Effects of final irrigation with CHX and EDTA based irrigant combined with detergents οn the surface of

dentin using surface analytical methods

by

Myrto Piperidou

A thesis submitted in conformity with the requirements

for the degree of Master of Science Endodontics

Faculty of Dentistry

University of Toronto

© Copyright by Myrto Piperidou 2018

ii

Effects of final irrigation with CHX and EDTA based irrigant combined with

detergents οn the surface of dentin using surface analytical methods

Myrto Piperidou

Master of Science Endodontics

Faculty of Dentistry

University of Toronto

2018

ABSTRACT This study evaluated the formation of precipitate or parachloroanaline (PCA) on the dentin surface

after irrigation with NaOCl and final irrigation with Smear OFF using of Time-of-Flight

Secondary-Ion-Mass-Spectrometry (TOF-SIMS) surface analysis and X- Ray Photoelectron

spectroscopy (XPS). Dentin blocks from human maxillary molars were used and the dentinal

tubules were exposed in a perpendicular orientation. The blocks were divided in CHX group:

Irrigation with 6% NaOCl 17% EDTA 6 % NaOCl and 2% CHX, Smear OFF group: Irrigation

with 6% NaOCl and Smear OFF. TOF – SIMS and XPS analysis of the blocks followed.

Precipitation with PCA and occlusion of the dentinal tubules were noted on the surface of the

dentin in the control group (CHX). No precipitate or PCA were detected on the surface of dentin

in the experimental group (Smear OFF) and the dentinal tubules were open.

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ACKNOWLEDGEMENTS

I would like to acknowledge my scientific advisory committee members for their real support:

Drs Bettina Basrani, Rana Sodhi and Kamil Kolosowski. Their insight and guidance were very

valuable for the completion of this project. I would also like to acknowledge Dr Anil Kishen for

his support and contribution to it.

I would like to add a great thank to my main classmates Jackie, and Aaron, being next to me during

all these three years and to the rest of my co residents supporting me with their own ambition and

persistence for learning and succeeding.

I would like also, to acknowledge Dr Shimon Friedman, whose approach reflected responsibility,

sincerity, dedication to excel in my goals and acceptance of possible unique personality traits of

me coming from another continent. His door was always open for me and my numerous questions

were never left without an answer.

Endodontic staff and assistants, made my life enjoyable and efficient, supporting me with their

wide smiles and dedication to my needs throughout the clinical sessions and the experimental part

of my project. Among them, I would like to separately thank Lilia Kaganovsky, who introduced

me to the conditions of the clinic with patience and smile and helped me adapt to this environment

with real care.

Last but not least, I cannot express in words, the support and love I received from my family

members, Rita, Anestis, Alexia, Margie and Bety, as well as from precious friends, Maria and

Penelope, who would smile to me on the video calls, patiently listen to me, and show me how

strong I can be with real and sincere love.

This study was granted from Canadian Academy of Endodontics Endowment Fund and Endo

Tech.

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TABLE OF CONTENTS ACKNOWLEDGEMENTS .......................................................................................................iii

TABLE OF CONTENTS ........................................................................................................... iv

LIST OF APPENDICES ............................................................................................................ vi

INTRODUCTION ...................................................................................................................... 1

Apical periodontitis ................................................................................................................ 1

1.1 Etiology........................................................................................................................... 1

1.2 Treatment ........................................................................................................................ 2

1.2.1 Mechanical instrumentation-objectives ................................................................ 2

1.2.2 Root Canal Irrigants ............................................................................................. 3

1.3 Sodium Hypochlorite (NaOCl) ........................................................................................ 3

1.3.1 Mechanism of action ............................................................................................ 3

1.3.2 Concentrations ..................................................................................................... 4

1.3.3 Cytotoxicity ......................................................................................................... 4

1.4 Chlorhexidine (CHX) ...................................................................................................... 5

1.4.1 Mechanism of action ............................................................................................ 5

1.4.2 Concentrations ..................................................................................................... 6

1.4.3 Cytotoxicity ......................................................................................................... 6

1.5 Ethylenediaminetetraacetic Acid (EDTA) ........................................................................ 6

1.5.1 Mechanisms of action .......................................................................................... 7

1.5.2 Concentrations ..................................................................................................... 7

1.5.3 Cytotoxicity ......................................................................................................... 7

1.6 Irrigant Solutions with Detergents ................................................................................... 8

1.6.1 NaOCl with Detergent ......................................................................................... 8

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1.6.2 EDTA with Detergent .......................................................................................... 9

1.6.3 Antibiotic with Detergent ................................................................................... 10

1.6.4 CHX with Detergent .......................................................................................... 12

Interactions of NaOCl with irrigants proposed for final rinse ................................................ 14

2.1 Interaction between NaOCl and CHX ............................................................................ 14

2.1.1 Chemical analysis and detection of precipitate and para-chloroanaline (PCA) .... 15

2.1.2 Clinical significance, complications and prevention of precipitate and PCA ....... 16

2.2 Interaction between NaOCl and QMiΧ .......................................................................... 16

Time-of- Flight Secondary Ion Mass Spectrometry (TOF-SIMS).......................................... 17

3.1 Principles of function .................................................................................................... 17

X- Ray Photoelectron Spectroscopy (XPS) ........................................................................... 18

4.1 Principles of function .................................................................................................... 19

RATIONALE & AIMS ............................................................................................................. 20

Rationale .............................................................................................................................. 20

Aim….. ................................................................................................................................ 20

ARTICLE ................................................................................................................................. 21

DISCUSSION ........................................................................................................................... 36

Aim and Methodology .......................................................................................................... 36

Results ................................................................................................................................. 38

CONCLUSIONS & FUTURE DIRECTION: ............................................................................ 40

APPENDICES .......................................................................................................................... 54

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LIST OF APPENDICES Appendix 1: Letter of ethics approval from Health Sciences Research Ethics Board (REB),

University of Toronto

Appendix 2: Molecular structures

Figure A) Molecular structure of Chlorhexidine (CHX)

Figure B) Molecular structure of Ethylenediaminetetraacetic acid (EDTA)

Appendix 3: Pictures

Picture 1: Sequence to create the dentin blocks

Picture 2: Dentin blocks in resin

Picture 3: Dentin polishing with glass knife with Leica EM UC6/FC6 Ultracryomicrotome

Picture 4: Brown precipitate after irrigation with NaOCl followed by CHX in plastic cup & tooth

Picture 5: Minor change of color in a yellowish hue after combination of NaOCl and Smear OFF in plastic cup & tooth

Picture 6: Samples being analyzed in TOF – SIMS

Picture 7: Leica EM UC6/FC6 Ultracryomicrotome

Picture 8: TOF- SIMS- ION – TOF GmbH

Picture 9: ThermoFisher Scientific Escalab 250Xi

Appendix 4: Figures

Figure 1: Sample 4: Positive ions – TOF- SIMS selected spectra

Figure 2: Sample 4: Negative ions – TOF-SIMS selected spectra

Figure 3: Sample 4: Positive ions- TOF-SIMS images-selected spectra

Figure 4: Sample 4: Negative ions: TOF-SIMS images- selected spectra

Figure 5: Sample 10: Positive ions: TOF-SIMS selected spectra

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Figure 6: Sample 10: Negative ions: TOF-SIMS selected spectra

Figure 7: Sample 10: Positive ions: TOF – SIMS images- selected spectra

Figure 8: Sample 10: Negative ions: TOF – SIMS selected spectra

Figure 9: Sample 8: Positive ions: TOF-SIMS selected spectra

Figure 10: Sample 8: Negative ions: TOF-SIMS selected spectra

Figure 11 Sample 8. Positive ions: TOF-SIMS images- selected spectra

Figure 12: Sample 8- Negative ions: TOF- SIMS images- selected spectra

Figure 13: Sample 1: XPS analysis - Survey spectra, CHX group

Figure 14: Sample 2: XPS analysis- Survey spectra, CHX group

Figure 15: Sample 3: XPS analysis - Survey spectra, CHX group

Figure 16: Sample 1: XPS analysis- Survey Spectra, Smear OFF group

Figure 17: Sample 2: XPS analysis- Survey Spectra, Smear OFF group

Figure 18: Sample 3: XPS analysis - Survey Spectra, Smear OFF group

Figure 19: Sample 1: XPS analysis, Carbon (C1s) fit – Smear OFF group

Figure 20: Sample 2: XPS analysis, Carbon (C1s) fit – Smear OFF group

Figure 21: Sample 3: XPS analysis, Carbon (C1s) fit – Smear OFF group

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INTRODUCTION

Apical periodontitis

1.1 Etiology Apical periodontitis is an inflammation of the periapical tissues mainly resulting from bacterial

infection in the root canal system (1, 2). It has been described as a dynamic encounter between the

host immune defense and the bacteria which constitute the etiologic agents (3). The endodontic

environment serves as a beneficial habitat for the nutrition of the bacteria thus, allowing them or

their by- products to ingress deeper in the root canal system. The host immune system reacts to the

bacteria invasion by excreting multiple molecules and immune cells. More specifically, the innate

and adaptive immune system are induced by the released bacterial toxins. The innate response is

activated by the recognition of pathogen molecular patterns (PAMPS) found on bacteria, from

various pattern recognition receptors (PRRs) or toll like receptors (TLRs) on the phagocytic cells.

This results in increased vasodilation and expression of cytokines such as TNF–α and IL-1β.

