Qualitative Analysis of Precipitate Formation on the Surface and in the Tubules of

Transcription

1 Qualitative Analysis of Precipitate Formation on the Surface and in the Tubules of Dentin Irrigated with Chlorhexidine or QMiX as a Final Rinse by Kamil P. Kolosowski A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Faculty of Dentistry University of Toronto Copyright by Kamil P. Kolosowski 2014

2 Qualitative Analysis of Precipitate Formation on the Surface and in the Tubules of Dentin Irrigated with Chlorhexidine or QMiX as a Final Rinse Kamil P. Kolosowski Master of Science Faculty of Dentistry University of Toronto 2014 ABSTRACT This study examined the presence of precipitate and para-chloroaniline (PCA) in dentinal tubules (DT), after irrigation with NaOCl and CHX, or with NaOCl and QMiX, using of Time-of-flight secondary ion mass spectrometry (TOF-SIMS). Human upper molars dentin blocks were embedded in resin. Group 1 was irrigated with 2.5%NaOCl, followed by 17%EDTA, 2.5%NaOCl, and 2%CHX. Group 2 was irrigated with 2.5%NaOCl, saline and QMiX. TOF-SIMS spectra were obtained. Longitudinal sections of the irrigated blocks were exposed and examined with TOF-SIMS. All samples and analyses were triplicated for confirmation. Group 1 samples had irregular precipitate containing PCA on the surface and extending into DT. In group 2, no precipitate was seen on the surface or in DT. PCA and precipitate were present in the tubules of dentin irrigated with NaOCl followed by CHX. No precipitate or PCA were detected in the tubules of dentin irrigated with NaOCl followed by saline and QMiX. ii

3 ACKNOWLEDGEMENTS This thesis is the result of a scientific research study that has been carried out from 2011 to 2014 in fulfillment of a Masters of Science degree in the Discipline of Endodontics, Faculty of Dentistry, University of Toronto, Canada. I would like to thank my scientific advisory committee members without whom this project would not be possible: Drs. Bettina Basrani, Anil Kishen, and Rana Sodhi. They provided me with guidance and support to complete this thesis. Moreover, I would like to express my gratitude to Dr. Bettina Basrani, my principal supervisor, who spent countless hours helping me prepare for and lead me through this task, and whose office door was always open when I needed guidance. Without her, this project would not have started. Also, I would like to acknowledge the effort of Dr. Rana Sodhi, who introduced me to and operated TOF-SIMS apparatus, in addition to teaching me how to prepare samples, analyse and present the results. I would also like to thank Drs. Anil Kishen, Shimon Friedman, Cal Torneck and, again, Bettina Basrani for the opportunity and honour to be a part of Graduate Endodontics, learn from them and prepare me to be an endodontist. In addition, I appreciate the sacrifices of all faculty staff including Drs. Malkhassian, Plazas, and Cherkas iii

4 My appreciation goes to all my co-residents over the three years: Aly, Andrew, Luis, Anousheh, Pavel, Raj, Andrei, Jason, Karine, Brent, Craig, Helena, and especially my classmates Gillian and Peter for their encouragement and shared joys. I wish to add that my time at the faculty would not be as pleasant without the full support of the endodontic staff, who provided the assistance throughout the clinical sessions and made it easier for me to complete the clinical component of the program. This project was possible with the help of Surface Interface Ontario, its equipment for the sample preparation and computer lab for the data analysis. Words cannot express how much I appreciate my wife, Gisela, and my son, Ryan, for helping to bring meaning into my life, encouraging me to apply to the program and to persevere. This study was supported by grants from Canadian Academy of Endodontics Endowment Fund and Endo Tech. iv

5 TABLE OF CONTENTS Abstract..... ii Acknowledgments iii Table of Contents..... v List of Appendices.... viii List of Abbreviations... xi I. Review of Literature Introduction Dentin Composition Structure Dentinal Tubules Dentinal Tubule Fluid Bacterial Invasion of the Dentinal Tubules Irrigants Sodium Hypochlorite Chemistry and Mode of Action Antimicrobial Activity Effect on Hard and Soft Tissue Cytotoxicity Methods to Increase the Effectiveness Ability to Penetrate into Dentinal Tubules Deactivation Chlorhexidine Chemistry and Mode of Action Antimicrobial Activity Effect on Hard and Soft Tissue Cytotoxicity Substantitvity Ability to Penetrate into Dentinal Tubules Methods to Increase the Effectiveness v

6 3.2.7 Deactivation Breakdown Ethylenediaminetetraacetic Acid Chemistry and Mode of Action Antimicrobial Activity Effect on Soft and Hard Tissue Cytotoxicity Ability to Penetrate into Dentinal Tubules Methods to Increase the Effectiveness Deactivation Mixed Irrigation Solutions Effect of Detergent on Irrigation QMiX Antimicrobial efficacy Effect on Hard and Soft Tissue Cytotoxicity Ability to Penetrate into Dentinal Tubules Deactivation Interactions of Irrigation Solutions NaOCl and CHX Precipitate and PCA Detection Methods Chemistry of PCA Formation Effect of Precipitate on Dentin Toxicity of PCA and Precipitate Prevention NaOCl and EDTA Chemistry of Reaction Effect on Hard and Soft Tissue Prevention CHX and EDTA Chemistry of Reaction vi

7 5. Methods of Studying Dentin and Irrigation Dentin, Tubule, Infection, and Irrigation Interactions of Irrigating Solutions Time-of-Flight Secondary Ion Mass Spectrometry Principles of TOF-SIMS Modes of Operation Dentin, CHX, and Precipitate Studies Penetration of Precipitate into Dentinal Tubules.. 39 II. Rationale and Aims Rationale Aims III. Article IV. Discussion Methodology Results.. 65 V. Future Directions VI. Conclusion. 69 VII. References VIII. Appendices vii

8 LIST OF APPENDICES Appendix 1. Letter Grant of ethics approval from Health Sciences Research Ethics Board (REB), University of Toronto, dated February 28, Appendix 2. Molecular structures Molecular structure of Chlorhexidine. Molecular structure of Ethylenediaminetetraacetic acid. Molecular structure of para-chloroaniline. Appendix 3. Tables Table 1. Samples and experimental irrigation protocols. Table 2. TOF-SIMS spectra mass peaks of investigated ions. Appendix 4. Figures Figure 1. S1 sample selected positive ion TOF-SIMS spectra. Figure 2. S1 sample selected negative ion TOF-SIMS spectra. Figure 3. S1 sample selected positive ion TOF-SIMS images. Figure 4. S1 sample selected negative ion TOF-SIMS images. Figure 5. S2 sample selected positive ion TOF-SIMS spectra. Figure 6. S2 sample selected negative ion TOF-SIMS spectra. Figure 7. S2 sample selected positive ion TOF-SIMS images. Figure 8. S2 sample selected negative ion TOF-SIMS images. viii

9 Figure 9. S3 sample selected positive ion TOF-SIMS spectra. Figure 10. S3 sample selected negative ion TOF-SIMS spectra. Figure 11. S3 sample selected positive ion TOF-SIMS images. Figure 12. S3 sample selected negative ion TOF-SIMS images. Figure 13. S7 sample selected positive ion TOF-SIMS spectra. Figure 14. S7 sample selected negative ion TOF-SIMS spectra. Figure 15. S7 sample selected positive ion TOF-SIMS images. Figure 16. S7 sample selected negative ion TOF-SIMS images. Figure 17. S8 sample selected positive ion TOF-SIMS spectra. Figure 18. S8 sample selected negative ion TOF-SIMS spectra. Figure 19. S8 sample selected positive ion TOF-SIMS images. Figure 20. S8 sample selected negative ion TOF-SIMS images. Figure 21. S9 sample selected positive ion TOF-SIMS spectra. Figure 22. S9 sample selected negative ion TOF-SIMS spectra. Figure 23. S9 sample selected positive ion TOF-SIMS images. Figure 24. S9 sample selected negative ion TOF-SIMS images. Figure 25. S4 sample selected positive ion TOF-SIMS images of longitudinal section. Figure 26. S4 sample selected negative ion TOF-SIMS images of longitudinal section. Figure 27. S5 sample selected positive ion TOF-SIMS images of longitudinal section. Figure 28. Selected positive ion TOF-SIMS spectra of part highlighted in Figure 27. Figure 29. S6 sample selected positive ion TOF-SIMS images of longitudinal section. Figure 30. S6 sample selected negative ion TOF-SIMS images of longitudinal section. Figure 31. S10 sample selected positive ion TOF-SIMS images of longitudinal section. ix

10 Figure 32. S10 sample selected negative ion TOF-SIMS images of longitudinal section. Figure 33. S11 sample selected positive ion TOF-SIMS images of longitudinal section. Figure 34. S11 sample s elected negative ion TOF-SIMS images of longitudinal section. Figure 35. S12 sample selected positive ion TOF-SIMS images of longitudinal section. Figure 36. S12 sample selected negative ion TOF-SIMS images of longitudinal section. Appendix 5. Photographs Figure 1. Leica EM TXP Target Sectioning System. Figure 2. Leica EM TXP Target Sectioning System tooth sectioning. Figure 3. Prepared dentin blocks. Figure 4. Dentin blocks poured in resin. Figure 5. Leica EM UC6/FC6 Ultracryomicrotome. Figure 6. Leica EM UC6/FC6 Ultracryomicrotome dentin exposure. Figure 7. Prepared dentin blocks prior to placement in TOF-SIMS apparatus. Figure 8. TOF-SIMS apparatus ION-TOF GmbH. x

11 LIST OF ABBREVIATIONS Bi Ca CHX DEJ DT EC50 EDTA ESIQ-TOF-MS Bismuth Calcium Chlorhexidine Dentino-enamel junction Dentinal tubules Half maximal effective concentration Ethylenediaminetetraacetic acid Electrospray ionization quadrupole time-of-flight mass spectrometry ESI-MS HOCl LC50 LMIG mm mm μm Na NaOCl nm NMR OCl - Electrospray ionization mass spectrometry Hypochlorous acid Median lethal dose Liquid metal ion gun Micromolar Millimetre Micrometre Sodium Sodium hypochlorite Nanometre Nuclear magnetic resonance Hypochlorite ion xi

12 PCA PCU - PO 2 - PO 3 SEM TOF-SIMS u Para-chloroaniline (4-chloroaniline) Para-chlorophenylurea Phosphinate ion Phosphonate ion Scanning Electron Microscopy Time-of-flight secondary ion mass spectrometry Unified atomic mass unit xii

13 I. REVIEW OF LITERATURE 1. Introduction It has been established that primary Apical Periodontitis (AP) is caused by a microbial invasion of a root canal system which has spread beyond the apex of the affected tooth (1-3). Over the years, the number of described detectable agents that have the ability to infect a root canal has grown to over 700 (4, 5). The presence of bacteria, viruses, fungi, and archae has been found to be associated with AP, although their presence is not necessarily an indication of being a causative factor (3, 6-14). The goal of an endodontic treatment is to eliminate a cause of or to prevent a periapical disease. This is most often accomplished by a non-surgical root canal therapy, which reduces the number of pathogenic bacteria in the root canal, and may be achieved by shaping the canals with endodontic files and cleaning the canals with antimicrobial solutions (15). Elimination of bacteria with instrumentation alone is reported to be achieved in 28% to 47% of cases (16-18). Addition of an irrigant such as sodium hypochlorite (NaOCl) or ethylenediaminetetraacetic acid (EDTA) further increases the efficacy against microorganisms (19, 20). Irrigation of root canals with liquid solutions is expected to further clean the system. NaOCl has proven its effectiveness in killing bacteria as well as fungi and viruses, in addition to removing vital and necrotic soft tissue (12, 19, 21-23). EDTA is a chelating agent and used to remove the smear layer (24, 25). Chlorhexidine digluconate (CHX) is also an effective antimicrobial agent. Due to its ability to cationically bind to dentin, it seems to have substantivity effect in the roots (26-28). Among recent developments, QMiX is said to combine the benefits of two or more of the irrigants. 1

14 With its EDTA, CHX, and a detergent content it may be as efficacious as 17% EDTA and 6% NaOCl, and exhibit better biocompatibility (29-31). Though, those reports have not been independently confirmed. Irrigants can lead to dentin erosion, affecting bonding, sealing, cell adhesion, and thus healing potential (24, 32-35). Reactions can occur when two or more irrigants are mixed together. This is evident when NaOCl is mixed with CHX (36). Their reaction forms a precipitate that contains para-chloroaniline (4-chloroaniline, p-chloroaniline, PCA) (36-39). Although QMiX also reacts with NaOCl, no precipitate is seen beside a change of colour of the solution. This is based on an unpublished pilot study. In root dentin, these precipitates can block dentinal tubules (DT) and thus may interfere with dentin bonding, cell attachment, and lead to reduction in healing rates (40). Many techniques have been employed to measure the effect of irrigation on dentin. SEM analysis, dye bleaching measurements, and bacteria culturing are frequently used (41). Time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis can be used to distinguish and identify different components, whether they are inorganic such as calcium hydroxyapatite, organic such as protein, and other chemical compounds. It can also distinguish between structures that are minutely different in their chemical compositions. TOF-SIMS combines the visual effects of SEM and the information of mass spectroscopy (42-44). In this study we explored the formation of a precipitate, when NaOCl is mixed with CHX, on the surface of dentin and in DT. To do this, we irrigated human dentin blocks with NaOCl, EDTA, NaOCl and CHX, or NaOCl, saline and QMiX. We used TOF-SIMS, with its great surface and elemental sensitivity and the ability to create 2

