened and synergistic antimicrobial effect when employed as a final rinse following 2% NaOCl. 33 References 1. American Association of Endodontists. Glossary of endodontic terms. 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Thesis/Dissertation
eng
Craniofacial Science
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Shen, Ya
Antimicrobial effectiveness of an all-in-one endodontic irrigation solution (Triton) : an in vitro study
Text