Despite that, the necrotic pulp is hard to be reached, as there is no longer blood supply, making

the host system incapable of controlling the infection (4). RANKL/OPG, the ratio that is

responsible for the homeostasis of bone remodelling is altered and osteoclasts are activated, which

are responsible for the bone resorption. Consequently, the manifestation of apical periodontitis

occurs as a destruction of the tissues located in the interface of the infected radicular part and the

periodontal ligament (3). Infections of the root canal system usually consist of a number of

different species of organisms. The interactions between them and the immune system can result

in by-products which can contribute to the nutrition and growth of other species (5). These species

are agglomerated and organized in a form of biofilm. A biofilm is a composed of multiple bacteria

in different layers embedded in extracellular polymeric substance (EPS). This organization is

associated with different resistance mechanisms ie: i) resistance associated with the extracellular

polymeric matrix, ii) resistance associated with growth rate and nutrient availability and iii)

resistance associated with adoption of a resistance phenotype (6). Thus, eliminating the infection

and addressing the etiologic agents appears to be challenging and requires proper debridement of

the root canal.

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1.2 Treatment Successful treatment of root canal infection requires elimination of the bacteria in the root canal

and alteration of the environment in order to prevent further colonization of other species (7). This

can be accomplished by the biomechanical endodontic treatment, which consists mainly of

mechanical instrumentation and irrigation of the root canal system. The goals of instrumentation

and irrigation are to remove the pulp tissue and debride the dentinal walls of the canals in order to

facilitate anatomical shape where the irrigants, intracanal medicaments and root fillings can

achieve the maximum of their efficacy (8). After the completion of the treatment emphasis should

be placed on achieving an adequate and effective coronal seal to protect the new environment from

future bacterial ingress (9, 10). Haapasalo and colleagues (11), have shown that all the currently

available techniques have their own inherit limitations, therefore to achieve the reduction of

bacteria in a level lower than the threshold of provoking disease, a combination of multiple

techniques is necessary.

Although the efficacy of the current treatment methods is not the optimal, the treatment of

endodontic disease has shown to reach very good prognosis of success. Outcome studies assessing

the efficacy of current treatment protocols, have shown favorable response ranging from 75% to

85 % (12-14). Effective coronal seal is of paramount importance preserving the environmental

balance achieved in the host after the completion of the endodontic treatment (9, 15, 16)

1.2.1 Mechanical instrumentation-objectives

Hülsmann and colleagues (8) have described the following as the objectives of root canal

instrumentation: i) removal of vital and necrotic tissue from the main root canal space ii) creation

of sufficient space for irrigation and medication ii) preservation of the integrity and location of the

apical anatomy iv) avoidance of iatrogenic damage to the canal system and root structure v)

facilitation of canal filling vi) avoidance of further irritation and/or infection of the periradicular

tissues vii) preservation of sound root dentin to allow long-term function of the tooth. These can

be accomplished by prudent use of the various hand instruments and rotary files that exist in the

market. Ostravik et al. (17) showed 10- fold of bacterial decrease with reaming while Matsumiya

and Kitamura (18) reported that with larger sizes instrumentation, the number of bacteria is

decreased. Although mechanical instrumentation alone can achieve reduction of microbial factors

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in the root canal system, there are a number of in vitro studies emphasizing the important role of

the irrigation as an adjunct. Byström and Sundqvist (19) demonstrated that hand instrumentation

with saline resulted in decrease of root canal bacteria numbers from 102 to 103. Similarly,

Shupping et al. (20) concluded that instrumentation with NaOCl irrigation was superior in bacterial

reduction to instrumentation with sterile saline.

1.2.2 Root Canal Irrigants

Many in vitro studies (21-23) have described the complex anatomy of the root canal system with

the multiple lateral, accessory canals and isthmuses. Peters and colleagues (24), assessing the

cleaning efficacy of four NiTi preparation techniques, concluded that regardless of the technique

of instrumentation, 35% or more of the canals' surface area was untouched. Hence, emphasis

should be placed on combination of mechanical and chemical disinfection of the canals to achieve

elimination of the etiological factors of the infection and to result in favorable outcomes. The goals

of irrigation can be classified as mechanical, chemical and biological. The mechanical are: removal

of any debris from the canal walls and adequate lubrication, whereas the chemical is dissolution

of organic and inorganic tissue. Regarding the biological properties, the irrigants should have

broad antibacterial properties, be able to inactivate endotoxins, not provoke any systematic toxicity

or anaphylaxis (25, 26).

1.3 Sodium Hypochlorite (NaOCl) It was first produced in France in 1789 and was mainly used in hospitals as antiseptic. In

endodontics it was introduced by Collidge as a root canal irrigant (27).

1.3.1 Mechanism of action When NaOCl is dissolved in an aqueous solution it results into sodium hydroxide and

hypochlorous acid formation following the following chemical reaction:

NaOCl + H2Oà NaOH + HOClà Na+ + OH- + H+ + OCl-

(28)

Hypochlorous acid acts as an oxidizer and together with the hypochlorite ions (OCl-), lead to

4

amino acid degradation, neutralization and hydrolysis. When NaOCl is in contact with fatty acids,

it results into fatty acid salts and glycerol formation. This is the saponification reaction that is

responsible for the reduction of surface tension in the solution (28). At pH values of 9 and above,

the hypochlorite ion (OCl−) predominates and the tissue dissolution capacity is the highest. At

neutral pH, hypochlorous acid (HOCl) is present and its antibacterial action is the highest, while

below pH 4, chlorine gas starts to form. (29). Its high alkalinity interacts with the bacteria’s

cytoplasmic membrane and results in enzymatic inhibition. Enzymatic inhibition is also the result

of the formation of chloramines by the released chlorine. This chemical reaction results in the

irreversible oxidization of the SH (sulphydryl group) of the enzymes thus interfering with their

cellular metabolism (28). NaOCl has also been shown to be effective against bacterial endotoxins’

however, not in the same degree as against the bacteria themselves (30).

1.3.2 Concentrations

NaOCl is used in concentrations ranging from 0.5% to 6%. The higher the concentration, the better

the organic tissue dissolution capacity of the irrigant (31). However, in lower concentrations,

higher volumes of the irrigant can significantly increase its efficacy (32, 33). In the current

literature, the studies regarding the relationship between the concentration of NaOCl and its

antimicrobial efficacy, are controversial. Although in vitro studies have shown that it reduces more

effectively Enterococcus faecalis when used in higher concentrations (34), clinical studies have

shown that even in lower concentrations it can exhibit antibacterial action that can reduce the

bacteria in the root canal system (35). Haapasalo and colleagues (36) showed that the presence of

dentin caused marked delay in the killing of E. faecalis by 1% NaOCl. To counteract the impact

of the dentin and the environment such as inflammatory exudate, or organic tissue, emphasis

should be placed on the frequent intervals of large volumes of the irrgant as well as substantial

time of exposure to achieve adequate antibacterial efficacy (33).

1.3.3 Cytotoxicity

Various studies have analyzed the cytotoxicity of NaOCl. Hidalgo et al.(37) assessed the

cytotoxicity in human dermal fibroblasts and found that cell ATP depletion occurred at

concentrations of 0.00005% but cell death was concentration dependent and was observed at

concentrations greater than 0.01 %. Inhibition of DNA synthesis was also reported (37), however,

5

no genotoxicity was found in murine fibroblasts (38) . NaOCl cytotoxicity was evaluated also on

the survival of stem cells of human apical papilla which is an important step for the regenerative

procedures. It was found that the mean percentage of viable cells was decreased significantly over

a time of 1, 5 and 15 min of exposure to 5.25% NaOCl (39). Another study, evaluated the combined

and independent cytotoxicity of NaOCl, EDTA and chlorhexidine (CHX) on human lung

fibroblasts (MRC5 cells). Interestingly, the authors concluded that NaOCl was less cytotoxic than

CHX but more than EDTA and that the combined use of them did not produce any significant

increase in their cytotoxicity (39).

1.4 Chlorhexidine (CHX) It was first produced in UK where it was used as a cream for antiseptic purposes and later for

treatment of eye, skin and throat infections (40). In dentistry, it has been used for many years as a

mouth rinse for the treatment of periodontal disease and as a root canal irrigant in endodontics

(41).

1.4.1 Mechanism of action

Chlorhexidine (CHX)- C22H38N10Cl2 is cationic bisbiguanide. It consists of two symmetric four-

chlorophenyl rings connected by a hexamethylene chain to two bisbiguanide groups, (Figure A,

Appendix 2) (42). Its mechanism of action is based mainly to its cationic nature. This makes it

capable to electrostatically bind to the surface of negatively charged bacteria and damage their cell

wall by increasing their permeability. More specifically, the penetration of chlorhexidine, results

in precipitation of the cytoplasm thus preventing the repair of the cell membrane – bactericidal

effects (43-46). Regarding its antimicrobial properties, they are considered to be wide, including

Gram positive and Gram-negative bacteria and yeasts (44, 47, 48). Its effect can be bacteriostatic,

when it is used in low concentrations or bactericidal when used in higher concentrations. In

bacteriostatic effect, the cell is not irreversibly damaged since it only causes low molecular weight

substances to leak out (46).The efficiency of chlorhexidine’s action also depends on the pH. In

acidic pH, its bactericidal effect is reduced, whereas in neutral pH is absorbed quickly and the free

chlorhexidine available in the solution is low (46).

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1.4.2 Concentrations

In dentistry CHX is used in concentrations between 2% and 0.12%. In endodontics, as a root canal

irrigant it is mainly used in 2%. It was reported that 2% of CHX has better antibacterial efficacy

than 0.12 % (49) and it was found that this concentration was effective against E. faecalis biofilm

(26, 50). Concentration and time of CHX can have influence on its substantivity. In concentrations

0.005% to 0.01%, a monolayer is formed on hydroxyapatite. This cannot provide any release of

CHX since it is absorbed very quickly. However, in higher than 0.02 % concentrations, the tooth

surface is covered by a multilayer of CHX which can be released into the environment (51).