15 images of samples, to analyze the surface and longitudinal sections of those blocks. The following sections describe in detail the dentin, irrigating solutions, their interactions, and the methodology of TOF-SIMS. 2. Dentin 2.1 Composition By weight, dentin is composed of approximately 70% inorganic material, mainly in the form of calcium hydroxyapatite Ca 10 (PO 4 ) 6(OH) 2 (45). This percentage is lower than in overlying enamel. Other minerals and elements are also found in dentin, although in lower concentrations. The list includes nitrogen with its content ranging between 2.42% to 3.64%, carbonate between 2.99% and 3.25%, and zinc at around 300 parts per million. Fluoride concentration of approximately 100 to 500 parts per million depends on the fluoridation status of the area in which a particular individual lives in, and whether the tested dentin has been previously carious or not affected by the decay. Secondary dentin, in general, shows a lower content of fluoride (46, 47). About 20% of the mass is made of organic material, which includes various collagenous and non-collagenous proteins, proteoglycans, and growth factors. The remaining 10% constitutes water, which can be found as free or bound (45). Most of the collagen is of the type I, but type III and V are also found in smaller amounts. The ratio of an inorganic to organic component is not constant throughout the dentin. For example, a lower content of collagen and higher mineral component is present nearer the DT (48). Numerous non-collagenous proteins include dentin phosphoprotein, dentin and bone sialoproteins, osteopontin, dentin matrix protein, and dentin matrix protein 1 (49, 50). Water is primarily found in DT. Since the 3

16 dentinal tubule decreases in size, as it travels farther from the canal, the water content in dentin also similarly decreases (51). 2.2 Structure Dentin structure can be divided into primary, secondary, and tertiary dentin based on the time of formation and the presence of certain triggers (52). Primary dentin is formed by odontoblasts during the formation of the tooth, which is followed by a slow, secondary dentin deposition after the eruption of the tooth. Under the stress of abrasion, erosion, caries, restorative preparations, and fillings, the tertiary dentin can be formed (45, 52). First layer of primary dentin to be formed and the one closest to the dentino-enamel junction (DEJ) is named mantle dentin. It is between 5-30μm thick and has a lower mineral density (45, 53). Circumpulpal dentin constitutes the rest of the primary and secondary dentin and it is present in two forms, intertubular, which is lower in mineral content and higher in collagen, and peritubular (or intratubular) dentin with more mineralized content (48, 54). Unmineralized, 10-30μm thick, predentin is present at the area closest to the odontoblast layer of the pulp (45). It contains collagen I, II, III and VI, dentin phosphoprotein, and it is where mineralization of secondary and tertiary dentin occurs (49, 55). Tertiary dentin is subdivided further into reactionary and reparative dentin. Reactionary is formed by the original odontoblasts and the tubules remain continuous. Reparative dentin, formed by newly differentiated odontoblasts, has DT with disturbed continuity (56, 57). Tertiary dentin is usually present in the area closest to the irritant. Occasionally, cellular inclusions are found, and with time, they convert into open spaces within the dentin (52). 4

17 2.3 Dentinal Tubules Peritubular dentin surrounds the dentinal tubule. The content of the tubule in a vital tooth is an odontoblast process, present in the inner third of the tubule. The process can reach deeper into the dentin in the coronal dentin compared to the root dentin. The rest of the tubule is filled with fluid (58-60). The density of the tubules vary, but approximately 45,000/mm 2 are found in the coronal area of the tooth, and decrease significantly to around 8,000/mm 2 in the apical third, with the measurements done at the pulpal surface of the dentin. The coronal tubule density decreases to around 10,000/mm 2 at the DEJ (61-63). By volume, the tubules can make up for 10% of the volume of dentin. However, closer to the pulp this may account for 28%, and closer to the DEJ it decreases to 4%. The diameter of the tubule also differs as it travels throughout the length of the dentin. It has a shape of a cone, with the base at the pulpal region, where the size is almost 3μm, decreasing to less than 1μm at the junction with enamel (51, 62). It has been shown that the tubules do not travel in a straight line through the 3-3.5mm thickness of dentin, but rather exhibit an S shape. At the DEJ, they may be seen at a right angle but change the direction within 0.5mm from the junction. On average, maxillary teeth show a smaller change of direction. At the pulpal surface the tubules are present at a right angle (64, 65). The DT are not separate entities. There are multiple braches within the dentin complex, and where the density of the tubules is lower, there appears to be an increased number of branches. The size of the branches range from 25nm to 300nm for the microbranches that course at right angles to the main dentinal tubule, 300nm to 700nm for fine branches that run at 45 degrees, and 0.5μm to 1μm for major branches present peripherally. In the root area, most of the branches are 300nm to 700nm in diameter (65). When dental sclerosis 5

18 occurs, as a result of normal aging or stress such as attrition, caries, trauma, or filling, the dentinal tubule can become obliterated after continuous deposition of mineral. In roots, however, it appears that only aging is responsible for this process, progressing in a direction from apical to coronal (66, 67) Dentinal Tubule Fluid The tubule in non-sclerotic dentin of a non-infected tooth is mostly an empty space, filled with fluid. Therefore, on average this fluid takes up to 10% of the dentin volume, and up to more than 20% closer to the pulp. The fluid can move freely in the tubule. Although the presence of enamel, restoration or cementum prevents the flow, in absence of such barrier, the movement is usually in an outward direction. The speed of the flow decreases with aging and may completely disappear when caries are present (68). Temperature changes, restorative procedures and physical stress can cause this current to reverse and flow inward towards the pulp (51). It has been shown that the fluid in dentinal tubule most closely resembles interstitial fluid. The concentration of ions, and the fact that it lacks cellular component, show that most of the fluid is not present intracellulary in the odontoblast processes (69) Bacterial Invasion of the Dentinal Tubules Due to the size of dentinal tubule and usual outwards flow of the dentinal fluid, the colonization of the root canal by bacteria may be inhibited but not completely prevented. If untreated, caries bacteria and their byproducts reach and cause necrosis of the pulp. This may eventually lead to apical periodontitis (1-3). Once the root canal becomes 6

19 infected, the tubules become colonized shortly after (70). In younger teeth, due to the size of bacteria and their byproducts, which even at their largest are still smaller than the smallest size of patent dentinal tubule, penetration occurs when opportunity arises (70-73). The likeliness of this process decreases with age as dentinal sclerosis occurs (74). The depth of penetration of bacteria has been intensively investigated. Tubules of teeth without a vital pulp, therefore without outward flow of the tubular fluid, were able to be infected at a faster rate than those with vital pulps (75). The penetration of bacteria into the tubules is visible within a week of exposure. The depth of penetration increases with time, and bacterial products can reach the pulp, therefore transverse the entire length of the tubule, also within a week (76-79). Depending on the bacteria species and the measuring technique, the depth of invasion into the tubule can vary anywhere starting from 50μm to 375μm, up to 600μm for E. coli, and entire 1000μm length of the tested tubule for E. faecalis and S. sanguis (78-84). The presence of multiple species, in studies of co-cultures, the depth and rate of penetration was further increased (71). Even when the instrumentation and irrigation completely remove the bacteria from the root canal, bacteria in the infected tubules may still proliferate and become an infection source for persistent problems (18, 84, 85). It would be beneficial for a root canal therapy if the entire dentinal tubule fluid was replaced with an ideal endodontic irrigant able to eliminate the bacteria (86). 3. Irrigants Due to the inability of the mechanical preparation to reach all areas of the canal surface and to remove biofilms and debris material from the canal, irrigation should be used to 7

20 facilitate cleaning and disinfection of the root canals (41, 85, 87). The ideal irrigant is one that is an stable, effective, long acting antimicrobial, capable of reaching all root canal areas without causing any adverse effect on teeth or health, in addition to being inexpensive and easily obtainable (86). Unfortunately, a single irrigant solution is not able to possess all those characteristics. Most common irrigants in endodontics are NaOCl, EDTA, CHX, and saline, with new combination products such as QMiX or MTAD constantly being developed (87, 88). 3.1 Sodium Hypochlorite Originally used as a bleaching product, it has been extensively used for wound disinfection and sterilization (89). In solution, NaOCl has been successfully introduced and is used in endodontics as the most common irrigant (90-93) Chemistry and Mode of Action In aqueous solution, NaOCl is in equilibrium with sodium hydroxide and hypochlorous acid (HOCl), which in turn are dissociated into their respective ions. The reaction responsible is: NaOCl + H2O NaOH + HOCl Na + + OH - + H + +OCl - (94). NaOCl is a strong base with ph of about 11. Upon contact with amino acids, hydroxyl ion causes their neutralization, hypochlorite (OCl - ) and HOCl cause their hydrolysis and formation of chloramines. When in contact with fatty acids, NaOCl results in formation of glycerol and fatty acid salts. Multiple enzymes can get oxidized by the chlorine ion (Cl - ) released when HOCl contacts organic tissue. This reaction causes a drop in ph. 8

21 NaOCl in a solution can also act as a solvent for all products of those reactions (94). Unfortunately, the dissolving action is inhibited by the release of chlorine, which can easily escape the solution, especially when ph decreases. This can occur in as little as two minutes of contact with organic tissue (95) Antimicrobial Activity Multiple studies have been performed to test the effect of NaOCl on microorganisms, whether they are in a planktonic form or arranged in biofilms. It has been found that NaOCl is the only agent able to disrupt and dissolve the biofilm when used as a 1%, 5.25%, or 6% solution (96-99). At lower concentrations disruption of the biofilm can be noted, but a complete killing of the bacteria does not occur (97, 98, 100). Even in small concentrations, it is an effective antimicrobial agent against cultivable bacteria in root canals (19). It can also kill Candida albicans, however, when compared against fungal cultures, the time of contact must be extended when root canals are irrigated (12, 21, 101). Bacterial endotoxins can also be inactivated, although that effect is minor compared to the bacterial elimination ( ) Effect on Hard and Soft Tissue Complete dissolution of a soft tissue is the final result of the tissues' contact with NaOCl (94, 105, 106). NaOCl can dissolve vital organic material, but the efficacy against necrotic material is even greater (23, 88). This effect is present even if the NaOCl used is not a pure solution (107, 108). Due to the effect on soft tissue, NaOCl should be used with caution to reduce the possibility of extrusion through the apex. In case of extrusion, 9

22 the effect might not be pain alone, but also a profuse and long term tissue damage, edema, hemorrhage, and even paresthesia (109, 110). There are also some negative effects on the hard tissue dentin and bone. When in contact with the bone for prolonged time, NaOCl causes cratering in cancellous bone, with areas that appear to be demineralized. Cortical bone is less affected (111). When dentin is subjected to NaOCl, a reduction in microhardness, flexular strength, and elasticity is seen, which is mostly due to degradation of collagen and glycosaminoglycans ( ) (117). Irreversible erosion of dentin can also be detected, and may be responsible for the detrimental effect on the physical characteristics of the dentin ( ) Cytotoxicity When compared to its superior antimicrobial effect, NaOCl has been found to be very destructive to cell cultures (121, 122). In the human fibroblast cell cytotoxicity study, LC50 of NaOCl was found to be 0.79mM (123). The cytotoxic effect on fibroblasts is not seen with concentration at or below 0.005%, but is evident at concentrations above %, when cell detachment can be seen after an exposure of two hours. When increasing concentrations are used, cell death becomes more apparent. When concentration of NaOCl is greater than 0.01%, less than 10% of fibroblasts survive, and total fibroblast cell death is commonly found in studies ( ). In contrast to the cytotoxicity, there are conflicting conclusions on whether DNA damage can occur. No DNA damage effects are noted in some studies (128, 129). However, genotoxicity is seen in others, including one that explores the effect on human lymphocytes (130, 131). 10

23 3.1.4 Methods to Increase the Effectiveness When a solution with increased concentration or temperature is used, or an addition of sonic or ultrasonic activation is present, the effect of NaOCl increases. This effect is seen on tissue dissolution and antimicrobial effects, although an increased time of contact or increased volume can overcome the limitation of lower concentration (12, 20, 22, 87, 94, 100, ). The time, however, cannot be used infinitely as the active HOCl gets inactivated when Cl - and a chlorine gas are released (95). Sonic or ultrasonic irrigation also seem to increase the effect of the dissolution and cleaning efficacy (22, 95, 137). In contrast to advertising claims, an addition of an agent to increase the efficacy of NaOCl, such as sodium bicarbonate buffered solution or Chlor-XTRA, seems to play no significant role in terms of the effects (107, 108). However, this is not always the conclusion. In one study, the tissue dissolution ability was increased when NaOCl was supplemented with a surfactant (22) Ability to Penetrate into Dentinal Tubules Since the tubules can be infected within days or weeks, it is important for an irrigant to be able to penetrate into DT (76, 77, 86). Only a limited number of studies have been published exploring the penetration of NaOCl. Through the ability of NaOCl to bleach a dye solution that penetrated the dentin, it was measured that the penetration into DT occurred up to 300μm (138, 139). This depth was achieved with 6% NaOCl in a twentyminute dentin incubation. Lower concentrations or shorter time significantly decreased the penetration, with the lowest 77μm depth achieved with 1% NaOCl and a two-minute incubation (139). An addition of EDTA or citric acid to increase this depth resulted in an 11

24 opposite effect with lowered penetration. Only the combination of peracetic acid-naocl was able to increase the depth (138). Regardless whether the irrigant can be seen bleaching the dye in DT, it has been shown that the bacteria present in the dentinal tubule can be effectively killed by irrigation with NaOCl. The depth where dead bacteria are seen can vary from 200μm to 500μm, depending on whether 1% or 6% NaOCl is used respectively (30, ). Those studies concluded that NaOCl penetration occurred, but no direct measurement of this was performed Deactivation Soft tissue and dentin in the tooth can inactivate NaOCl quite quickly, this can occur in as little as two minutes (95, 146, 147). Most likely culprit is the release of Cl - and a chlorine gas, which occurs when soft tissue and organic component of the dentin are subjected to an attack by HOCl (94, 117). Therefore, a constant replenishment is necessary to ensure effective disinfection and dissolution. Another problem with deactivated NaOCl can be encountered upon mixing it with EDTA containing solutions or pastes. Their reaction causes a formation of bubbles and almost instant deactivation of NaOCl and the result is a loss of active chlorine component. Even the smallest concentrations of EDTA can cause this effect ( ). A similar reaction causing loss of Cl - is present when citric acid is added to NaOCl (148, 150, 151). Therefore, it is prudent to avoid mixing NaOCl with any other product, as it may cause the loss of its efficacy. It has been noted that EDTA will reduce the antimicrobial efficacy of NaOCl (152). Also, the tissue dissolution ability is significantly lowered (153). 12

25 3.2 Chlorhexidine Chlorhexidine Digluconate (CHX), C 22 H 30 Cl 2 N 10 2(C 6 H 12 O 7 ), has been used in health care for many years as a disinfecting solution (90). In dentistry, it has been successfully incorporated as a periodontal rinse agent, used as an endodontic irrigant for many years, and remains a third most common irrigant used in the United States (93, 154). One recent study has shown that high success rates can be obtained if vital teeth are irrigated with CHX during treatments, when the use of NaOCl is contraindicated (155). However, when tested as an adjunct irrigant, CHX was shown to lower the success rate of root canal treatments (156) Chemistry and Mode of Action Chlohexidine is a polybiguanidine antimicrobial with structure of two symmetric 4- chlorophenyl rings and a pair of bisguanidine groups connected by a haxametylene chain. It is strongly basic, has a cationic potential, and can form a stable salt that is easily soluble in water (26, 157). When it contacts a negatively charged surface of bacteria, the cationic CHX is able to electrostatically bind to it, increasing permeability and damaging the cell wall ( ). CHX is bacteriostatic in low concentration, causing leakage of cytoplasmic potassium and phosphorous. In high doses it is bactericidal, damaging the cell wall and causing precipitation of cytoplasm, it also has a detrimental effect on bacterial metabolism (26, 157, 159, 162). As a wide-spectrum antimicrobial agent, it is effective against Gram positive and Gram negative bacteria, in addition to yeast, although the effect against Gram positive is the strongest (79, 159, 161). 13

26 3.2.2 Antimicrobial Activity Antimicrobial activity of CHX has been tested extensively. In most studies, its effect compared to saline and NaOCl was examined. Controversies do exist, and no consensus has been reached in studies of antimicrobial effects of CHX compared to NaOCl in-vitro. In some studies, CHX was better, in others worse, and in some, the effects were not statistically different (12, 30, 79, 141, 142, ). In in-vivo studies, it was noted that addition of CHX was more effective than addition of saline to the irrigant protocol (166, 167). However, NaOCl was able to reduce the bacteria with greater antimicrobial effectiveness compared to CHX (168). Occasionally, it was seen that an addition of CHX provided no benefit (169). Moreover, combining both irrigants together seemed to increase the effectiveness (170). When a higher level of evidence study was conducted, it indicated that the ability to culture the bacteria was reduced to one quarter with NaOCl and to one half with CHX, and the assessment of bacterial loads with molecular techniques resulted in similar findings (168). This, however, was not reproduced in a subsequent study with a lower concentration of CHX (171). When the effects on biofilm was examined in an in-vitro experiment, CHX was seen to effectively remove endodontic biofilm bacteria (172). However, this effect was exerted on artificially grown biofilm. When compared to other irrigants, such as NaOCl, CHX outcome was much lower, indicating that CHX might not be effective as a single irrigant (30, 99, 164). When the biofilm was grown in an in-vivo situation and in an intraoral environment, it was noted that the disruption of the biofilm did not occur with CHX irrigation (96, 97). Although, due to the damage of a cell wall, the killing of the bacteria can take place, the studies that measured the effect against endotoxins did not find CHX significantly different compared 14

27 to NaOCl in its ability to inactivate LPS (103, 104). When effectiveness against yeast is tested, 2% CHX effectiveness equals that of 1.3% NaOCl (101) Effect on Hard and Soft Tissue In contrast to NaOCl, soft tissue dissolution does not occur when irrigation with CHX is used. There was no effect shown when bovine pulp tissue and pig palate tissue were subjected to the irrigant (173, 174). When dentin is subjected to CHX, no detrimental effects are seen (116). This most likely is due to its inability to dissolve soft and hard tissue. In an in-vitro study, it was seen that CHX had an inhibitory effect on matrix metallo proteinase 2 (gelatinase A), 9 (gelatinase B), and 8 (collagenase 2) activity, and a suggestion was made that the mechanism responsible for this action might be a cationchelation (175). This may explain why resin bond strength to dentin is more stable when pretreatment with CHX is performed (176, 177). However, in a clinical situation this effect is not found to be significant, as more factors may play a role in the stability of the resin-dentin interface (178) Cytotoxicity At concentrations commonly used in dentistry, CHX exhibits a low toxicity (154, 179). A study measuring cytotoxicity of CHX noted that 95% toxicity against human fibroblast was achieved with 0.01% concentration of CHX (180). Another study found that 0.005% of CHX produced total fibroblast cell death (181). In in-vitro culture studies some deleterious effects on fibroblasts were seen (154, 181, 182). Crevicular and peripherial neutrophils can be also affected by CHX, due to a lytic action and cell membrane 15

28 disruption. It inhibits the function of neutrophils at concentrations higher than 0.005% (183). No DNA damage or other genotoxic effects were noted in some studies that tested concentrations up to 1% (184, 185). Other studies, however, noted that DNA damage was possible, and that could even occur with 0.12% CHX oral rinse solutions, however no chromosomal changes occurred. Therefore, it was concluded that the DNA disruption could be transient (186, 187). In an in-vivo situation, when compared to NaOCl, the cytotoxicity of CHX is much lower, but it is noted that the inflammation in a short term and granulation changes in long term transpire (188). In another study, more severe tissue reactions were seen and their severity was dependent on the concentration of CHX (182) Substantitvity When used as a periodontal irrigant, 0.12% CHX antimicrobial action does not extend beyond the immediate use (189). As an endodontic irrigant, in an in-vitro study, the antibacterial effect extended up to 72 hours (27). Other studies confirm this and even suggest a longer time of action, ranging from 4 to 12 weeks (190, 191). In a clinical study, substantivity was said to last only 48 hours after performing instrumentation with CHX irrigation (192). The long term duration of the CHX action is explained by a reversible action of uptake and release of CHX. Due to the cationic nature of the CHX molecule, it can be absorbed onto hydroxyapatite and teeth. At low concentrations, less than 0.02%, a stable monolayer of CHX is formed on the surface, and at above 0.02%, a multilayer of CHX forms, allowing its release (193, 194). Some studies suggested that the subtantivity action could only take place after a long term CHX contact to allow the 16

29 dentin binding to ensue. This time of contact has been suggested to be anywhere from one hour to seven days (194, 195) Ability to Penetrate into Dentinal Tubules Multiple published studies have measured the penetration of CHX into the tubules. This is always tested by measuring the effect on bacteria in DT. Non-viable bacteria at certain distance in the tubules indicated the penetration of CHX to that depth. The results show that the distance of 100μm to 300μm remains the limit of the efficacy in the tubules (30, 79, 84, 141, 142, 144, 145, 163). The concentration of CHX seems to play no role in penetration (79). The addition of a detergent may enhance the antimicrobial effect of CHX (163). All published studies showing penetration of CHX into DT only demonstrate it indirectly and no available published studies measured the actual presence of CHX in the tubules (30, 79, 84, 141, 142, 144, 145, 163) Methods to Increase the Effectiveness When CHX is used as a gel or a solution, a greater antimicrobial effect was noted with 2% compared to 0.2% (196, 197). However, the mode of action remains the same regardless of concentrations (197). The 2% concentration is more effective and has a longer substantivity than the 0.12% (27). However, the action inside the tubules is not dependent of the concentration used (79). It has also been suggested that substantivity relies on increased time of contact between dentin and CHX, which should range anywhere from 1 hour to 7 days (194, 195). Formation of chlorhexidine-chloride has been proposed when CHX is mixed with NaOCl in the root canals. This also increased 17

30 the effectiveness of irrigation (170). Other studies may dispute that statement (36-39, 198). The addition of detergent reduced the time needed to kill bacteria (199) Deactivation It has been shown that dentin powder reduces the effectiveness of the antimicrobial action of CHX. This is more evident when a lower concentration of 0.05% is used. When concentrations greater than 0.5% are used, the reduction is minimal (147). The mechanism of this decline in effectiveness is unknown, but it is suspected that it differs from the dentin inhibition of calcium hydroxide or iodine potassium iodide (200, 201). Bovine serum albumin, heat killed bacteria and fungi, and dentin matrix can inhibit CHX even more than dentin (200). Collagen, on the other hand, is weakly inhibiting (201). The inactivation of CHX by dentin is inhibited by the addition of EDTA or cetrimide (199, 201) Breakdown Zong et al (202) investigated the fate of CHX when subjected to acidic or basic environment and concluded that the breakdown of CHX occurred fairly rapidly. It can also occur when CHX is subject to heating. In a solution with a low ph, CHX is seen to degrade either directly into PCA, or indirectly into PCA by an intermediate product of p- chlorophenyl-biguanide-amidino-p-chlorophenyl-urea. In high ph, a different, indirect pathway of breakdown into PCA is present, one that leads through p-chlorophenyl-urea (PCU) breakdown into PCA. The formation of PCA is ph dependent (202). 18

31 3.3 Ethylenediaminetetraacetic Acid CHX cannot dissolve dentin effectively, and NaOCl can produce some surface erosion (116, ). However, neither of those components can effectively dissolve hard tissue components of a smear layer formed during the root canal instrumentation. Only the addition of a demineralizing agent, such as ethylenediaminetetraacetic acid (EDTA) chelator, can perform this action (203). Thus, the main reason for the use of EDTA irrigation is to remove inorganic components of the dentin and the smear layer. This purpose makes it the second most common irrigant used by endodontists in the United States. EDTA has been an endodontic irrigation solution for more than half a century (204). Most common concentrations range from 10 to 17% (88, 93) Chemistry and Mode of Action EDTA has a chemical formula C 10 H 16 N 2 O 8. It forms a ring-shaped stable bond with ions when exposed to heavy metals or calcium. EDTA has more than one pair of free electrons and each of those pairs can form a bond to a centrally positioned metal ion (205). When distilled water is added to dentin, calcium hydroxyapatite dissociates into its ionic components, mainly calcium and phosphate, until equilibrium is established. When EDTA is added, it sequesters calcium by binding it and removes it from the solution, promoting further dissolution of calcium hydroxyapatite. Two simultaneous reactions occur: EDTA H 3- + Ca 2+ EDTA Ca 2- + H + and EDTA H 3- + H + 2- EDTA H 2 (205). 19

32 As those reactions continue, more dissociated acid forms and the rate of demineralization slows down, but the process continues to occur until all EDTA molecules contain bound calcium ( ) Antimicrobial Activity Although a major reason for the use of EDTA in endodontics is to remove the smear layer, one in-vivo study found that it was much more effective in eliminating root canal bacteria compared to saline (208). It has been suggested that the reason for the antimicrobial effect is the chelation and removal of the calcium component from the cell membrane of bacteria (205). Another study mentions that EDTA produces antibacterial effect if left in contact with bacteria for 24 hours (209). This can occur in a root canal if the solution is not completely removed, but sealed in without placement of intracanal medication. When the antibacterial action NaOCl and CHX is compared with the effect of EDTA, EDTA containing product shows a lower efficacy (165). If bacteria in the tubules are measured, one study found EDTA to be antimicrobial (144). However, a different study concluded that EDTA showed minimal or non-existent action (84). Similarly, when the efficacy against biofilm is measured, EDTA removes minimal number of bacteria, and fails to disrupt the integrity of the biofilm (99) Effect on Soft and Hard Tissue Since the main reason for the use of EDTA is to remove hard tissue components in the root canal, dentin dissolution also occurs (205, 206). Even 0.03% EDTA show a decalcifying effect on the dentin, whereas with 10% EDTA the effect is sizable (209). 20