Different concentrations of CHX have been tested for their antibacterial substantivity. The reports

in the literature are controversial. Other studies have found direct relationship between the

substantivity and the concentrations, showing that 4% CHX is more efficient than 0.2% for 5 mins

exposure (52), while other reported that time is more critical factor than concentrations (53).

1.4.3 Cytotoxicity

Cytotoxicity of CHX at the concentrations used in dentistry (0.12% and 0.2%) has been found to

be very low level (54, 55). Recent studies have reported CHX – induced cytotoxicity and

genotoxicity on macrophages which is associated with ROS (reactive oxygen species) generation

(56). Also, CHX exerted an inhibitory concentration-dependent effect on DNA synthesis on human

dermal fibroblasts from concentrations as low as 0.0001% with depletion of cell ATP in a time

and concentration dependent manner (57). CHX effects on gingival fibroblasts were examined and

concluded that chlorhexidine even in low concentrations equal of 0.12% can decrease the cellular

proliferation and reduce both the collagen and non-collagen protein production, affecting

negatively the wound healing process (58, 59).Toxicity effects on human osteoblastic cells with

inhibition of cell growth, proliferation and collagen synthesis (60, 61) as well as on human alveolar

bone cells (62) were also reported posing potential risks for periapical tissues toxicity.

1.5 Ethylenediaminetetraacetic Acid (EDTA) It was first produced in 1935 by Ferdinand Munz, who prepared the compound from

ethylenediamine and chloroacetic acid. (63). In endodontics, it was used in 1957 by Nygaard-

Østby, in cases of challenges in “cleaning and shaping” narrow and calcified root canals (64).

7

1.5.1 Mechanisms of action

Ethylenediaminetetraacetic acid (EDTA) - C10H16N2O8 (Figure B, Appendix 2), forms a stable

complex with ions when it is exposed to calcium and heavy metals. This is a result of the ring –

shaped bonds that it creates. EDTA has more than one pair of free electrons and it bonds with the

central metal ion with each of these pairs (64). More specifically, its chelating property derives

from its capacity to “sequester: di- and tri-cationic metal ions such as Ca2+ and Fe3+(26). When all

the available ions have been bound, an equilibrium is formed, and the dissolution stops.

Chemically, two reactions have been described,

EDTA H3- + Ca2+ à EDTA Ca2- + H+ and

EDTA H3- + H+ à EDTA H22- (64)

As this reaction proceeds, acid accumulated, and the rate of demineralization is reduced since

protonation of EDTA prevails.

1.5.2 Concentrations In endodontics EDTA is used in concentration of approximately 17%. EDTA has been used in

different concentrations and pH and its effect on the dentine has been assessed by various studies

(65-67). Serper and Calt (68), have analyzed the demineralizing effects of EDTA in concentrations

of 10% and 17% at pH 7.5 and 9.0 by the amount of liberated phosphorus at different time periods

of exposure. They concluded that the highest concentration of EDTA and increased time of

exposure were more efficient in pH 7.5. The phosphorus release effect rapidly rises to a level

within 1 min, and further exposure to EDTA only doubled this effect upon 15 min of further

administration(68). Similar results were reported from another study by Parmar and Chhatariya

(69) emphasizing that the maximum demineralizing efficiency of EDTA was found within the first

minute of exposure.

1.5.3 Cytotoxicity

Two in vitro studies by Segura JJ et al. (70, 71) have examined the effects of EDTA on macrophage

functions. They reported that EDTA in concentration lower than the commonly used in root canal

8

treatment reduced significantly the adherence capacity of macrophages. The toxicity of EDTA was

examined on fibroblasts as well. Moderate to severe cytotoxicity was reported depending on the

concentrations of EDTA with cell lysis and death (72, 73). However, these results are based on

vitro studies.

1.6 Irrigant Solutions with Detergents Various irrigant solutions that combine as an additive a detergent or a surface acting agent have

been used in endodontics. The role of the detergent is to reduce the surface tension of the irrigant

and fluid viscosity, as well as to improve its antibacterial efficacy (74). Increase in wettability of

a solution results in better adaptation with dentinal walls, which is very important for a thorough

cleaning and disinfection. In addition, it helps the irrigant to better penetrate into the dentinal

tubules and enable its antibacterial and chelating properties to be carried more easily to the full

length of the root canal (75, 76).

The efficacy of these combined irrigants against bacteria, could be also attributed to their ability

to disrupt the interactions involved in cross-linking the biofilm matrix (77) as well as to weaken

the coehesive forces of the extracellular matrix of the bacteria, making them less resistant to the

antibacterial action of the irrigants and their membranes more permeable (78). Some examples are,

Smear Clear (Sybron Endo, Orange, CA), Chlor-XTRA (Vista Dental, Racine, WI), Hypoclean

(Ogna Laboratori Farmaceutici, Milano, Italy), Tetraclean (Ogna Laboratori Farmaceutici, Milano,

Italy), MTAD, CHX-Plus (Vista Dental, Racine, WI), Canal Pro EDTA 17% (Coltène/Whaledent

Inc. Cuyahoga Falls, OH, USA) CanalPro CHX 2% (Coltène/Whaledent Inc. Cuyahoga Falls, OH,

USA), QMiX (Dentsply Tulsa Dental, Tulsa, OK, USA) and the new developed Smear OFF (Vista

Dental, Racine, WI) (26).

1.6.1 NaOCl with Detergent

Chlor- XTRA & Hypoclean

Chlor-XTRA is a sodium hypochlorite solution enhanced with surface disinfectants and wetting

agents as well as alkylating agents to increase its electrical capacity. According to the

manufacturer, it is 2.5 times more wettable, stable and digestive than the standard sodium

hypochlorite (79). Hypoclean, consists of 5.25 % sodium hypochlorite and two detergents,

9

cetrimide and polypropylene glycol (80). Two in vitro studies assessed the penetration of sodium

hypochlorite into the dentinal tubules and found that it increased with the addition of surfactants,

increase of temperature and exposure time. More specifically, it was found that Chlor-XTRA at

45oC for 20 mins was more efficient than NaOCl at all the concentrations, exposure times and

temperatures tested (81, 82). Opposite results were found in a study assessing the penetration of

the NaOCl compared with Hypoclean in the coronal and middle third of dentinal tubules, where

no difference was detected (83). Another in vitro study assessed the combination of Hypoclean

with Tetraclean in comparison to plain NaOCl and EDTA. It was shown that the use of adjunctive

agents reducing the surface tension associated with oxidant improved the antimicrobial activity of

irrigating solutions and the intratubular decontamination against E. faecalis (84). The antibacterial

efficacy of Chlor-XTRA and Hypoclean was also assessed in an ex vivo study in bovine dentine

and found that was greater than sodium hypochlorite (85). Regarding the organic tissue dissolution

capacity, Stojicic et al.(86) tested it on bovine meat and it was higher than NaOCl without

detergent and in various concentrations, temperatures as well as sonic and ultrasonic agitation.

However, in another study, Chlor-XTRA, was found to be less efficient compared to NaOCl in

dissolving in situ the pulp tissue (87). One possible explanation of that is the loss of the free

available chlorine which an in vitro study has shown that it was hastened by the presence of

surfactant (88) . As far as the increase of their efficacy is concerned, temperature increase from 20

to 45 oC increased the antibacterial efficacy of Chlor-XTRA and Hypoclean which was greater

than 5.25% NaOCl when the same temperature was used (89). However, ultrasonic activation of

NaOCl has been found to possibly compensate for the absence of surfactants and has been shown

to result in increased efficacy in porcine palatal mucosa dissolution from artificial grooves in the

root canals of human teeth (90).

1.6.2 EDTA with Detergent

Smear Clear

Smear Clear is an EDTA formula enhanced with a proprietary surfactant. More specifically, it is

composed of 17% EDTA, cetrimide and 2 additional surfactants (91). According to the

manufacturer, it aims to remove the smear layer from the root canals and result in patent dentinal

tubules, clearer of organic matter than the plain EDTA (92). Smear clear was evaluated by few in

10

vitro studies. One study that compared the efficacy of different root canal irrigants (NaOCl, Smear

Clear, 2% chlorhexidine, REDTA and BioPure MTAD) against E. faecalis biofilm concluded that

Smear Clear had greater efficacy than chlorhexidine, REDTA and MTAD (93). Also, by increasing

the application duration, its combination with NaOCl resulted in better smear layer removal in the

apical third than the combination with the plain 17% EDTA. However, other studies (94-98) have

shown that the addition of detergent did not result in better removal of smear layer. As far as the

antibacterial properties, organic tissue dissolution capacity is considered critical for a root canal

irrigant. Thus, Smear Clear or other such irrigants, alone, missing that capacity such as MTAD,

cannot be efficient against endodontic biofilm and should be used in combination with NaOCl

(99). Cytotoxicity of Smear Clear was also assessed in case of extrusion in the periapical tissues.

This in vitro study evaluated the expression of nitric oxide (NO) concentrations by murine

peritoneal macrophages after contact with MTAD Tetraclean Smear Clear and EDTA and

concluded that severe proinflammatory effects on murine-cultured macrophages were noted. Citric

acid-based solutions induce lower NO release than EDTA-based irrigants (100).

1.6.3 Antibiotic with Detergent

BioPure MTAD & Tetraclean

These two irrigants consist of antibiotics, specifically tetracycline, citric acid, and a detergent.

(63). Tetracycline targets the ribosome of the bacterial cell and its protein synthesis and it has

broad antibacterial spectrum, including both gram + and gram - bacteria. Depending on the

concentrations, tetracycline can exhibit bacteriostatic effects in low concentrations and bactericidal

in high (101).