33 The extent of demineralization is related to the amount of time EDTA is in contact with the substrate. Time of contact is also directly related to the decrease in hardness of the dentin and the depth of demineralization, which could reach up to 50μm (24, 204, 209). Hardness could drop 25% after a nine-minute application (209). Eventually, with time, the decalcifying effect stops when all EDTA molecules contain bound calcium ( ). It has been demonstrated that old dentin, from patients older than 60 years, experience greater demineralization or erosion. This may be explained by the fact that older dentin contains more inorganic component (210). Even with a short exposure to EDTA, microhardness of dentin is negatively affected (211, 212). This reduction of hardness leads to reduced fracture resistance of dentin (213). When EDTA is injected, and the effect on soft tissue is measured, inflammation is only moderate (209). EDTA, in concentrations above 0.5%, depresses cells metabolic activity. However, it is incapable of destroying collagen (214) Cytotoxicity In an in-vitro study, it has been concluded that EDTA is more cytotoxic than NaOCl (215). The reason might be that NaOCl quickly loses it efficiency with the loss of chlorine, but EDTA remains active until all calcium ions are sequestered (94, 205). Other studies have also shown the cytotoxic effect of EDTA on fibroblasts (127, 216). Macrophage function is also altered when tested in an in-vitro experiment (217, 218). The validity of measuring the cytotoxicity can be questioned, since in an in-vivo study no tissue damage with EDTA irrigation has been shown. That holds true with a periapical 21

34 tissue and pulpal tissue (204). EDTA extrusion through the apex has not been associated with endodontic accidents Ability to Penetrate into Dentinal Tubules Although the antimicrobial properties of EDTA are weak, a study has shown its ability to penetrate and kill bacteria in DT (144). This is disputed by another study showing minimal or no effect in the tubules (84). Nevertheless, the demineralizing action can expose the tubules and ETDA can rapidly penetrate up to 20-30μm. The limit of this penetration has been established to be 50μm, even over long term irrigation (204). This has been confirmed by multiple studies, measuring penetration or the exposure of patent tubules (24, 203, 209, 211, 219). Dentinal erosion can easily occur with excessive irrigation (210). This can affect intertubular and peritubular dentin (24) Methods to Increase the Effectiveness One way of increasing the effect of EDTA is to increase the amount of time a contact exists between the solution and the substrate (24, 204, 209). Increasing the concentration also increases the efficacy, however, the negative effects are also enhanced (220). Surfactant addition to EDTA can also potentiate the action (221) Deactivation EDTA action will continue until available acid molecules are bound to calcium (94, 205). Therefore, a long time factor may be necessary to completely inactivate EDTA. It has 22

35 been shown that while EDTA reduces the antimicrobial efficacy of NaOCl, the calcium binding efficacy of EDTA is not altered by NaOCl (149, 150, 152). 3.4 Mixed Irrigation Solutions Multiple irrigation solutions have been developed to contain more than one active component. Among those, the most common addition is a surfactant, a wetting agent. The surfactant is frequently added to NaOCl, CHX, or EDTA. Smear Clear, CHX-Plus, MTAD, and QMiX are some examples of mixed solutions (90). Although multiple studies investigate their in-vitro effectiveness, the in-vivo studies are lacking (29, 30, 141, 142, 164, ). One study that measured outcome of endodontic therapy using MTAD did not replicate the in-vitro efficacy of this irrigation solution (231) Effect of Detergent on Irrigation A detergent or a surface acting agent is usually added to irrigants to enhance their effectiveness which is usually achieved by decreasing the surface tension of the solution (232). It has been shown that the contact angle of NaOCl on dentin is significantly decreased even with a small addition of a surface acting agent. This addition does not negatively alter other properties of NaOCl, such as cytotoxicity, free chlorine content, and antimicrobial effects (232). Several other investigations of the effect of added detergent on irrigation have been performed. It has been presented that addition of a detergent to irrigants reduced the time required for killing E. faecalis and eliminated more bacteria than solutions without the detergent (163, 199, 233). In addition, their residual effectiveness was enhanced (233). Moreover, the tubules were open (221). Thus, the 23

36 ability to penetrate and kill bacteria in DT is increased (143, 163). However, some of those findings were disputed by later studies (228, 229). The deactivation of an irrigant by dentin can be decreased by the addition of a detergent (199, 201) QMiX QMiX, a novel irrigant sold by Dentsply Tulsa Dental, Tulsa, OK, USA, has recently entered the market. It contains CHX and EDTA, in addition to a detergent (90). It is said to combine the benefits of EDTA with a surfactant and CHX, while being gentler on dentin (234). Manufacturer s recommendation is to use it as a final irrigant. If NaOCl irrigation is used prior to the final rinse, then saline should be introduced into the canal to prevent a possible reaction (90, 235). Few studies are available examining the characteristics of this novel irrigant Antimicrobial Efficacy QMiX in-vitro antimicrobial efficacy against planktonic and biofilm E. faecalis, in addition to mixed plaque bacteria, has been tested against other irrigants. It has been concluded in one study that it is as effective in killing E. faecalis as 1% NaOCl, but more effective than 2% CHX. It acts faster against biofilm than 1% NaOCl and 2% CHX, but equal to 2% NaOCl (164). This conclusion is disputed by two studies measuring not only the number of killed bacteria, but also the number of live bacteria and the volume of the biofilm (228, 229). One conclusion stated that QMiX is effective in killing bacteria but is ineffective in reducing biofilm bacteria, making it ineffective overall. This is contrasting the effectiveness of NaOCl (229). Another study once again concluded that QMiX is 24

37 ineffective against biofilm. Although larger effect is seen as compared to CHX, it is significantly less effective than NaOCl, and as effective as distilled water (228) Effect on Hard and Soft Tissue When smear layer removal is tested, QMiX effectiveness equals to that of 17% EDTA (164, 224, 227). This is regardless of the ph of the QMiX solution tested (29). Another study concluded that QMiX formulations were superior to EDTA in smear removal and exposure of the tubules (226). The removal of smear layer by QMiX and EDTA might be related to the results that show similar dentin bond strengths of cements or sealers after the use of these solutions (224, 227). In addition to the smear layer removal, it has been shown that QMiX increases the wettability of dentin, with EDTA having worse wettability potential (225). This is most likely due to the presence of a detergent in QMiX. Although more erosion is noted with 17% EDTA, the microhardness of dentin does not differ to that of QMiX (223). With regards to soft tissue biocompatibility of QMiX in an in-vivo experiment, it is seen that it is less toxic to rat subcutaneous tissues, with less inflammatory cells induction than 17% EDTA, 2% CHX, and 3% NaOCl (31) Cytotoxicity Only one study is published investigating cytotoxic potential of QMiX. It reports that although both NaOCl and QMiX are toxic to mesenchymal stem cells, QMiX induces cell death at a slower rate and less aggressive fashion, without cell lysis occurring (222). 25

38 Ability to Penetrate into Dentinal Tubules Three studies explored the possibility of QMiX penetrating the tubules. All those were comparing antimicrobial efficacy at different irrigation time and measured dead bacteria in the tubules (30, 141, 142). It has been shown that QMiX was able to exert its antimicrobial action to the same depth as 6% NaOCl. Both of those irrigants killed bacteria in the entire length of DT tubules within three minutes. Even one minute of QMiX exposure was more effective than three minute use of 2% CHX or smaller concentrations of NaOCl (142). In addition, it has been shown that the proportion of one day old biofilm bacteria killed in the tubules was similar between QMiX and 6% NaOCl, but it was higher in QMiX than in lower concentrations of NaOCl and 2% CHX (30, 142). With a three week old biofilm tested, QMiX was less effective than 6% NaOCl, but still more effective than the other irrigating solutions (30). Another study investigated the ability to kill bacteria in the tubules when smear layer was present on the dentin. It has been shown that a ten-minute irrigation with 6% NaOCl, which was followed by QMiX, was as effective in killing bacteria in DT as 6% NaOCl followed by 17% EDTA and 2% CHX, and this effect was seen up to 300μm (141) Deactivation When the QMiX antimicrobial effect is tested and subjected to interaction with dentin powder, it is seen that its efficacy is delayed in time. However, the dentin does not completely eliminate the effect. Although the immediate antibacterial action is inhibited, with time the antimicrobial action does recover (236). 26

39 4. Interactions of Irrigation Solutions Multiple researchers recommend alternating irrigation solutions in order to increase their antimicrobial effect (152, 170, 221). However, mixing of the solutions can cause deactivation of at least one of them ( ). 4.1 NaOCl and CHX One of the first references that mentions the possibility of a reaction between NaOCl and CHX, proposes that a formation of chlorhexidine-chloride occurs, and this product in turn increases the effectiveness of irrigation (170). Other studies dispute that statement. The consensus is that the mixture results in a precipitation of a product that is toxic or potentially toxic (36-39, 148, 198, ). The reaction produces immediate colour change when 0.023% of NaOCl is mixed with 2% CHX, and an immediate brownish precipitate forms when 0.19% NaOCl is used (36). Even with the concentration of CHX being ten times lower at 0.2% CHX, the reaction with 0.5% NaOCl is also instantaneous (239, 240) Precipitate and PCA Detection Methods There is some discussion regarding the composition of the precipitate product found when CHX is mixed with NaOCl. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry (TOF-SIMS) studies have detected presence of chloroaniline, which has amine and a chlorine bound to benzene ring, in the precipitate (36). This can include any of the three isomers of aniline (Cl(C 6 H 4 )H 2 N), namely 2- chloroaniline, 3-chloroaniline, and 4-chloroaniline also named para-chloroaniline or 27

40 PCA. A confirmation test has been performed utilizing a technique of diazotization, which is used to detect the presence of an aromatic amine. It concluded that chloroaniline, an aromatic amine, is indeed present (38). In a subsequent study, using the gas chromatography-mass spectrometry method, the results have shown that 2- chloroaniline and 3-chloroaniline are not present. Only PCA is detected in the precipitate (37). A conflicting result has been obtained with the precipitate analyzed by the 1H nuclear magnetic resonance (NMR) spectroscopy and no measurable quantity of PCA has been detected (237). Another study using the Beilstein test and a solubility test confirmed the presence of chlorine and aniline, and by again NMR imaging has confirmed the para position occupied by chlorine (198). By using one-dimensional and two-dimensional NMR spectroscopy, one group has concluded that indeed a parasubstituted benzene compounds is present and it appears to be para-chlorophenylurea (PCU) and parachlorophenylguanidyl-1,6-diguanidyl-hexane (238). Since PCA, being aniline, is volatile, there is a possibility that the lack of detection is due to that characteristic. In an environment that allows the sample to evaporate, no PCA may be found. In the TOF- SIMS methodology, when the sample of the precipitate is cooled to prevent the vaporization, a large increase in the detection of PCA has been noticed, whereas at room temperature the detection is decreased (39). Electrospray ionization mass spectrometry (ESI-MS) once again confirmed the presence of PCA (242). Also, a gas chromatographymass spectrometry method has confirmed PCA presence using three part evidence. The chromatographic retention time, molecular ion with correct isotope pattern for chlorine element, and an electron impact spectrum with correct fragmentation pattern and ratios were noted (243). However, electrospray ionization quadrupole time-of-flight mass 28

41 spectrometry (ESIQ-TOF-MS) has failed to detect a signal consistent with PCA (148). Some of the conflicting results can be explained by the different detection sensitivities, and methods that require handling and dissolution of the precipitate affecting the recovery of the product. As failure to detect PCA in some of the studies may be related to the materials and methods used, and is not an evidence of absence, an assumption that PCA does form can be safely made Chemistry of PCA Formation The amount of PCA present in the solution is proportional to the concentration of NaOCl used in the mixture (36). Pure acetic acid can dissolve the precipitate but the colour change persists, explaining low polarity of the product and the fact that it can form a precipitate in an aqueous solution (239). The proposed initial reaction for the formation of the precipitate and PCA is an acid base reaction. CHX, a dicationic acid, donates protons, and alkaline NaOCl accepts those protons producing the precipitate, with PCA also forming and becoming a part of that precipitate (36, 202). An initial breakdown of CHX is proposed to be achieved by a chlorination of the guanidino nitrogen (148). Alternatively, in an acidic solution, a solvent attack on an electron-deficient carbon in CHX leads to a tetrahedral intermediate, with leaving groups being either PCA or ammonia. It is easier for PCA to leave the group, as ammonia is more basic in comparison to aromatic amine of PCA. Those reactions can form p- chlorophenylbiguanide-amidinourea or p-chlorophenylbiguanide-amidino-pchlorophenylurea. In basic environment, a tetrahedral intermediate is easily protonated and giving rise exclusively to PCU. The unsaturated carbon in PCU can be also attacked 29

42 to form a tetrahedral intermediate, which is able to degrade to PCA and carbamic acid (202) Effect of Precipitate on Dentin The precipitate can form when CHX is used after NaOCl, and allowed to mix with it in a root canal system. The precipitate covers the dentinal wall, with a thickness ranging from 139μm to 684μm (198). This thick precipitate, looking more like debris and unlike a smear layer, can lead to partial closure of the tubules on the inner surface of the dentin (240). It reduces the number of the open, patent DT, and is said to occlude the tubules (40, 244) Toxicity of PCA and Precipitate One study has suggested that PCA is non-toxic (184). However, when the precipitate was tested on rats to examine its cytotoxicity, it showed that it is more toxic than CHX or NaOCl, even more so in a short term testing (241). PCA has been shown to be carocinogenic and cytotoxic when tested on animals (245, 246). In a human subject, it can cause methemoglobinemia. This is regardless whether the exposure to PCA comes from PCA powder or from the spontaneous breakdown of CHX (247, 248). The cytotoxicity study of human fibroblasts subjected to PCA found its EC50 to be 14.8mM (249). It has been suggested that the precipitate formation and its effect on the periapical tissues may be responsible for a lower success of a root canal therapy when CHX is used as an adjunct in canals previously irrigated with NaOCl (156). 30

43 4.1.5 Prevention Since the reaction occurs when NaOCl is mixed with CHX, one way of preventing the formation is to avoid using both of these solutions together. However, this may not always be possible. Several studies investigated methods of prevention of the formation of the precipitate (40, 148, 198, 243, 250). When compared leaving NaOCl in the canal with the use of a paper point to remove NaOCl from the canal prior to CHX rinse, the paper point method could reduce, but not eliminate, the number of the tubules occluded by the precipitate in a root canal (40). Another method tested is to use an intermediate flush between the two irrigants (148, 198, 243, 250). It has been suggested to use citric acid before the rinse with CHX, and this allowed DT to remain open without the formation of precipitate (250). However, PCA is still present after this regimen (243). Distilled water is also suggested as another irrigant to prevent or at least reduce the formation of the precipitate (148). If saline or distilled water are used before CHX, then the thickness of the precipitate is decreased compared to the dentin where intermediate irrigation is not used. Ethanol, on the other hand, completely eliminates the formation of the precipitate, as determined by a visual inspection method (198). 4.2 NaOCl and EDTA When NaOCl is mixed with EDTA, immediate release of chlorine gas occurs. This can be easily seen as a bubbling action ( ). The loss of active chlorine is rapid. Even with small amounts of EDTA, up to 80% of chlorine is lost rapidly (149). Additionally, a drop in ph values can be detected (153). 31

44 4.2.1 Chemistry of Reaction The bubbling action, when EDTA is mixed with NaOCl, is seen as a consequence of the release of chlorine gas ( ). The following two reactions are responsible for this action: NaOCl + H2O NaOH + HOCl Na + + OH - + H + + OCl - 2HOCl + 2H + + 2e - Cl2 (g) + 2H2O (94, 148) Effect on Hard and Soft Tissue The loss of chlorine from NaOCl, and the solution, affects the ability to dissolve soft tissue. When tested, porcine and bovine tissues were not dissolved when EDTA was added to the NaOCl solution, in contrast to unmixed NaOCl that was able to completely liquefy them. This shows that the addition of the chelating agent leads to the deactivation of NaOCl (152, 153). However, the calcium sequestering ability of EDTA is not altered by NaOCl, even when tested over several hours. This can be seen in calcium titration experiments and testing of the effect on human teeth (149, 150, 152). Similarly, the smear layer removal ability of EDTA is not impaired by NaOCl (150) Prevention Intermediate flushes with distilled water are recommended to prevent or reduce the reaction between NaOCl and EDTA (148). Another way to avoid the reaction is using a copious irrigation with NaOCl after EDTA use (149, 150). This action eventually dilutes the EDTA component to a minute level that is unlikely to cause the reaction. 32

45 4.3 CHX and EDTA An instantaneous reaction occurs when CHX is mixed with EDTA (251). In one study, this reaction was seen to form an insoluble pink powdery precipitate (251). In other studies, it was described as white, or white and milky (148, 252). When further investigated, using atomic absorption spectrophotometry, it has been shown that the precipitate contains EDTA and CHX in a 1:1 ratio. Therefore, a proposed reaction, forming the precipitate, is the neutralization of the cationic CHX by anionic EDTA, resulting in salt formation (251). When additionally studied with a high performance liquid chromatography, the precipitate has been described to contain 90% of either EDTA or CHX, without any degradation of CHX and without any PCA formation (252). ESIQ- TOF-MS analysis of the product reveals the presence of the original compounds in the precipitate, once again suggesting an acid base reaction without the breakdown of CHX (148) Chemistry of Reaction The following equations are proposed to describe the reaction when EDTA is mixed with CHX: 2HEDTA 3- (aq) + 3H2CHX 2+ (aq) (HEDTA) 2 (H 2 CHX) 3 H 2 CHX 2+ (aq) + 2OH - (aq) CHX (s) + H2O (l) (252). Both reactions can lead to a formation of a solid component containing CHX. However the second reaction, during formation, would have to trap EDTA to contain it (252). 33

46 5. Methods of Studying Dentin and Irrigation 5.1 Dentin, Tubule, Infection, and Irrigation Multiple techniques have been employed to study microbial infections of dentin, canal and the tubules, and the effect of irrigation on dentin and microbes. Many imaging techniques have been developed to obtain detailed information, and those include light, transmission or scanning electron microscopy (3, 6, 24, 53, 65, 67, 82, 97, 119, 120, 142, 223, 224, 240, 244, 253). Those techniques have the ability of visualizing dentin and the position of the microorganisms in the canals and the tubules. Some modified techniques have the ability to determine whether the bacteria are alive or dead, such as confocal laser scanning microscopy (30, 141, 142, 163). This has led researchers to be able to indirectly analyze the depth of the penetration of an irrigant by studying the level of the dentinal tubule where the bacteria are vital. Shallower depths where bacteria are found dead indicate the depth limit of the penetration of the irrigant. Another indirect method to measure the depth is the use of a substrate that has the ability to react with the irrigant. The change in the substrate can then be observed indicating the depth of the penetration (138, 139). 5.2 Interactions of Irrigating Solutions When two solutions are mixed and a reaction occurs, it is possible that the event can be visually inspected. A reaction can be described, for example, as a formation of bubbles ( ). Another type of information can be obtained by looking at the clarity change of the solution, and the development of a precipitate (148, 251, 252). In addition, a colour change can be detected (36, 38, 40, 198, 237, 238, 242, 243, 251). These, however, do 34

47 not provide much evidence regarding the product that forms. This information can only be obtained via chemical and physical analytical methods and techniques. Using a solubility test, Beilstein test, diazotization, high performance liquid chromatography, gas chromatography-mass spectrometry, atomic absorption spectrophotometry, X-ray photoelectron spectroscopy, X-ray diffraction, NMR (one or two-dimensional) spectroscopy, ESI-MS, ESIQ-TOF-MS, TOF-SIMS, or the combination of any of those techniques can provide the composition and the molecular structure information of the product (36-39, 148, 198, 210, 237, 238, 242, 243, 251, 252). 6. Time-of-Flight Secondary Ion Mass Spectrometry For over a half a century, secondary ion mass spectrometry (SIMS) has been used to localize ions on a tested surface (254). An improvement on this method has been achieved by adding a time-of-flight analyzer. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a very sensitive method to determine masses of chemical compounds on a surface and to chemical map a surface (255). It has been used to measure the composition of products that form a precipitate (36, 37, 39). Also, it was utilized to study the hard tissue of the bone and dentin, and to obtain images and study the ion charge distribution of certain elements and functionalities such as Ca +, Na +, PO - 2, and PO 3 - in those tissues ( ). Its short analysis depth of around 1nm allows molecular orientation to be revealed (255). TOF-SIMS is ideal for compounds with molecular weights smaller than 1,500 Daltons, but can detect fragments up to 10K Daltons (260). 35

48 6.1 Principles of TOF-SIMS Primary ion source guns such bismuth (Bi + n ) liquid metal ion guns (LMIG) or fullerene (C + 60 ) direct a focus a beam of ions towards a point on a target surface of tested material under ultra-high vacuum (255, ). The primary ions encounter the surface and penetrate it to a certain depth, causing a collision cascade. Energy transfer allows atoms and fragments to escape. Most of those atoms and fragments are neutral, but a tiny percentage, 0.1% or less, may possess a charge (42, 255, 264, 265). The secondary ions are accelerated by the extractor to a set and common energy. Ions of smaller mass travel at higher velocity and are detected earlier than larger ions (264). The mass spectrum is obtained. It is linear in nature, but it becomes necessary to internally calibrate the data obtained. Internal mass calibration is usually carefully performed using common ions of known mass, such as C +, C 2 H + 4,CH + 3, C 2 H +, for positive spectra and CH, OH, and C 2 H for negative ones (261, 265). The mass spectrum of a target usually contains multiple peaks, resulting from the fragmentation pattern of the molecules. Generally, it can be said that the mass spectrum of a particular molecule forms a fingerprint pattern, which then can be used to identify the substance. Often an aid of SIMS library can be employed, where not only the presence of peaks can be helpful but their intensity relative to each other (264). When the SIMS library information is not available or the fingerprint has not been determined then the molecular and spatial information must be deduced from the available spectrum (42). To process a digital image of the target surface, a portion of it, a set of pixels, can be separated and its mass spectrum analyzed. This can be repeated for a different portion of the surface and a comparison can be made. Alternatively, a section of a mass spectrum can be chosen and the image of the surface 36

49 yielding that particular spectrum section displayed. Summation, subtraction, colour overlay, and other operations, such as Principal Component Analysis can be performed on those portions and sections (265) Modes of Operation Several modes of operation are possible with TOF-SIMS depending on the information required. When the primary ion beam bursts are short, high mass resolution results, and by the bunching of the pulses full detailed spectrum of secondary ion can be obtained. The problem with this mode is that the spatial resolution suffers. In order to counteract the loss of spatial resolution another mode can be used. The primary ion beam, usually sourced with LMIG, can be narrowed by a collimator aperture and directed towards a particular pixel at each burst. A mass spectrum is then obtained for each pixel, and only the top monolayer is sampled. The number of secondary ions is diminished. This greatly improves spatial resolution at a cost of lower mass resolution (42, 264). A different mode of operation is used when a 3D depth profiling is performed. The surface is first sputtered to remove the molecules from the surface before the analysis is performed on deeper layers. When repeated multiple times, a 3D image can be obtained. This can be performed with the same primary ion source, such as C + 60, used for sputter erosion and analysis. Alternatively and most commonly, two different guns are used, such as fullerene for sputter erosion and Bi n + LMIG for analysis. Initially, the un-sputtered surface is analyzed with LMIG, followed by a surface erosion with C The cycle continues until desired depth is reached. The data is then used to produce a 3D image of the target sample ( ). 37

50 6.2 Dentin, CHX, and Precipitate Studies Only three available published studies investigate dentin using the TOF-SIMS technology and all those studies look at bovine dentin ( ). The data shows that a detailed map of the bovine dentin surface distribution of main amino acids, in addition to elements, can be obtained and analyzed (256). It was found that in a positive ion image of dentin, calcium (40u), sodium (23u), potassium (39u), magnesium (24u), and a fragment common to many proteins, CH 4 N (30u), were present in significant amounts, with calcium being the most intense and magnesium the least. Calcium intensity is more concentrated in the peritubular dentin compared to the intertubular dentin; however, the calcium counts are decreased in a narrow band surrounding the tubule. The tubule itself also holds sodium containing components. The fragments specific to multiple aminoacids are also noted and can be visualised in the dentin sample. For example, glutamic acid and serine are more intense in the peritubular dentin. In the negative ion view, protein indicators CN - and OCN - peaks are clearly observed while carboxylate (HCO - 2 ) intensity is quite weak. CN - and OCN - are more intense in the peritubular dentin. Phosphates (P -, PO -, PO - 2 ) signals are strong and are distributed evenly across the peritubular and intertubular dentin. This indicates that they are part of the hydroxyapatite signal. Phosphate PO - 3 is present in a greater intensity in intertubular dentin and is thought to originate from proteins (256). TOF-SIMS can show colocalization of calcium with glutamic acid within bovine peritubular dentin, and calcium with proline, hydroxyproline, and glycine in the intertubular dentin (257, 258). A conclusion is reached that the TOF- SIMS technique may be the best way to examine the structure of the intact tooth (258). All three studies provide validation for the use of TOF-SIMS technology to study dentin. 38

51 TOF-SIMS is also used to study the composition of precipitate formed while mixing NaOCl and CHX (36, 37, 39). It has been shown that CHX has characteristic peaks present at 505u, 195u, 170u, 153u, and 127u, which are consistent with the parent molecule (C 22 N 10 H 31 Cl + 2 ), Cl(C 6 H 4 )C 2 H 4 N + 4, Cl(C 6 H 4 )CH 5 N + 3, Cl(C 6 H 4 )CH 2 N + 2, and PCA (Cl(C 6 H 4 )H 2 N + ) respectively. There is also peak at 111u consistent with Cl(C 6 H 4 ). When mixed with NaOCl and the precipitate tested at room temperature 505u, the peak is absent, but 153, 170, and 195 are still present, with possibly increased 127u peak, and an increased 111u peak. When the precipitate is cooled, the 127u peak assigned to PCA is significantly enlarged. The residual CHX signal may be still present when the precipitate forms, it may arise from the precipitate or CHX adsorbed into the precipitate (39). Another TOF-SIMS study looking at CHX adsorption into the skin has utilized not only the positive signal for CHX 505u but also Cl - at 37u, and Cl(C 6 H 4 )CN - 2 at 151u. TOF- SIMS mapping, performed using PO 2 - and CNO - ions, has been utilized to compartmentalize a sample of tissues into histological components. Images obtained with high resolution mode highlight the presence of CHX within a small layer of the sampled tissue (269). 7. Penetration of Precipitate into Dentinal Tubules It is well established that a precipitate forms when NaOCl is mixed with CHX (36-39, 148, 198, 237, 238). In addition, PCA can be detected in this precipitate using TOF-SIMS methodology (36-39, 198). Previous publications showed that the precipitate and PCA could form inside the root canal and occlude the DT, however, the identification of the product formed was performed after the precipitate was removed from the canal (40, 39