In 2003, Torabinejad and Johnson at Loma Linda University patented a new irrigant, MTAD. It

contains 3% doxycycline, 4.25% citric acid, a demineralizing agent and 0.5% polysorbate 80

detergent (Tween 80). It is intended to be used as a final irrigant and in contrast with Tetraclean,

it is not premixed (102). More specifically, the suggested protocol of use is: 1ml MTAD for 5

minutes in the canal then 1.3 % NaOCl and a final flush with 4 ml of MTAD (103). Regarding its

antibacterial efficacy, one clinical trial by Malkhassian and colleagues (103), concluded that the

11

final rinse with MTAD and medication with CHX did not reduce bacterial counts beyond levels

achieved by canal preparation with NaOCl. Furthermore, its efficacy against E. faecalis was

assessed in an in vivo study using qPCR in necrotic primary teeth and showed that it was equal

with NaOCl (104). Many in vitro studies also assessed the antibacterial efficacy of MTAD on E.

faecalis and other pathogens and the results were very promising (105-108). A number of in vitro

studies have assessed MTAD’s efficacy in removing the smear layer and the results were

controversial. Studies comparing it with final rinse of the combination of 5.25 % NaOCl and 17%

EDTA, QMiX and Smear Clear, have shown that it was less efficient in removing the smear layer

(109-113). However, a scanning electron microscopy study has shown that compared to 17%

EDTA, it was more efficient in removing the smear layer in the apical third of the canals (114).

The cytotoxicity of MTAD was evaluated in osteoblast like cells and L929 fibroblasts and found

that it was less cytotoxic compared to CHX, EDTA, NaOCl and Ca(OH)2 paste without affecting

the differentiation of osteoblast like cells into osteoblasts (115, 116). In another study, assessing

genotoxicity and cytotoxicity of MTAD, it was concluded that it did not cause cell death, but it

presented cytotoxicity effects in murine fibroblasts (38).

In 2004, Luciano Giardino introduced a new irrigant called Tetraclean. (Ogna Laboratori

Farmaceutici, Muggio, Italy). It is composed of doxycycline but one third of the amount that

MTAD contains, (50mg/5ml vs. 150mg/5ml of MTAD) polypropylene glycol, citric acid and

cetrimide. Various studies examined its antibacterial effect comparing it with the commonly used

irrigants. Neglia et al (117) compared the bactericidial activity of it with NaOCl using both in vivo

and ex vivo studies. Although both irrigants displayed very similar bactericidal activity against E.

faecalis in vitro, in the ex vivo model it was shown that only in the teeth irrigated

with Tetraclean bacterial burden gradually dropped until no bacteria were detectable a few days

post-irrigation. Regarding the NaOCl group, although the drop in the bacterial burden was rapid it

was only temporary and most of the teeth were re-colonized again by 48 hours post-irrigation.

Another in vitro study, comparing Tetraclean to MTAD and five experimental solutions that were

modifications of existing formulae including MTAD + 0.01% cetrimide (CTR), MTAD + 0.1%

CTR, MTAC-1 (Tween 80 replaced by 0.01% CTR in MTAD), MTAC-2 (Tween 80 replaced by

0.1% CTR) and MTAD-D (MTAD without the Tween 80 and no CTR added) has shown that it

was more effective than MTAD against E. faecalis in planktonic culture and in mixed-species in

an in vitro biofilm (118). It is also emphasized that the CTR probably improved the antimicrobial

12

properties of the solutions, whereas Tween 80 did not have an effect on their antimicrobial

effectiveness (118). In agreement with that, Poggio et al (119) compared in vitro the antibacterial

activity of Tetraclean (mixture of doxycycline, citric acid and polypropylene glycol), Niclor 5

(5.25% sodium hypochlorite solution), Cloreximid (0.2% chlorhexidine and 0.2% cetrimide

solution) and hydrogen peroxide 12 volumes on three endodontic pathogens associated with

primary endodontic infections. They concluded that Tetraclean, similarly with 50°C-preheated

hydrogen peroxide 12 volumes showed highest inhibition of the bacterial growth. The substantivity

of Tetraclean was found to be superior than MTAD and 5.25% NaOCl in an in vitro study in bovine

root dentin (120).

1.6.4 CHX with Detergent

CHX- plus

CHX–plus, according to the manufacturer, consists of chlorhexidine gluconate solution 2% and

surface modifiers for better antibacterial efficacy. Shen Y et al.(121) compared CHX-plus with

CHX 2% plain in an in vitro environment and different time periods, 1min, 3min and 10min of

exposure. They concluded that CHX-plus had higher bactericidal effect compared to CHX 2% at

all periods of time. Furthermore, the effect of mechanical agitation on these two irrigants was

evaluated. Sonic and ultrasonic agitation with CHX-plus and CHX 2% plain was compared and

found that sonic activation with CHX-plus was the most effective at all time periods (122).

Regarding the stage of the biofilm development and the efficacy of CHX, CHX-plus exhibited

higher bactericidal effect at all biofilm ages compared with CHX 2% (123). However, the

resistance of the multispecies biofilm to CHX 2% and CHX-plus was found to be similar (124).

QMiX:

QMiX is a new irrigant, which has entered the market in 2011 (26). According to the manufacturer,

it combines EDTA, CHX and a detergent and it is supposed to be used as a final irrigant. If NaOCl

is used before it, a saline rinse is required to prevent any possible harmful reaction.

13

Cytotoxicity

Until the time this manuscript was written, two studies have assessed the cytotoxicity of QMiX.

Alkahtani and colleagues (125), compared the cytotoxicity of QMiX and various concentrations

of NaOCl. They concluded that QMiX exposure resulted in a significantly higher percentage of

cell viability than NaOCl. Also, the death type associated with QMiX was apoptotic without cell

lysis and the number of live cells were more than in NaOCl. On the other hand, the death type

associated with NaOCl was necrosis, with substantial morphological changes to the cells and cell

lysis. Another study by Mollashahi et al. (39) evaluated the effects of Biopure MTAD Cleanser,

EDTA, CHX , NaOCl and QMiX on stem cells from the human apical papilla in different time

exposure periods. They concluded that the mean percentage of viable cells significantly decreased

over time in NaOCl, QMiX, EDTA and MTAD groups, but no significant reduction was noted in

CHX group.

Antimicrobial activity:

Stojicic et al. (126) tested the antibacterial activity of QMiX against biofilm in an in vitro study

and found that QMiX and 2% NaOCl were superior to 1% NaOCl, 2% CHX and MTAD in killing

E. faecalis and plaque bacteria in planktonic and biofilm culture in 1 and 3 min. However, at 3

min QMiX had killed more bacteria than any other solution, without statistically significant

difference from 2% NaOCl. Later on, Ordinola – Zapata et al. (99), reported that in the previously

mentioned study only the percentage of dead cells was assessed. Hence, they evaluated the effect

of MTAD, QMiX, Smear Clear, 7% maleic acid, 2% iodine potassium iodide, 4% peracetic acid

and 2.5% and 5.25% NaOCl on the architecture and viability of mixed biofilms attached to dentin.

Their conclusion, contrary to the previous group, was that there were no changes in the biofilm

structure after irrigation distilled water, MTAD, QMiX, Smear Clear, 2% IKI or 7% maleic acid

but NaOCl and PAA resulted in some changes of the biofilm organization (99). Recent in vitro

studies though, showed again, the efficacy of QMiX against E. faecalis and Candida alibicans

proposing it as a potential alternative to the routinely used root canal irrigants (21, 127, 128)

14

Smear OFF

Smear OFF is an irrigant from Vista Dental US and has been recently released into the market. It

contains CHX gluconate <1% wt, tetrasodium ethylenediaminetetraacetate dihydrate 18% wt and

a surface-active detergent as the active components. According to the manufacturer, it has

antibacterial properties and it effectively removes the smear layer. Also, it is formulated with

wetting agents and surface modifiers. Moroever, it claims to achieve superior chelation and better

calcium suspension. The manufacturer recommendation is to use it as a final irrigant and if NaOCl

is used before, no additional rinse is needed (129). Very few studies have assessed the properties

of Smear OFF so far. One study evaluated the free available chlorine after the interaction of Smear

OFF with NaOCl and it was suggested that NaOCl lost the free available chlorine after interaction

with EDTA and Smear OFF (130) . Thus, the paper suggests that Smear OFF should only be used

as a final irrigant. Another in vitro study assessed the canal wall smear layer removal of maleic

acid, Smear OFF and 18% EDTA. Smear OFF and maleic acid have shown to be more efficient in

removing the smear layer than 18% EDTA (113).

Interactions of NaOCl with irrigants proposed for final rinse

2.1 Interaction between NaOCl and CHX In order to increase the antimicrobial efficacy of chemical irrigation, Zehnder et al.(25) suggested

an irrigation regimen which combines NaOCl, EDTA and CHX. He proposed, irrigation with

NaOCl for disinfection, organic tissue dissolution and lubrication, during shaping, then use of the

chelating agent EDTA to remove the inorganic tissue and final rinse with CHX because of its

substantivity and broad antimicrobial activity. However, if NaOCl is still in the canal and CHX is

added, there is an instant change of color that can vary from peach to brown which does not change

with time and formation of precipitation (25, 131, 132). The formation of the precipitation can be

noted even with concentrations of 0.19% NaOCl and it is directly proportional with the

concentration of NaOCl while the change of the color starts from peach hue in concentrations of

0.023% NaOCl to more brownish as the concentrations increase (131).

15

2.1.1 Chemical analysis and detection of precipitate and para-chloroanaline (PCA)

Although there is an agreement on the formation of precipitate and the color change after

alternating NaOCl with CHX among the various researchers, there is a controversy regarding the

composition of the precipitate and specifically the formation of parachloroanaline (PCA).

Precipitate formation is a result of acid and base reaction when CHX is mixed with NaOCl. CHX

is a dicationic acid with pH: 5.5 to 6 and it gives protons to the alkaline NaOCl resulting in the

formation of the precipitate (131). Basrani et al. (79), have shown the presence of PCA in the

precipitate by using XPS and TOF-SIMS analysis. More specifically, TOF-SIMS testing showed

an increase in mass 127 u relative to other characteristic peaks of chloroanaline. It was further

confirmed that this corresponds to the toxic 4-chloroanaline or parachloroanaline (PCA) in later

studies (133) by use of gas chromatography- mass spectrometry (GC-MS) method, which excluded

the presence of the other two isomers of chloranaline (2- and 3-), these having different levels of

toxicity (81). Moreover, a technique of diazotization confirmed that chloranaline, which is an

aromatic amine, is present in the precipitate (134).

In agreement with these results, Kolosowski et al.(135) examining dentin samples from human

maxillary molar teeth irrigated with NaOCl, EDTA, NaOCl and final rinse of CHX, showed the

presence of PCA in the precipitate using TOF-SIMS. On the contrary, other researchers using

different analytical methods did not support the formation of PCA. Thomas et al.(136) using H

NMR spectroscopy, found that the exact location of the two aromatic peaks in the PCA doublet

region when examined the PCA alone, was not visible in the assessment of precipitate from the

combination of NaOCl and CHX, thus concluding the absence of PCA from the precipitate (136).

Another group, using one dimensional and two dimensional NMR spectra, found that the

precipitate contains 2 different parasubstituted benzenesystems present (PCGH and PCU) and not

PCA (137). The absence of PCA was reported by other studies as well including electrospray

ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) (138), Beilstein test and

solubility test (139). In addition, one recent study by Orhnan et al.(140) , reported that it is the

“cutoff proof for the argument on PCA formation” showing the absence of PCA with various

methods: high performance liquid chromatography (HPLC), proton nuclear magnetic resonance

(1H-NMR) spectroscopy, gas chromatography (GC), thin layer chromatography (TLC), infrared

16

(IR) spectroscopy, and gas chromatography/ mass spectrometry (GC/MS) tests. The conflicting

results can be explained by the different methodologies and handling of the samples which can

affect the ability of detection of PCA. More specifically, in the last study, regarding the TLC

analysis the brown precipitate is dissolved in ethyl acetate. Thus, it was the latter being analyzed.

PCA could have been adsorbed on the surface and concerns are raised about what component was

analyzed. Also, PCA is volatile in nature, and in methods that do not include cooling of the sample

and use room temperature, the PCA may be evaporated and not detected. Thus, inability to detect

the PCA, cannot undoubtedly be a conclusion that it is absent from the sample.

2.1.2 Clinical significance, complications and prevention of precipitate and PCA

It has been reported that the formation of precipitate and the color change may have some clinical

complications associated with the staining that it provokes, the sealing ability of the root filling

materials (132) and the occlusion of the dentinal tubules in the coronal and middle third of the

canal, which can prevent the penetration of intracanal medicaments (141). Most important though,

is the associated cytotoxicity that has been found with the formation of PCA. Haemolytic anemia,

metheaemoglobinaemia and carcinogenicity induced from PCA were reported in rats and mice

(142, 143). Moreover, human in vitro and in vivo studies have shown severe

metheamoglobinaemia in neonates and in a middle- aged patient due to parachloroanaline (144-

146). Hence, the prevention of the precipitate and PCA formation, has been investigated by various

studies in which the use of paper points between the two irrigants (25) or a flush with citric acid

(147), distilled water, saline or ethanol (139) have been proposed. However, only the use of ethanol

can completely diminish this formation whereas the other methods result in reduction of it (139).

2.2 Interaction between NaOCl and QMiΧ Few in vitro studies have examined the interaction between NaOCl and QMiX with controversial

results. Kolosowski et al. (135), have analyzed the precipitate and PCA formation after irrigating

dentinal samples with NaOCl, saline and QMiX according to the manufacture protocol. They

found that no color change and no precipitate was formed. TOF-SIMS analysis revealed that no

PCA was evident in the solution, which was concluded by the absence of the characteristic peak

of PCA (127 u) and the presence of peaks more consistent with the detergent that is present in

17

QMiX (135). They also analyzed the interaction between NaOCl and CHX. The TOF-SIMS

analysis confirmed that the precipitate contains the toxic PCA as seen by an intense peak at 127 u

in comparison to the low parent peak of CHX (505 u) and its associate fragments (135). Arslan

et al. (148), using H NMR, revealed the formation of orange brown precipitate after the interaction

of NaOCl and QMiX, but agreed that no PCA was detected. Precipitate formation was also

detected by Barkley et al.(149), who compared the interaction of QMiX and NaOCl using both a

fresh and old bottle of QMiX. According to them, the precipitation is caused by the ionic strength

of sodium ions and color change by the oxidation of detergents by NaOCl. Regarding the PCA

formation, they reported that a compound with a molecular weight around 500 g/mol was detected

using 1 H NMR and 2D NMR (DOSY) spectra analysis but the rest of the peaks were due to the

surfactants from QMiX, concluding that further research is needed until the molecular structure of

the precipitate is identified.

Time-of- Flight Secondary Ion Mass Spectrometry (TOF-SIMS)

Time-of- Flight Secondary Ion Mass Spectrometry (TOF- SIMS) is a surface sensitive analytical

method which is used to analyze the compounds on a surface without significant damage on it

(150). Gotliv BA and colleagues, have used TOF-SIMS to analyze the composition and structure

of hard tissue from bovine dentin and bone. They focused on mapping the molecular distributions

of specific ionized elements and amino acid fragments in order to provide images that show distinct

patterns of these tissues (151-153). Moreover, it has been used in a number of different in vitro

studies, to analyze the different elemental fragments in the precipitate that forms in dentin teeth

after using multiple irrigant solutions in the root canal system (131, 133-135).

3.1 Principles of function In TOF-SIMS, a primary ion gun is used to fire a pulsed beam of ions onto the target surface (154).

When an ion impinges on the surface of a solid, it reaches to a specific depth, while depositing

energy. This deposited energy produces collision cascades and results in fragmentations and

breaking of the bonds between the atoms of the bulk. As a consequence, some intact molecules are

emitted from the top monolayer of the surface since their energy is greater than the binding energy

to the surface (154). A small fraction may be charged positively or negatively, and these secondary

18

ions are separated according to their mass/charge (m/z) ratio. Since a pulsed ion beam is used,

these can be detected by measuring their time of flight in reaching the detector after being

accelerated by an extraction voltage to a common energy. The heavier the fragment, the longer it

takes to reach the detector, resulting in the mass spectrum. As well as the parent ion, various

fragments are obtained characteristic of the particular substance resulting in a fragmentation

pattern or “fingerprint”. Charge-compensation can be provided between the pulses by means of a

flood gun, thereby allowing spectra of insulating materials to be obtained without having to coat

the samples (154).

By following the distribution of the fragments using a finely-focused ion beam, a chemical

mapping of the surface can be obtained. Liquid metal ion guns (LMIGs) are the standard primary

ion source since they can easily be focused to a small spot size (~20 – 30 nm). Initially Ga was the

source, however, these have been superseded by Au and Bi cluster sources, which can also increase

the secondary ion yield by several orders of magnitude (154).

TOF-SIMS can work in several modes of operation depending on required information. In order

to achieve good mass resolution, the pulse of ions that impinges on the target surface should have

short duration approximately 1ns. This can be achieved by bunching the pulsed primary ion with

electrostatic fields. This mode however results in low spatial resolution. For high spatial

resolution, a non-bunched mode is required, however, this results in only nominal mass resolution

(154).

Most TOF- SIMS instruments are also equipped with other ion sources which can be used for

sample cleaning and depth profiling in a dual beam mode. Typical sources are electron ionization

sources (Ar, Ar cluster and O) and Cs.

X- Ray Photoelectron Spectroscopy (XPS) X- Ray Photoelectron Spectroscopy (XPS) is another surface analytical tool, used to study

properties of atoms, molecules, solids and liquids Whereas TOF -SIMS provides chemical and

molecular structure information, XPS gives quantitative elemental and chemical state information

of the surface constituents to a depth of ~ 10 nm (155). In dentistry, it has been used in various

studies, to analyze the chemical composition of different surfaces, such as amalgam (156) and

19

dental implants (157-159). Moreover, XPS has been used in analyzing the enamel and dentin

surface after rinse with various irrigants (160) or combination of them (131) .

4.1 Principles of function Irradiation of the sample with incident X-rays causes photoemission of electrons from the surface.

Regarding the type of X-rays used, monochromatic Al Ka X-rays are the most common source,

though non-monochromatic Al Ka and Mg Ka X-rays have also been used.

The photoemitted electrons are energy analyzed, allowing the binding energy of the emitted

electron to be obtained from the following equation:

EB= hν – (EK + Φ)

Where EΒ= binding energy

EK= kinetic energy

hν = photon energy

Φ= work function (155)

The binding energies are characteristic of each element and can thus be used for elemental

identification. Some of the advantages of this analytical tool are that it is very surface sensitive

and requires minimal sample preparation. Furthermore, the escape depth of electron makes it

surface specific (3-10nm). Straightforward quantification (±5%) to a detection limit of ~ 0.1 – 1

at. % can also be obtained by integrating the peak and dividing by the appropriate instrumental

sensitivity factor.

The main strength of XPS is its ability to provide chemical information by means of a chemical

shift. The binding energy of a particular electron is affected by the environment the atom is

situated in and thus, information can be obtained on the type of chemical bonding that the element

participates in and also its particular oxidation state (155). By applying curve-fitting routines

quantifiable information can also be obtained.

20

RATIONALE & AIMS

Rationale

It has not been examined before whether Smear OFF alternated with NaOCl solution on human

dentin can result in a formation of precipitate and PCA. Precipitate formation and occlusion of

dentinal tubules can affect the distribution of medicaments and root filling materials (141) while

PCA has been found to be carcinogenic in animals (142) and cause methemoglobinemia in humans.