52 198). To date, no available studies have attempted to identify the precipitate without removing it from the dentin surface, and none have investigated the ability of PCA and the precipitate to penetrate the DT. Moreover, no information is available on the interaction between NaOCl and QMiX other than a pilot study showing a colour change when these two solutions are mixed together, thus, a potential for PCA formation exist. Due to its high toxicity and potential to lower the success rate of a root canal therapy, it is important to investigate any possible formation of PCA in a human body (156, 241, ). The potential for PCA formation in DT exist when NaOCl and CHX or QMiX are allowed to mix while in contact with dentin, since it has been shown that NaOCl, CHX, and QMiX can independently penetrate the tubules (30, 141, 142). 40

53 II. RATIONALE AND AIMS 1. Rationale It is unknown whether the precipitate and PCA can extend into or form inside DT. If the precipitate or PCA can be found in DT, then those DT may act as a reservoir. Attempts to remove the already formed precipitate from the root canal walls may not be completely successful in eliminating it from the tubules. In addition, if PCA can form inside DT then efforts to completely prevent the formation in the canal by using intermediate irrigants may also be unsuccessful in DT. 2. Aims The aim of this study was to determine if the precipitate and PCA can form in DT, when dentin is irrigated with NaOCl, EDTA and a final irrigation with CHX or saline and QMiX. This study was looking to analyze the surface and the longitudinal sections of the irrigated dentin utilizing TOF-SIMS. The specific aim was to investigate whether there is a detectable presence of Cl - (35u and 37u), PCA (127u), other products of CHX breakdown (153, 170u, 195u), and CHX (505u), on the surface, operating under the high mass and spatial resolution mode and penetrating into DT utilizing the high spatial resolution mode. 41

54 III. ARTICLE (Submitted for publication) Qualitative analysis of precipitate formation on the surface and in the tubules of dentin irrigated with chlorhexidine or QMiX as a final rinse. Kamil P. Kolosowski, HBSc, DDS, Rana N.S. Sodhi, BSc, MSc, PhD*, Anil Kishen, BDS, MDS, PhD, Bettina R. Basrani, DDS, MSc, PhD**, Discipline of Endodontics, Faculty of Dentistry, University of Toronto, Toronto, Canada *Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto Canada **Corresponding author Dr. Bettina Basrani Associate Professor, Co-Director MSc Endodontic Program University of Toronto, Faculty of Dentistry 124 Edward Street, Room 348C Toronto, ON, M5G 1G6, Canada Bettina.basrani@dentistry.utoronto.ca 42

55 Abstract Introduction Interaction of sodium hypochlorite (NaOCl) mixed with chlorhexidine (CHX) produces a brown precipitate containing para-chloroaniline (PCA). When QMiX is mixed with NaOCl no precipitate forms but color change occurs. The Aim The aim of this study was to qualitatively assess the formation of precipitate and PCA on the surface and in the tubules of dentin irrigated with NaOCl, followed either by ethylenediaminetetraacetic acid (EDTA), NaOCl and CHX, or by saline and QMiX, using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Materials and Methods Dentin blocks were obtained from human maxillary molars, embedded in resin and crosssectioned to expose dentin. Specimens in Group 1 were immersed in 2.5% NaOCl, followed by 17% EDTA, 2.5% NaOCl, and 2% CHX. Specimens in Group 2 were immersed in 2.5% NaOCl, followed by saline and QMiX. The dentin surfaces were subjected to TOF-SIMS spectra analysis. Longitudinal sections of dentin blocks were then exposed and subjected to TOF-SIMS analysis. All samples and analysis were performed in triplicate for confirmation. 43

56 Results TOF-SIMS analysis of Group 1 revealed an irregular precipitate, containing PCA and CHX breakdown products, on the dentin surfaces, occluding and extending into the tubules. In TOF-SIMS analysis of Group 2, no precipitate, including PCA, were detected on the dentin surface or in the tubules. Conclusion Within the limitations of this study, precipitate containing PCA was formed in the tubules of dentin irrigated with NaOCl followed by CHX. No precipitate or PCA were detected in the tubules of dentin irrigated with NaOCl followed by saline and QMiX. Key words: Sodium hypochlorite; chlorhexidine; QMiX; parachloroanilline; precipitate; dentin; Endodontics; Dentistry; TOF-SIMS 44

57 Introduction Microbial infections are the most common cause of an endodontic disease (1, 2). To critically reduce bacterial loads, endodontic treatment regimens combine mechanical instrumentation with chemicals, namely irrigation and medication (3). The most commonly used irrigation solution is sodium hypochlorite (NaOCl). It is antimicrobial and an effective tissue dissolving agent (4-6). It is used at a concentration varying between 0.5% and 6% (4). One disadvantage of NaOCl is its ineffectiveness in removing the smear layer (7). Chlorhexidine Digluconate (CHX) has also been suggested as a possible irrigation solution in endodontic treatment (4, 6). It exhibits antimicrobial activity in addition to substantivity (4, 6, 8). The concentrations used vary from 0.12% to 2% (4, 6). Its mode of action is a cationic binding, which causes bacterial cell membranes disruption (9). As a disadvantage, CHX is ineffective in removing the smear layer and possesses no tissue dissolving ability (10). Ethylenediaminetetraacetic acid (EDTA) is a chelating agent that dissolves inorganic components of the dentin but not the organic components (4, 6, 7). Concentration used is normally 17% (4). Although its main function is to remove smear layer, dentin erosion can occur with prolonged exposures of 10 minutes (4, 11). QMiX is a novel irrigation compound containing EDTA, CHX and a non-specified detergent (12). It is antimicrobial and has been shown to remove smear layer. The detergent s function is to decrease surface tension and increase surface wettability (12, 13). In vitro studies showed that NaOCl and CHX can diffuse into dentinal tubules (DT) to a depth of 300μm (14, 15), where they can exert a detrimental effect on bacteria 45

58 (16). Interestingly, the effect of QMiX on bacteria was reported to extend deeper into the tubules than CHX and similar to NaOCl, up to 500μm (16, 17). In endodontics, irrigation solutions are used in succession, and it is crucial to discern that irrigation solutions used in succession can react with each other. This is especially evident when NaOCl is mixed with CHX. This interaction breaks down CHX, produces a brown precipitate containing para-chloroaniline (PCA) (18-20), and can occlude dentinal tubules (DT) (21). The amount of PCA is directly proportional to the concentration of NaOCl used (18). A reaction also occurs when QMiX is mixed with NaOCl, resulting in a visually detectable color change but without precipitate formation (pilot study). Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a method used in surface chemistry that has been successfully adapted to analyse mineralized tissue such as dentin (22). It consists of ion gun pulsing high energy ions into the target surface. This bombardment causes collision cascades, disrupting atoms in the top 1 or 2 monolayers, causing an ejection and emissions of secondary ions from the surface. The charged ion mass is determined by the flight time from the surface to the detector (23). Mass spectra with multiple peaks that arise as a result of molecular fragmentations can be then analysed or compared with SIMS library to obtain parent molecular information. When subjected to TOF-SIMS analysis, NaOCl/CHX precipitate yields peaks at 127u, 153u, 170u and 195u, PCA yields a specific 127u peak, and CHX yields a parent molecule peak at 505u (18). 46

59 The penetration of the precipitate and PCA into dentinal tubules has not been determined. If PCA is formed inside dentinal tubules, this may act as a reservoir of PCA even if attempts are made to eliminate its formation in the main root canal. Therefore, the aim of this study was to determine the formation of precipitate and PCA on the surface and in the tubules of dentin irrigated with NaOCl, EDTA and subsequent irrigation with either CHX, or saline and QMiX, using qualitative time-of-flight secondary ion mass spectrometry analysis. Materials and Methods Preparation of samples The study was approved by the university s Health Sciences Research Ethics Board. Thick 2mm horizontal slices of mid root dentin were obtained from 4 extracted noncarious human upper molars that had been previously stored in methyl alcohol, using a diamond coated saw (Leica EM TXP Target Sectioning System, Leica Microsystems GmbH, Vienna, Austria). The slices were split vertically, to create 12 dentin blocks. Blocks were embedded in low viscosity epoxy resin (Epo-Thin, Buehler, Lake Bluff, USA), and allowed to set for 24h. The resin covering the root canal aspect of the embedded block was then carefully removed with a diamond blade (Leica EM UC6/FC6 Ultra-cryomicrotome, Leica Microsystems GmbH, Vienna, Austria), to expose dentin with the dentinal tubules oriented approximately perpendicular to the exposed surface. 47

60 Treatment of samples Blocks were randomly divided into two groups. Group 1 (G1 - CHX) - 6 blocks were immersed in 5 ml 2.5% NaOCl (Sodium hypochlorite, Lavo inc., Montreal, QC, Canada) for 3 min, followed immediately by 5 ml 17% EDTA (EDTA, Vista Dental Products, Racine, WI, USA) for 1 min, fresh 5 ml. 2.5% NaOCl for 2 min. and a final immersion in 5 ml 2% CHX (chlorhexidine digluconate BP, Medisca, Montreal, QC, Canada) for 1 min. Group 2 (G2 - QMiX) - 6 blocks were immersed in 5 ml 2.5% NaOCl for 3 min. followed immediately by immersion in 5 ml sterile saline (Aqualite, Hospira, Montreal, QC, Canada) (as per manufacturer s recommendation) then 5 ml QMiX (Dentsply Tulsa Dental, Tulsa, OK, USA) for 1 min. Samples were dried on a bench top overnight. TOF-SIMS analysis Cross Sections The surface analysis of 3 blocks from each group was performed by TOF-SIMS (TOF- SIMS IV, ION-TOF GmbH, Münster, Germany). A bismuth (Bi) liquid metal ion gun was used as the primary ion source utilising the Bi ++ 3 cluster. The gun was operated in a high-mass-resolution bunched mode over an area of 500μm x 500μm for 100 sec. In addition, spectra were obtained in a high-spatial-resolution imaging mode ( burstalignment (BA)). Images (256 pix x 256 pix) were obtained from 20 scans over an area of 150μm x 150μm (23). Charge neutralization was achieved using pulsed electron flood gun. Both positive and negative polarity spectra were obtained. Mass scale calibration was obtained by clearly identifiable and well spaced peaks for both the positive and negative spectra. 48

61 TOF-SIMS analysis - Longitudinal Sections The remaining 3 blocks from each treatment group were removed from the resin with the diamond coated saw, turned 90 o, and re-embedded in low viscosity resin with the DT now oriented parallel to the surface. The resin was again shaved from the surface to expose DT in length, to determine presence and depth of precipitate. The surface of the longitudinal sections prepared for each group (G1 - CHX and G2 - QMiX) where again analysed with TOF-SIMS in BA imaging mode using the previously stated conditions. Results Precipitate formation A brown precipitate was visually observed on the cross sectional surfaces of all G1 samples immediately after CHX immersion. No color change or precipitate was visualized on the cross sectional surface of G2 samples after immersion in QMiX. TOF-SIMS analysis Cross sections In positive ion mass spectra analysis, intense peaks for residual CHX, and CHX breakdown products, including PCA, were detected on the surface of all G1 (CHX) blocks (Figure 1A). In negative ion mass spectra analysis, intense peaks of chlorine (Cl - ), but no PO - 2 and PO - 3, were detected on the surface (Figure 1B). An irregular precipitate occluding nearly all DT could be seen on the surface in BA mode. (Figure 2A, B). In positive ion mass spectra analysis, no precipitate, CHX, or CHX breakdown products, including PCA, were detected on the dentin surface of all G2 (QMiX) blocks (Figure 1C). 49

62 In negative ion mass spectra analysis, significant peaks were, however, seen for PO 2 -, PO 3 -, and Cl - (Figure 1D). Patent DT with an absence of precipitate could be seen on the surface in BA mode (Figure 2C, D). TOF-SIMS analysis Longitudinal sections In positive BA imaging, breakdown products of CHX, and a consistent reading for PCA, were visualized on the surface and in the DT of all G1 (CHX) blocks (Figure 3A). In negative BA imaging, Cl - was present on the surface and in the DT of all G1 (CHX) blocks (Figure 3B). In positive BA imaging, no breakdown products of CHX, including PCA, was visualized on the surface or in the DT of all G2 (QMiX) blocks (Figure 3C). Under negative BA imaging, Cl - could not be visualized on the surface or in the DT of all G2 (QMiX) blocks (Figure 3D). Discussion Up until the time this manuscript was written, there had been no published studies of TOF-SIMS analysis of human dentin subjected to new irrigation modalities. In addition, no published papers explored the presence or absence of PCA inside DT, or on dentin surface without first removing the precipitate for testing (24). QMiX is a novel root canal irrigant formulated to remove smear layer and enhance root canal disinfection (12, 13). It is recommended that it be used as the final irrigant, following irrigation with NaOCl and saline (25). Its composition is proprietary, 50