Hence, it is considered crucial to further investigate the Smear OFF interaction with NaOCl and

rule out any possibility of its use being hazardous.

Aim…..

The aim of this study was to examine the formation of precipitate and PCA on the surface of

dentin irrigated with NaOCl followed by Smear OFF.

21

ARTICLE (Submitted for publication)

Effects of final irrigation with Smear OFF οn the surface of dentin using

surface analytical methods

Myrto Piperidou, DDS*, Rana N.S. Sodhi, BSc, MSc, PhD**, Kamil P. Kolosowski, HBSc,

DDS* and Bettina R. Basrani, DDS, MSc, PhD*

*MSc Endodontic program, Faculty of Dentistry, University of Toronto,**Department Of

Chemical Engineering And Applied Chemistry, University of Toronto

Abstract:

Introduction: Smear OFF is an irrigation solution containing chlorhexidine (CHX),

Ethylenediaminetetraacetic acid (EDTA) and a surfactant. This study examined the chemical

interaction of Smear OFF with sodium hypochlorite (NaOCl) on the dentin surface, specifically

the formation of precipitate and/or parachloroanaline (PCA).

Methods: Dentin blocks prepared from human maxillary molars were mounted in resin. Dentinal

tubules were exposed in a perpendicular orientation using an ultra cryomicrotome. The blocks

were divided into two groups: CHX Group: Irrigation with 6% NaOCl, 17% EDTA, 6% NaOCl

and 2% CHX; Smear OFF Group: Irrigation with 6% NaOCl and Smear OFF. The dentin surface

was analyzed with Time-of-Flight Secondary-ion Mass Spectrometry and X-ray photoelectron

spectroscopy to determine the formation of precipitate or / and PCA on the surface of dentin.

Results: Precipitation with PCA and occlusion of the dentinal tubules were noted on the dentin

surface in the CHX group. No precipitate and no PCA were detected on the surface of dentin in

the Smear OFF group.

Conclusion: Interaction of Smear OFF with NaOCl on the dentin surface did not result in

formation of precipitate or PCA.

22

Introduction

Chemical irrigation, combined with mechanical instrumentation, is a fundamental step in

endodontic therapy, aimed at achieving debridement of pulp tissue and disinfection of the root

canal space (1). Sodium hypochlorite (NaOCl), the principal irrigation solution used, possesses

antibacterial and organic tissue dissolving properties, but it does not efficiently remove the smear

layer (2, 3). Also, it is cytotoxic to the periapical tissues (4) and it lacks substantivity.

Chlorhexidine (CHX), a suggested adjunct irrigation solution (3, 5) may exert a substantive

antibacterial effect (6-8) and has low grade toxicity (9), but it does not dissolve organic tissue or

remove the smear layer (2, 3). Removal of the smear layer is commonly achieved by adjunctive

irrigation with ethylenediaminetetraacetic acid (EDTA) which is a chelating agent (2, 10). It has

been established that the combination of irrigation solutions can improve the antibacterial activity

(11); thus, the final rinse of root canals with CHX after irrigation with NaOCl followed by EDTA,

has been proposed (3, 5). However, the application of CHX after NaOCl results in the formation

of a brown precipitate (12-21), shown in several studies to contain the presence of toxic

parachloraniline (PCA) (12-14, 22). Apart from potential toxic effects, the formation of precipitate

can lead to partial occlusion of dentinal tubules and prevention of lateral penetration of irrigation

agents, medicaments and filling materials (23). Also, PCA can lead to methemoglobin formation

in humans and is carcinogenic in animals (24, 25).

Two standard surface analytical techniques were utilized to examine the effects of the irrigation

agents on the dentin surface, namely Time-of-Flight Secondary-ion Mass Spectrometry

(TOFSIMS) and X-ray photoelectron spectroscopy (XPS) (26, 27). In TOF-SIMS, a primary ion

beam is used to generate a secondary ion mass spectrum. It has been used in dentistry to

qualitatively analyze the dentinal surface in various studies (17, 28-30). Complementary

examination of the chemical composition of a surface can be obtained with XPS. Irradiation of the

sample by X-rays results in the emission of photoelectrons characteristic of the elements present

and via chemical shifts, their chemical environment. Unlike TOFSIMS, XPS is readily

quantifiable. Previous studies utilizing TOF-SIMS and XPS have examined the adsorption of CHX

to hydroxyapatite and analyzed as well as quantified the precipitate formed by combinations of

NaOCl with CHX (14, 31).

23

Irrigation solutions may be combined with detergents to reduce surface tension and improve

antibacterial efficacy (32). QMiX (Dentsply Tulsa Dental, Tulsa, OK) and Smear OFF (Vista

Dental Products, Racine, WI) are two proprietary irrigation agents containing EDTA, CHX and

detergent. They are both proposed for final rinse of the root canals with the additional step of saline

irrigation after NaOCl when QMiX is used (17). Similarly, with QMiX (17), mixing Smear OFF

with NaOCl (pilot studies) resulted in a minor color change without detectable precipitation;

however, the manufacturer claims that Smear OFF can be applied directly after NaOCl without the

need for in-between saline irrigation. Thus, the aim of this study was to examine the formation of

precipitate and PCA on the surface of dentin irrigated with NaOCl followed by Smear OFF, using

TOF-SIMS and XPS analyses.

Materials and Methods

Specimens preparation

This study protocol was approved by the University of Toronto, Health Sciences Research Ethics

Board (#: 00033125). Following a previously published protocol (17), six extracted human

noncarious maxillary molars were stored in thymol solution before use. Two horizontal slices were

obtained from the cervical part of each tooth with a nominal thickness of 2 mm, using a diamond

coated saw (Leica EM TXP Target Sectioning System, Leica Microsystems GmbH, Vienna,

Austria). The 12 dentin blocks were embedded in low viscosity epoxy resin (Epo-Thin, Buehler,

Lake Bluff, USA) for 24 hours. In order to expose the dentinal tubules in a perpendicular

orientation to the surface, the surface was further ground with either a diamond or glass

microtomes (Leica EM UC6/FC6 Ultra-cryomicrotome, Leica Microsystems GmbH, Vienna,

Austria).

Exposure to irrigation agents

The dentin block specimens were randomly divided into two groups. CHX Group: Six specimens

were immersed in 5 ml of 6% NaOCI (Sodium Hypochlorite, Lavo inc., Montreal, Quebec) for 1

min, immediately followed by 5 ml of 17% EDTA (EDTA, Vista Dental Products, Racine, WI,

USA) for 1 min, then in fresh 5 ml of 6% NaOCI for 1 min and a final immersion in 5 ml of 2%

CHX (chlorhexidine digluconate Vista Dental Products, Racine, WI, USA) for 1 min. Smear OFF

24

Group: Six specimens were immersed in 5 ml of 6% NaOCI for 1 min, then in 5 ml of Smear OFF

for 1 min (as per manufacturer’s recommendation). After completion of the immersion protocol,

the samples were left on a bench top to dry overnight.

TOF-SIMS analysis

Three specimens from each group were analyzed using an IonTOF TOFSIMS V (IonTOF GmbH,

Münster, Germany) equipped with a bismuth (Bi) liquid metal ion gun. The Bi3 ++ cluster was used

as the primary ion source. The gun was operated in both high-mass and high-spatial resolution

modes. High mass resolution spectra were obtained on the initial surface (500 µm x 500 µm, 100

sec), followed by high mass resolution spectra on a 150 µm x 150 µm area, within a 450 µm x 450

µm cleaned area using an Ar cluster source. Images (256 x 256 pixels, 20 scans) were also obtained

from this region in high spatial resolution mode. Both positive and negative spectra were obtained.

XPS analysis

XPS spectra from the remaining three specimens were obtained on a ThermoFisher Scientific

ESCALAB 250Xi (Thermofisher Scientific, East Grinstead, UK). A monochromatic Al Kα source

with a spot size of 400 µm was used. Photoelectrons were collected at take-off angle of 90⁰ relative

to the specimen surface using a pass energy of 100 eV for the survey spectra and 20 eV for the

spectral regions. Charge compensation was applied using the combined Ar+/e-flood gun and the

peaks were shifted to place the main C 1s peak at 285.0 eV. Composition was obtained from the

latter spectra by integrating the peaks after subtracting a Shirley type background (27) and

applying the supplied sensitivity factors using the instrument’s software (Avantage 5.926—

Thermofisher Scientific, East Grinstead, UK). To ascertain depth of coverage, the surfaces were

Ar cluster sputter-cleaned followed by more intense Ar+ depth profiling using the dual-mode Ar

mono/cluster source provided with the instrument. The beam was rastered over a 2 mm x 2 mm

area. The XPS spectra were obtained from a 400 µm spot at the crater’s center.

25

Results

Brown precipitate formation was detected in all specimens of the CHX group, whereas no

precipitate and no color change were observed in any specimens of the Smear OFF group.

TOF- SIMS analysis

The positive ion TOF-SIMS spectra for the CHX and the Smear OFF groups are shown in Fig. 1A

and 1B, respectively, for mass ranges 100 – 200 u and 500 – 510 u. The negative ion spectra for

CHX and Smear OFF groups, for mass range 0 – 100 u, are shown in Fig. 1C and 1D, respectively.

The position of the characteristic positive fragments for CHX (127 u, 153 u, 170 u and 195 u) and

that of the parent ion for CHX (505.24 u), are indicated in Figures 1A and B. It should be noted

that the peak at 127 u also corresponds to the main peak of PCA. Characteristic peaks in the

negative ion spectra, CN- and Cl for the additive (CHX or Smear OFF), and the phosphate peaks

PO2 - (63 u) and PO3 - (79 u) for the substrate, are indicated in Figures 1C and D.