63 but the manufacturer has disclosed that it contains EDTA, CHX and a surface-active detergent (12). In view of the previous reports highlighting the interaction between CHX and NaOCl (18-20, 24, 26) and the formation of PCA with its known toxicity (27-29), this study set out to examine whether a potential for such precipitation existed when QMiX inadvertently came in contact with NaOCl during clinical use. TOF-SIMS is mostly used as a qualitative study tool with fragmentation pattern peaks or fingerprinting analysis. TOF-SIMS technique was chosen for this study due to the advantages of great surface sensitivity, which can measure composition of 1-2 monolayers. It exhibits good spatial and mass resolutions, and provides chemical mapping of the samples with analysis of the images. TOF-SIMS however, is sensitive to contamination and demonstrates a difficulty in data quantification. This latter disadvantage implies that the intensities of individual peaks of the ions cannot be compared among different samples (23, 26). In dentin blocks treated in accordance with the protocol used in Group 1 (CHX) a precipitate formed (18-20, 24, 26, 30-32) and was present on the dentin surface as had been described in previous publications (21, 24). The precipitate occluded the DT (21), to - the extent that it prevented detection of dentin as evidenced by lack of emission of PO 2 and PO - 3 ions from the surface. This precipitate was shown to contain PCA in some studies (18-20, 24, 26), but not in others (30-32). It could be argued that the inability to identify PCA was related to the use of different detection techniques, with a reduced 51

64 sensitivity to low molecular weight products or the use of solvents that may have undermined PCA detection (30-32). TOF-SIMS analysis of Group 1 (CHX) blocks yielded a 127u peak characteristic of PCA. An analysis of CHX alone yields intense peaks at 127u, 153u, 170u, and 195u, in addition to 505u, its parent molecule. This fingerprint of CHX and the relative intensities of its different peaks are altered when CHX is broken down (18, 26). The precipitate formed on dentin when NaOCl/CHX were mixed showed a low parent peak, and a more intense peak assignable to PCA. This highlights the previously identified degradation of CHX exposed to NaOCl and the formation of its degradation products (18, 26). In Group 2 (QMiX ) no precipitate could be detected on the dentin surface indicating its absence or its presence below detectable levels. The absence of a PCA containing precipitate could be considered a positive finding when QMiX is used, considering its toxic potential (27-29). It also represents a confirmation of its protocol for use, which advises a saline rinse following NaOCl and prior to QMiX (25). We found the presence of PCA and Cl - containing products, which would include CHX and PCA in the DT of blocks exposed to CHX after NaOCl (G1). This finding was not unexpected given that both NaOCl and CHX have the ability to enter DT as has been shown in previous studies that have used dyes (14, 15), or measured their antibacterial penetration (16, 17, 33). In this study we were not able to detect a peak characteristic of CHX, or its breakdown products inside the DT of all the blocks in the QMiX (G2) group. This was surprising since previous studies have shown that it was effective in 52

65 destroying bacteria in DT 500μm below the surface (16, 33). We also found a consistent absence of Cl - in the DT of the QMiX group. This could reflect a low concentration of CHX in QMiX or possibly that a product consistent with detergent, as reflected by the presence of peaks at 128u, 142u, 156u, 170u, 184u, and 198u, on the dentin surface (Figure 1C) blocked detection of CHX or inhibited its penetration into the DT. With TOF-SIMS analysis, it needs to be stressed that artifacts can occur due to manner in which the dentin blocks were handled. Regardless of this possibility, however, the finding of a PCA containing precipitate in all Group 1 (CHX) blocks and none in all Group 2 (QMiX ) blocks left little doubt that the findings were correct. Finally, even though there is still debate whether a precipitate containing PCA is present in the root canal after it is irrigated with CHX and NaOCl, its potential as a carcinogen in animal models, its cytotoxicity on human cells, and its ability to cause methemoglobinemia in neonates (27-29) makes it imperative that every precaution be exercised to avoid its production. It is for that reason that we endorse the manufacturer s recommendation that a saline rinse be used in the root canal after NaOCl and prior to the final rinse with QMiX (25), or that distilled water and ethanol as an intermediate alternative (24). Conclusion Within the limitations of this study, PCA containing precipitate was formed on the surface and in the tubules of dentin irrigated with NaOCl followed by CHX. No 53

66 precipitate or PCA was detected in the tubules of dentin irrigated with NaOCl followed by saline and QMiX. Acknowledgments This study was supported by a grant from Canadian Academy of Endodontics Endowment Fund and Endo Tech. The authors thank the Surface Interface Ontario for its assistance in the conduct of these experiments, and also Dr. Torneck and Dr. Friedman for their feedback on the manuscript. The authors deny any conflicts of interest related to this study. 54

67 References 1. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 1965;20: Peters LB, van Winkelhoff AJ, Buijs JF, Wesselink PR. Effects of instrumentation, irrigation and dressing with calcium hydroxide on infection in pulpless teeth with periapical bone lesions. Int Endod J 2002;35(1): Shuping GB, Orstavik D, Sigurdsson A, Trope M. Reduction of intracanal bacteria using nickel-titanium rotary instrumentation and various medications. J Endod 2000;26(12): Haapasalo M, Shen Y, Qian W, Gao Y. Irrigation in endodontics. Dent Clin North Am 2010;54(2): Byström A, Sundqvist G. Bacteriologic evaluation of the effect of 0.5 percent sodium hypochlorite in endodontic therapy. Oral Surg Oral Med Oral Pathol 1983;55(3): Zehnder M. Root canal irrigants. J Endod 2006;32(5): Baumgartner JC, Mader CL. A scanning electron microscopic evaluation of four root canal irrigation regimens. J Endod 1987;13(4): White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. Journal of endodontics 1997;23(4): Davies A. The mode of action of chlorhexidine. J Periodontal Res Suppl 1973;12: Naenni N, Thoma K, Zehnder M. Soft tissue dissolution capacity of currently used and potential endodontic irrigants. J Endod 2004;30(11): Calt S, Serper A. Time-dependent effects of EDTA on dentin structures. J Endod 2002;28(1): Stojicic S, Shen Y, Qian W, Johnson B, Haapasalo M. Antibacterial and smear layer removal ability of a novel irrigant, QMiX. Int Endod J 2012;45(4): Eliot C, Hatton JF, Stewart GP, Hildebolt CF, Jane Gillespie M, Gutmann JL. The effect of the irrigant QMix on removal of canal wall smear layer: an ex vivo study. Odontology Kuga MC, Gouveia-Jorge É, Tanomaru-Filho M, Guerreiro-Tanomaru JM, Bonetti- Filho I, Faria G. Penetration into dentin of sodium hypochlorite associated with 55

68 acid solutions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;112(6):e Zou L, Shen Y, Li W, Haapasalo M. Penetration of sodium hypochlorite into dentin. J Endod 2010;36(5): Ma J, Wang Z, Shen Y, Haapasalo M. A new noninvasive model to study the effectiveness of dentin disinfection by using confocal laser scanning microscopy. J Endod 2011;37(10): Wang Z, Shen Y, Haapasalo M. Effect of smear layer against disinfection protocols on Enterococcus faecalis-infected dentin. J Endod 2013;39(11): Basrani BR, Manek S, Sodhi RN, Fillery E, Manzur A. Interaction between sodium hypochlorite and chlorhexidine gluconate. Journal of endodontics 2007;33(8): Basrani BR, Manek S, Mathers D, Fillery E, Sodhi RN. Determination of 4- chloroaniline and its derivatives formed in the interaction of sodium hypochlorite and chlorhexidine by using gas chromatography. J Endod 2010;36(2): Basrani BR, Manek S, Fillery E. Using diazotization to characterize the effect of heat or sodium hypochlorite on 2.0% chlorhexidine. J Endod 2009;35(9): Bui TB, Baumgartner JC, Mitchell JC. Evaluation of the interaction between sodium hypochlorite and chlorhexidine gluconate and its effect on root dentin. J Endod 2008;34(2): Gotliv BA, Robach JS, Veis A. The composition and structure of bovine peritubular dentin: mapping by time of flight secondary ion mass spectroscopy. J Struct Biol 2006;156(2): Sodhi RN. Time-of-flight secondary ion mass spectrometry (TOF-SIMS):-- versatility in chemical and imaging surface analysis. Analyst 2004;129(6): Krishnamurthy S, Sudhakaran S. Evaluation and prevention of the precipitate formed on interaction between sodium hypochlorite and chlorhexidine. J Endod 2010;36(7): Dentsply Tulsa Dental Specialities. QMiX 2 in 1 Irrigating Solution, Directions for Use. In.; Sodhi R, Manek S, Fillery E, Basrani B. Tof-SIMS studies on chlorhexidine and its reaction products with sodium hypochlorite to ascertain decomposition products. Surface and Interface Analysis 2011;43(1-2):

69 27. Chhabra RS, Huff JE, Haseman JK, Elwell MR, Peters AC. Carcinogenicity of p- chloroaniline in rats and mice. Food Chem Toxicol 1991;29(2): van der Vorst MM, Tamminga P, Wijburg FA, Schutgens RB. Severe methaemoglobinaemia due to para-chloraniline intoxication in premature neonates. Eur J Pediatr 1990;150(1): Lueken A, Juhl-Strauss U, Krieger G, Witte I. Synergistic DNA damage by oxidative stress (induced by H2O2) and nongenotoxic environmental chemicals in human fibroblasts. Toxicol Lett 2004;147(1): Prado M, Santos Júnior HM, Rezende CM, Pinto AC, Faria RB, Simão RA, et al. Interactions between irrigants commonly used in endodontic practice: a chemical analysis. J Endod 2013;39(4): Thomas JE, Sem DS. An in vitro spectroscopic analysis to determine whether parachloroaniline is produced from mixing sodium hypochlorite and chlorhexidine. J Endod 2010;36(2): Nowicki JB, Sem DS. An in vitro spectroscopic analysis to determine the chemical composition of the precipitate formed by mixing sodium hypochlorite and chlorhexidine. J Endod 2011;37(7): Wang Z, Shen Y, Haapasalo M. Effectiveness of endodontic disinfecting solutions against young and old Enterococcus faecalis biofilms in dentin canals. J Endod 2012;38(10):

70 Figure Legends Figure 1. Selected TOF-SIMS mass spectra of dentin surfaces with high-mass-resolution: (A) positive ion of G1 (CHX); (B) negative ion of G1 (CHX); (C) positive ion of G2 (QMiX); (D) negative ion of G2 (QMiX). Marked are the positions of peaks assignable to: CHX + at 505u (right inset); breakdown products of CHX at 153u, 170u, 195u, including PCA + at 127u; Cl - at 35u and 37u; PO - 2 at 63u; PO - 3 at 79u. Note the relative abundance of: PCA + in G1, absence of PO - 2 and PO - 3 in G1, absence of PCA + in G2; and abundance of Cl - in G1 and G2 Figure 2. High-spatial-resolution (BA) TOF-SIMS images of ion distribution on dentin surface: (A) positive ions of G1 (CHX); (B) negative ions of G1 (CHX); (C) positive ions of G2 (QMiX): (D) negative ions of G2 (QMiX). Total shows raw image, ClC 6 H 4 NH 2 + shows distribution of PCA. Note the irregular precipitate, containing PCA, on the surface of G1, and smooth appearance with patent dentinal tubules in G2. (mc maximum ion count in one pixel, tc total ion count in the entire image) Figure 3. High-spatial-resolution (BA) TOF-SIMS images of ion distribution in longitudinal sections of dentin: (A) positive ions of G1 (CHX); (B) negative ions of G1 (CHX); (C) positive ions of G2 (QMiX); (D) negative ions of G2 (QMiX). Pulp space is on topmost and dentin bottommost in each image. Total shows raw image, ClC 6 H 4 H 2 N+ + ClC 6 H 4 CH 2 N 2 + shows distribution of PCA and CHX breakdown products, and Cl Cl - show distribution of chlorine. Note the irregular precipitate on the surface 58

71 (green arrows), the extension of PCA and CHX breakdown products, in addition to chlorine, into DT in G1 (yellow arrows), and lack thereof in G2. (mc maximum ion count in one pixel, tc total ion count in the entire image) 59

72 Figure 1. 60

73 Figure 2. 61

74 Figure 3. 62

75 IV. DISCUSSION 1. Methodology This in-vitro study was performed because extensive search failed to show any studies that directly examined the infiltration of CHX into DT. No available studies were testing the formation or penetration of the precipitate and PCA in DT. Although a conclusion was previously reached that QMiX is able to penetrate the tubules, this statement was made after testing the antimicrobial effect of this product in DT (30, 141, 142). An outcome study showed that irrigation with CHX, lowered the success of a root canal therapy, and a suggestion was made that the precipitate might be responsible for the persistent inflammation at the apex of a treated tooth (156). Initially, we made an attempt to test a canal wall for the formation of the precipitate. This has proven to be an ineffective method as we noted that the curved canal surface was contaminated with dentin powder during the sectioning of the tooth. In addition, TOF- SIMS imaging and analyses are best done with a flat surface (42, 255, 260, 261, 264, 270). Therefore, a decision was made to study the precipitate on dentinal blocks. This reduced the possibility of topographical effects of the surface to factor into the results, and eliminated a number of variables, such as different radii of curvature, uneven surface that could cause lingering of the irrigant sample, cracks and groves that could harbor tissue or contaminants. Upper molars were chosen for this study, as it has been published that maxillary teeth possess DT that exhibit smaller change in the direction (64). This was an important characteristic, as the preparation of the samples involved the exposure of DT in perpendicular direction for the irrigation and TOF-SIMS analysis. In addition, the testing 63

76 of the penetration involved sectioning the dentin in a parallel direction to the DT. Although, this was not perfectly accomplished in all the samples, it was nonetheless shown that the penetration could be detected. The age of the teeth could play a role, since older dentin is more likely to be mineralized (52, 56, 66). This study used the teeth that were anonymously donated and no attempt was made to determine their age. However, all samples yielded similar results indicating either that the effect of the age on the results is negligible or that the teeth used had similar mineral deposition in the tubules. Mid-root dentin was chosen to reduce the chance of using both a age related sclerotic dentin and tertiary dentin formed due to occlusal irritation (52, 66). During the use of the Leica instrument to section the teeth, saline irrigation was avoided to prevent the contamination of the sample with chlorine containing solution. It was, however, still possible that the contamination of the samples occurred. Therefore, multiple samples were prepared in case of a sample loss. Concentrations of the solutions used for the irrigation were set at 2.5% for NaOCl, 2% for CHX, and 17% for EDTA. Those concentrations were not varied for this study. However, since no independent test was performed to confirm that those concentrations were indeed factual in this study, it is possible that small variations were present. The QMiX group varied from the CHX group in that no EDTA or second NaOCl irrigation was performed; instead saline was used as a rinse. This change was made following the manufacturer s recommendation that saline is used between NaOCl and QMiX and no EDTA rinse is necessary (234). Since we wanted the precipitate to form, and be able to penetrate into DT, we used EDTA to expose the tubules before second NaOCl irrigation in the CHX group. NaOCl was used for three minutes initially in both groups, but for two 64

77 minutes prior to CHX. EDTA, CHX, and QMiX were used for one minute each time, and thirty seconds application of saline was used. This was based on the estimated time that the final irrigation procedure is performed in a clinical practice. The samples had to be manually manipulated during the study, as teeth had to be sectioned, embedded in resin, irrigated, and resectioned in multiple samples. An attempt was made to limit the handling to prevent the contamination. Since PCA is volatile, the vacuum environment during testing could reduce the detection of this product (39). However, preparation and testing while cooling was not attempted, as a previous study has shown that PCA still could still be detected at room temperature (39). Previously shown, TOF-SIMS, performed on tissue, allowed the analysis using certain ions to compartmentalize it into histological components, and permitted the detection of the position of CHX ions within these compartments (269). Although in this previous study, to visualize the position of CHX within the tissue, chlorine position was analyzed, in the current study, due to extensive use of NaOCl and saline, chlorine was used for confirmation only and not as a primary method of detection. 2. Results The CHX irrigation group exhibited a formation of the precipitate on the surface and the penetration of a product consistent with PCA into the tubules. High intensity PCA and precipitate signal was found to a depth of approximately 50μm, with a lower intensity but still detectable signal reaching beyond 100μm inside DT. This was expected as previous studies have shown independently that NaOCl and CHX can penetrate into DT up to 300μm, in addition to the fact that NaOCl and CHX react when mixed together (30, 36-65

78 39, 79, 84, , 148, 163, 198, ). The formation on the surface can be explained easily as the sample had NaOCl on the surface when CHX was added. However, two explanations for the detection in DT exist. One would require the formation of the precipitate on the surface, followed by its diffusion into the tubules. The second explanation can be described by the diffusion of CHX into DT, still full of NaOCl. The reaction would then occur within the tubule. This study did not measure the kinetics and dynamics of the reaction. The detection of PCA and the precipitate on the surface of the dentin was accomplished by examining the high mass resolution results. There could be some discussion due to the fact that 127u, 153u, 170u, and 195u peaks are common for more than one molecule each. In fact, the 127u peak is present when PCA is tested, the precipitate is tested, and also when CHX is tested (39). The difference between those three substances is the relative heights of the peaks themselves. Since this study s experimental peak heights were not similar to CHX as tested previously, it was concluded that they arose from the precipitate and PCA. The presence of the 505u peak indicated that some residual CHX was also adsorbed into the precipitate during the formation. It is possible that if the TOF-SIMS analysis were to be performed while cooled, different results would be obtained (39). When the longitudinal sections of the dentin samples were tested, only a high spatial resolution scan was performed. This was to visually examine the penetration of the precipitate into the tubules. In a pilot study, in the high mass mode, the tubules were not clearly visualized and no conclusion could be reached. In the QMiX group, what was well visualized on the surface of the dentin was a compound that contained a carbon chain. This was consistent with the presence of a detergent. Inability to detect PCA and the precipitate could be related to the fact that no 66

79 precipitate formed since saline rinse was used before QMiX. It could be that no precipitate formed because EDTA and/or the detergent had already reacted with CHX, possibly preventing the reaction with residual NaOCl (148, 251, 252). Furthermore, the detergent could have formed a micelle or a layer on top of the sample. TOF-SIMS would therefore analyze the detergent layer and not the deeper layer. This last possibility may also be responsible for the lack of detection of CHX in the sample irrigated with QMiX. Alternatively, the amount of CHX is so low that the detection with TOF-SIMS was not possible. Also, low concentration of CHX in QMiX and the formation of a detergent layer may be mutually responsible for the absence of the CHX signal in the results. The same explanation can be provided for the lack of detection of PCA, precipitate, and CHX in the tubules. This study did not attempt to explore the penetration of the compound consistent with the detergent in the tubules as it was not the aim. The information regarding the type of detergent in QMiX is not available. Therefore, it is unknown at this time which other peaks are responsible for the compound consistent with the detergent. Although the precipitate and PCA were present in the tubules when CHX was used after NaOCl and none were detected with QMiX, it is not possible to extrapolate this information to determine which group would result in a better outcome of a root canal therapy. It was determined previously that CHX irrigation lowered the success of the therapy (156). No QMiX outcome studies are available; therefore, any statements regarding the in-vivo benefits of this product are purely speculative in nature. 67

80 V. FUTURE DIRECTIONS As this study only measured a precipitate formation under a strict irrigation protocol, it would be of interest to use different strengths of the irrigants to see if the results can be applied across a wide range of concentrations. In addition, the use of distilled water, saline, alcohol, or citric acid before irrigation with CHX can be investigated (148, 198, 243, 250). This would then indicate if those intermediate rinses completely prevent or just lower the formation of the precipitate in the main root canals and what the effects are on DT. Another option is to use apical and coronal root dentin as a different tubule diameter may alter the penetration of the irrigants. If possible, irrigation of a root canal in a root that has not been sectioned could be performed and then the examination of the cross section of the tooth may yield results that are more likely to mirror an in-vivo situation. Ultimately, a tooth scheduled for extraction can be rinsed with those irrigants and then examined after the extraction. A TOF-SIMS analysis can also be done under different modes. Sputter erosion can be performed on the CHX and QMiX samples. In QMiX samples, erosion of the detergentlike product can expose the contents which may remain covered while examined with the current methodology. In CHX samples, different concentrations of PCA and other breakdown products of CHX can be detected within the precipitate and may be related to their sedimentation potential. Nevertheless, it is unknown at this time if the precipitate that can form in the tubules is toxic to periapical tissues. A test of the cytotoxic potential of the leakage of the product from the tubules can be executed and compared to the potential cytotoxic leakage of NaOCl, CHX, EDTA, and QMiX. 68

81 VI. CONCLUSION - This in-vitro study utilizing dentin blocks has shown that the precipitate, resulting from NaOCl being mixed with CHX, may form on a dentin surface and occludes dentinal tubules, as observed with TOF-SIM. - TOF-SIMS has revealed this precipitate to contain PCA, and to extend into dentinal tubules when dentin blocks are irrigated with 2.5% NaOCl, followed by 17% EDTA, 2.5% NaOCl and 2% CHX. - It has been noted that the precipitate is not detected with TOF-SIMS when dentin is irrigated with 2.5% NaOCl, followed by saline and QMiX, neither on the dentin surface, nor in dentinal tubules. - Whether the formation of this precipitate is detrimental to tissues and outcomes of a root canal therapy remains to be tested. 69

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103 VIII. APPENDICES Appendix 1. Letter Grant of ethics approval from Health Sciences Research Ethics Board (REB), University of Toronto, dated February 28,

104 Appendix 2.Molecular structures Molecular structure of Chlorhexidine. Molecular structure of Ethylenediaminetetraacetic acid. Molecular structure of para-chloroaniline. 92

105 Appendix 2. Tables Table 1. Samples and experimental irrigation protocols. Samples 2.5% NaOCl 3 minutes Irrigation protocol in the progressing order of irrigant 17% EDTA 1 minute 2.5% NaOCl 2 minutes 2% CHX 1 minute Saline 30 seconds QMiX 1 minute S1 V V V V S2 V V V V S3 V V V V S4 V V V V S5 V V V V S6 V V V V S7 V V V S8 V V V S9 V V V S7 V V V S8 V V V S9 V V V Table 2. TOF-SIMS spectra mass peaks of investigated ions. Ion Mass (u) Calcium: Ca Para-chloroaniline: ClC 6 H 4 H 2 N Chlorhexidine: C 22 H 31 Cl 2 N + 10 parent molecule Chlorhexidine: C 22 H 31 Cl 2 N + 10 fragmentation pattern 127.0, 153.0, 170.0, 195.0, Chlorine: Cl Chlorine: 37 Cl Phosphinate: PO Phosphonate: PO TOF-SIMS data of the dentin blocks, irrigated according to Table 1 protocol and analysed for the presence of peaks presented in Table 2, is shown in Appendix 3 on pages It includes TOF-SIMS spectrum and images of each sample. 93

106 Appendix 3. Figures Graphs display TOF-SIMS spectra of samples irrigated according to the protocol in Table 1, Appendix 2, page 93. Spectra were analyzed for the presence of peaks indicated in Table 2, Appendix 2, page 93. Pictographs display TOF-SIMS images of samples irrigated according to the protocol in Table 1, Appendix 2, page 93. Total ion and selected ion images are presented. Figure 1. S1 sample selected positive ion TOF-SIMS spectra. Figure 2. S1 sample selected negative ion TOF-SIMS spectra. 94

107 Figure 3. S1 sample selected positive ion TOF-SIMS images. Figure 4. S1 sample selected negative ion TOF-SIMS images. 95

108 Figure 5. S2 sample selected positive ion TOF-SIMS spectra. Figure 6. S2 sample selected negative ion TOF-SIMS spectra. 96

109 Figure 7. S2 sample selected positive ion TOF-SIMS images. Figure 8. S2 sample selected negative ion TOF-SIMS images. 97

110 Figure 9. S3 sample selected positive ion TOF-SIMS spectra. Figure 10. S3 sample selected negative ion TOF-SIMS spectra. 98

111 Figure 11. S3 sample selected positive ion TOF-SIMS images. Figure 12. S3 sample selected negative ion TOF-SIMS images. 99

112 Figure 13. S7 sample selected positive ion TOF-SIMS spectra. Figure 14. S7 sample selected negative ion TOF-SIMS spectra. 100

113 Figure 15. S7 sample selected positive ion TOF-SIMS images. Figure 16. S7 sample selected negative ion TOF-SIMS images. 101

114 Figure 17. S8 sample selected positive ion TOF-SIMS spectra. Figure 18. S8 sample selected negative ion TOF-SIMS spectra. 102

115 Figure 19. S8 sample selected positive ion TOF-SIMS images. Figure 20. S8 sample selected negative ion TOF-SIMS images. 103

116 Figure 21. S9 sample selected positive ion TOF-SIMS spectra. Figure 22. S9 sample selected negative ion TOF-SIMS spectra. 104

117 Figure 23. S9 sample selected positive ion TOF-SIMS images. Figure 24. S9 sample selected negative ion TOF-SIMS images. 105

118 Figure 25. S4 sample selected positive ion TOF-SIMS images of longitudinal section. Figure 26. S4 sample selected negative ion TOF-SIMS images of longitudinal section. 106

119 Figure 27. S5 sample selected positive ion TOF-SIMS images of longitudinal section. Figure 28. Selected positive ion TOF-SIMS spectra of part highlighted in Figure

120 Figure 29. S6 sample selected positive ion TOF-SIMS images of longitudinal section. Figure 30. S6 sample selected negative ion TOF-SIMS images of longitudinal section. 108

121 Figure 31. S10 sample selected positive ion TOF-SIMS images of longitudinal section. Figure 32. S10 sample selected negative ion TOF-SIMS images of longitudinal section. 109

122 Figure 33. S11 sample selected positive ion TOF-SIMS images of longitudinal section. Figure 34. S11 sample selected negative ion TOF-SIMS images of longitudinal section. 110

123 Figure 35. S12 sample selected positive ion TOF-SIMS images of longitudinal section. Figure 36. S12 sample selected negative ion TOF-SIMS images of longitudinal section. 111

124 Appendix 5. Photographs Figure 1. Leica EM TXP Target Sectioning System. Figure 2. Leica EM TXP Target Sectioning System tooth sectioning. 112

125 Figure 3. Prepared dentin blocks. Figure 4. Dentin blocks poured in resin. 113

126 Figure 5. Leica EM UC6/FC6 Ultracryomicrotome. Figure 6. Leica EM UC6/FC6 Ultracryomicrotome dentin exposure. 114

127 Figure 7. Prepared dentin blocks prior to placement in TOF-SIMS apparatus. Figure 8. TOF-SIMS apparatus ION-TOF GmbH. 115