For the CHX group, the characteristic peaks for CHX are clearly observed whereas the substrate

phosphate peaks are greatly reduced, indicative of substantial coverage. The reverse occurs for

Smear OFF in that the substrate peaks are prominent and peaks assignable to CHX are greatly

reduced.

This observation is confirmed in the high imaging resolution mode, for both the positive and the

negative ions. An irregular precipitate was observed occluding the dentinal tubules in specimens

from the CHX group (Figs. 2A and C), whereas no precipitate and patent dentinal tubules were

seen in specimens from the Smear OFF group (Figs. 2B and D).

XPS analysis

The XPS survey spectra for the Smear OFF group (Fig. 3B) show an increase in the relative

intensity of Ca + and PO4 - peaks arising from the dentin substrate, compared to the spectra for the

CHX group (Fig. 3A), suggesting that the dentin in the Smear OFF group is not covered by

precipitate or thick (> 10 nm) film.

From the molecular formula of CHX (C22H30Cl2N10 ) the ratio of N/Cl is 5 and in PCA it is 1.96

(14). The ratio of N/Cl in the CHX group in all samples was smaller than 5. While not conclusive,

26

this finding was consistent with the presence of PCA. For the Smear OFF group, this ratio was not

assessed since nitrogen from the substrate would also be present.

The Cl 2p spectra (Figs. 3C and D) show the presence of covalent Cl and the chloride (Cl -). For

the Smear OFF group, the amount of Cl -relative to the covalent Cl was greater than that for the

CHX group. This implied a much thinner layer, as did the presence Ca and P peaks in the spectrum

and indicated that the over layer was less than the escape depth of the photoelectrons (<10 nm)

(31).

The C 1s envelope for Smear OFF (Fig. 3F) implied the presence of CHX since a shake-up feature,

characteristic of aromatic structures, was observed and the overall peak shape was similar to that

of CHX (Fig. 3E).

Discussion

Interaction between CHX and NaOCl has gained attention with the controversial finding of the

formation of PCA (12-14, 17-20). Even if PCA does not form, mixing these two irrigation agents

produces a visible insoluble precipitate which has been shown to occlude dentinal tubules and

discolor teeth (23), in addition to the possibility of its being toxic (24, 25). Therefore, it is

imperative that any new CHX-containing irrigation agent applied as a final root canal flush after

use of NaOCl should be assessed for its interaction with NaOCl. This study examined the

interaction of NaOCl and Smear OFF by assessing by-product formation on the dentin surface.

Smear OFF is an irrigation agent containing CHX gluconate (<1% wt),

tetrasodiumethylenediaminetetraacetate dihydrate (18% wt) and a surface-active detergent as its

active components (33). According to the manufacturer, it is formulated to have antibacterial effect

and to remove the smear layer of the root canal walls. An earlier report on its interaction with

NaOCl suggested that NaOCl lost the free available chlorine after alternating with either Smear

OFF or with EDTA (33).

TOF-SIMS analysis provides the composition, distribution and molecular information of the

analyzed surface. Analysis of the fragmentation pattern can result in the identification of the

various components (26). In a previous study (34) from our group, the fingerprint of CHX was the

peaks of 127 u, 153 u, 170 u, 195 u and its parent 505 u. The characteristic peak of PCA was 127

27

u (34). Since the precipitate in the present study showed peaks at 127 u, 153 u, 170 u and 195 u,

the peak at 127 u could not solely be used as characteristic of PCA. Therefore, comparison of the

relative heights and ratios of these peaks was used. In the precipitate, the peaks of 153 u, 170 u

and 195 u were less intense than the 127 u, in contrast to CHX where the signal of 127 u was less

intense than the rest of the peaks. Normally, from the CHX spectra, the peak height ratio for 170

u/127 u is 3:1, and for 153 u/127 u it is 3:2. In the present study, the 153 u/127 u ratio was more

than 1:1 and the 170 u/127 u ratio was 3:2, indicating a stronger signal coming from the 127 u

component suggestive of PCA. This fragmentation pattern was absent from the Smear OFF and

presence of PO2 - (63 u) and PO3 - (79 u) was noted, suggesting that the dentin was not covered by

a thick layer or precipitate. While Kolosowski et al. (17) analyzed cross sections of the dentin with

TOF-SIMS to assess penetration of precipitate into dentinal tubules, a similar analysis was not

performed for Smear OFF in the present study because no precipitate was observed.

XPS analysis was utilized to further investigate the source of the chlorine in the Smear OFF group,

which could be either CHX or NaOCl. For both PCA and CHX, chlorine should appear as the

covalent species, whereas the chloride reflects the presence NaOCl. Even though small amounts

of CHX exist in Smear OFF, the observed combination of both forms of chlorine suggested that

CHX was concentrated on the surface. Specific profiling was performed using a combined

mono/cluster Ar source. The absence of detectable changes for the CHX group implied the

formation of a thick layer over the dentin surface, the quick reduction in the amount of Cl for the

Smear OFF group implied that the surface was covered with just a few monolayers. These results

were consistent with presence of Ca and P peaks in the survey spectra for the Smear OFF group

and not in the CHX group.

The lack of precipitate formation in the Smear OFF group could be attributed to various reasons.

The surfactant forms a tertiary structure with CHX which protects the CHX from hypochlorite

anion (OCl-) attack and eliminates the formation of a precipitate. Moreover, the layer that the

detergent forms may be the one analyzed by TOF-SIMS. However, lack of detection of chlorine

could also be attributed to the possible small amount of CHX in the irrigation agent.

28

The results of the present in vitro study suggest that Smear OFF can be safely applied after NaOCl,

as a final irrigant, without the need of an extra step of saline rinse. However, assessment of its

antibacterial properties is a research question that is prudent to be assessed in future studies.

Conclusion:

Within the limitations of this in-vitro study, the exposure of dentin surfaces to NaOCl followed by

Smear OFF did not result in formation of precipitate or PCA.

Acknowledgements

We acknowledge the Ontario Centre for the Characterisation of Advanced Materials (OCCAM)

for the conduction of the experiments, as well as Dr. Anil Kishen for valuable contribution to the

study design and Dr. Shimon Friedman and Dr Calvin Torneck for assistance with the manuscript.

29

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2. Basrani B, Haapasalo M. Update on endodontic irrigating solutions. Endod Topics 2012;27(1):74-102.

3. Zehnder M. Root canal irrigants. J Endod 2006;32(5):389-398.

4. Hidalgo E, Bartolome R, Dominguez C. Cytotoxicity mechanisms of sodium hypochlorite in cultured human dermal fibroblasts and its bactericidal effectiveness. Chemico-Biological Interactions 2002;139(3):265-282.

5. Ng YL, Mann V, Gulabivala K. A prospective study of the factors affecting outcomes of nonsurgical root canal treatment: part 1: periapical health. Int Endod J 2011;44(7):583-609.

6. Souza M, Cecchin D, Farina AP, Leite CE, Cruz FF, da Cunha Pereira C, et al. Evaluation of Chlorhexidine Substantivity on Human Dentin: A Chemical Analysis. J Endod 2012;38(9):1249- 1252.

7. Komorowski R, Grad H, Yu Wu X, Friedman S. Antimicrobial Substantivity of Chlorhexidine-Treated Bovine Root Dentin. J Endod;26(6):315-317.

8. White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Endod;23(4):229-231.

9. Löe H, Rindom Schiøtt C. The effect of mouthrinses and topical application of chlorhexidine on the development of dental plaque and gingivitis in man. J Periodontal Res 1970;5(2):79-83.

10. Haapasalo M, Shen Y, Qian W, Gao Y. Irrigation in endodontics. Dent Clin North Am 2010;54(2):291-312.

11. Kuruvilla JR, Kamath MP. Antimicrobial activity of 2.5% sodium hypochlorite and 0.2% chlorhexidine gluconate separately and combined, as endodontic irrigants. J Endod 1998;24(7):472-476.

12. 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):1296-1299.

13. 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):312-314.

14. Basrani BR, Manek S, Sodhi RN, Fillery E, Manzur A. Interaction between sodium hypochlorite and chlorhexidine gluconate. J Endod 2007;33(8):966-969.

30

15. Krishnamurthy S, Sudhakaran S. Evaluation and Prevention of the Precipitate Formed on Interaction between Sodium Hypochlorite and Chlorhexidine. J Endod 2010;36(7):1154-1157.

16. Mortenson D, Sadilek M, Flake NM, Paranjpe A, Heling I, Johnson JD, et al. The effect of using an alternative irrigant between sodium hypochlorite and chlorhexidine to prevent the formation of para-chloroaniline within the root canal system. Int Endod J 2012;45(9):878-882.

17. Kolosowski KP, Sodhi RN, Kishen A, Basrani BR. Qualitative analysis of precipitate formation on the surface and in the tubules of dentin irrigated with sodium hypochlorite and a final rinse of chlorhexidine or QMiX. J Endod 2014;40(12):2036-2040.

18. 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):505-510.

19. 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):983-988.

20. Thomas JE, Sem DS. An In Vitro Spectroscopic Analysis to Determine Whether Para- Chloroaniline Is Produced from Mixing Sodium Hypochlorite and Chlorhexidine. J Endod 2010;36(2):315-317.

21. Arslan H, Uygun AD, Keskin A, Karatas E, Seçkin F, Yıldırım A. Evaluation of orange-brown precipitate formed in root canals after irrigation with chlorhexidine and QMix and spectroscopic analysis of precipitates produced by a mixture of chlorhexidine/NaOCl and QMix/NaOCl. Int Endod J 2015;48(12):1199-1203.

22. Nocca G, Ahmed HMA, Martorana GE, Callà C, Gambarini G, Rengo S, et al. Chromographic Analysis and Cytotoxic Effects of Chlorhexidine and Sodium Hypochlorite Reaction Mixtures. J Endod 2017;43(9):1545-1552.

23. 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):181-185.

24. Matsumoto M, Aiso S, Senoh H, Yamazaki K, Arito H, Nagano K, et al. Carcinogenicity and Chronic Toxicity of para-chloronitrobenzene in Rats and Mice by Two-Year Feeding. J Environ Pathol Toxicol Oncol. 2006;25(3):571-584.

25. Chhabra RS, Huff JE, Haseman JK, Elwell MR, Peters AC. Carcinogenicity of p chloroaniline in rats and mice. Food Chem Toxicol 1991;29(2):119-124.

26. Sodhi RN. Time-of-flight secondary ion mass spectrometry (TOF-SIMS):--versatility in chemical and imaging surface analysis. Analyst 2004;129(6):483-487.

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27. Ratner BD, Castner DG. Electron Spectroscopy for Chemical Analysis. In: Vickerman JC, Gilmore IS, editors. Surface Analysis: The Principal Techniques. 2nd. ed. Chichester, U.K.: John Wiley & Sons, Ltd; 2009.

28. 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):320-333.

29. Gotliv BA, Veis A. The composition of bovine peritubular dentin: matching TOF-SIMS, scanning electron microscopy and biochemical component distributions. New light on peritubular dentin function. Cells Tissues Organs 2009;189(1-4):12-19.

30. Gotliv BA, Veis A. Peritubular dentin, a vertebrate apatitic mineralized tissue without collagen: role of a phospholipid-proteolipid complex. Calcif Tissue Int 2007;81(3):191-205.

31. Sodhi RN, Grad HA, Smith DC. Examination by X-ray photoelectron spectroscopy of the adsorption of chlorhexidine on hydroxyapatite. J Dent Res 1992;71(8):1493-1497.

32. Bukiet F, Couderc G, Camps J, Tassery H, Cuisinier F, About I, et al. Wetting Properties and Critical Micellar Concentration of Benzalkonium Chloride Mixed in Sodium Hypochlorite. J Endod;38(11):1525-1529.

33. Krishnan U, Saji S, Clarkson R, Lalloo R, Moule AJ. Free Active Chlorine in Sodium Hypochlorite Solutions Admixed with Octenidine, SmearOFF, Chlorhexidine, and EDTA. J Endod 2017;43(8):1354-1359.

34. 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 InterfaceAnalysis 2011;43(1-2):591-594.

32

Legends to Figures

Figure 1: Selected TOF-SIMS high mass resolution spectra of the treated dentin: Positive ion

spectra for (A) CHX group; (B) Smear OFF group; and the corresponding negative ion spectra

for (C) CHX and (D) Smear OFF. Peaks assignable to PCA, CHX and the substrate are indicated

– see text for details.

Figure 2: Selected TOF-SIMS high spatial resolution images of dentin surfaces: Negative ion

images for (A) CHX group; (B) Smear OFF; and the corresponding positive ion images for (C)

CHX and (D) Smear OFF. Presence of precipitate is observed in the CHX group whereas open

and patent dentinal tubules are observed in the Smear OFF group.

Figure 3: Survey spectra with relative atomic percentages for (A) CHX; (B) Smear OFF. Also

shown are the fitted high resolution peaks for the Cl 2p region (C and D) and the C 1s region (E

and F) for CHX and Smear OFF respectively – see text for details.

33

Figure 1

34

Figure 2

35

Figure 3:

36

DISCUSSION

Aim and Methodology

This in vitro study was conducted in order to examine whether precipitation occurs after rinse with

NaOCl followed by final rinse of Smear OFF and if PCA can be detected in the precipitate. Up

until this manuscript was written, this was the first study assessing formation of precipitate or PCA

using Smear OFF as a final rinse protocol. Previous studies from our group have assessed the

interaction after rinse with NaOCl followed by CHX (131, 133-135) and after final rinse with

QMiX (135). The results revealed that precipitate with PCA occurred after final rinse with CHX

whereas in case of QMiX no precipitation occurred.

A pilot study was first performed in order to assess the end result of combining 6% NaOCl

followed by Smear OFF in a plastic cup according to the manufacturer’s protocol. A reaction was

noticeable, with bubbling action occurring, probably because of the surfactant that the irrigant

contains and a color change of the solution to a more yellowish hue (pic. 4). No precipitate was

noted. However, the end result of a mixture between two or more products is not necessarily only

related to the products themselves. Dentin, inorganic components and proteins contained within,

can act as a catalyst allowing a reaction to occur, where the reaction would not otherwise occur in

the absence of dentin. Hydrogenation reactions are examples where a surface can act as a catalyst.

Therefore, a decision was made to study the precipitate on dentin. Following Kolosowski’s study

(135), dentinal blocks were created in order to eliminate the different confounding factors such as

grooves, curvatures and irregularities in the topographical aspect of the surface which could

potentially be source of contaminants. In addition, TOF-SIMS analysis requires flat surface

otherwise topographical shape will influence the direction of the ions hitting the surface of the

sample and emitted from the surface and hence the number of the ions reaching the detector.

Therefore, upper maxillary teeth were chosen for the experiments, since the dentinal tubules

exhibit more consistency in terms of the direction (161, 162). More specifically, dentinal tubules

travel through the dentin exhibiting an S shape. The shape and the size of them depends on the

location. They are conical in shape with the base being close to the pulp and they present with a

right angle and size of 3 µm whereas in the junction of the enamel the size is less than 1µm (161,

163, 164). The cervical dentin was chosen in order to have the maximum size dimeter of the

dentinal tubules and better assess the results. In Kolosowski’s et al. study (135), regarding the

37

CHX group analysis of penetration of the precipitate with cross sections of the dentinal samples

was also performed and penetration of the precipitate was confirmed. In the present study, TOF –

SIMS analysis of the cross sections was not pursued since no precipitate was found. The age of

the teeth could be an influencing factor. In that study, a bank of teeth anonymously donated was

used. Aging, progresses from the apical to the coronal part of the teeth and it results to sclerosis.

Previous study from our group using midroot dentin has shown that the results among the samples

were similar, possibly meaning that the effect of age was not very substantial.

The irrigation protocol that was used in the control group was based on the outcome study of Ng

et al.(12), penultimate EDTA increases the success rate. Thus, the irrigation protocol used 6 %

NaOCl, 17 % EDTA, 6 % NaOCl and final rinse of 2% CHX, and for the experimental group 6%

NaOCl and Smear OFF as per manufacture instructions. The concentrations used for this study,

followed the protocol that is used for EDTA and CHX. Regarding the concentration of NaOCl,

there is a variability in the concentration being used among dentists and endodontists. The

maximum concentration of 6% was decided to be used in that study, in order to maximize the

precipitate formation, since the amount of precipitate has been shown to be proportional to the

concentration of NaOCl (131). All the reported irrigants were used for 1 minute each for

standardization purposes. However, clinical times vary among the different clinicians. Regarding

the surface that was analyzed, cross sections of the dentinal samples were not prepared, since in

the Smear OFF group precipitate was not found and the penetration in the CHX group of the

precipitate was tested from previous study (135).

During the preparation of the dentinal blocks, various technical steps took place, which could have

provoked contamination of the analyzed surface. TOF-SIMS can be performed in various modes.

In our study it was decided to use sputter erosion with an Ar cluster source in order to remove any

surface contamination thereby simplifying the mass spectrum and making the surface composition

clearer. PCA is a volatile in nature and in high temperatures, such as in a vacuum environment it

may be sublimated before being detected. Thus, liquid nitrogen was used in order to cool the

samples(165). However, it has been shown, that even in ambient temperature it could be detected.

In order to further investigate the source of the chlorine in the Smear OFF group, which could be

either CHX or NaOCl, XPS analysis was utilized as well. XPS is another surface sensitive

analytical tool, however, in contrast to TOF-SIMS, it is readily quantifiable. Another advantage in

38

comparison to the TOF-SIMS, is that the analyzed surfaces do not have to be so precisely polished,

allowing for minor handling inaccuracies not to result into significant artifacts. Theoretically both

in PCA and in CHX, chlorine should be covalent (Clo). The chloride (Cl-) reflects the presence

NaOCl. There is a chemical shift between these components with the covalent Cl appearing at a

higher binding energy. Thus, it was possible to confirm the presence of CHX in Smear OFF group.

Results

PCA detection in the precipitate was done by assessing the fragmentation pattern of the high mass

resolution analysis. Previous study from our group has shown that the fingerprint of CHX are the

peaks 127 u, 153 u, 170 u, 195 u as well as the parent ion at 505 u (165). The precipitate also

shows these features. It can be argued that since the peak of 127 is fragment of both CHX as well

as being a characteristic peak for PCA, it cannot solely be used to determine the presence of PCA

(165). Therefore, comparison of the relative heights and ratios of these peaks was used. More

specifically, in the precipitate, the peaks of 153 u, 170 u and 195 u were less intense than the 127

u on the contrary to the CHX alone where the signal of 127 u is less intense than the rest of the

peaks. Normally, from the CHX spectra, there is a ratio of peak height 3:1 170 u to 127 u and 3:2

looking at 153 u to 127 u. In my study, the ratio 153u to 127u was more 1:1 and ratio of 170 u to

127 u was 3:2 which indicates stronger signal coming from 127 u component (meaning PCA).

Interestingly, in the precipitate we could also identify the 505 u peak, which is parental peak of

CHX, showing that possibly there was residual CHX in the precipitate.

With reference to the XPS analysis, addition of NaOCl to the CHX group should result in the

presence of some amounts of chloride (Cl). This reacts with the CHX and forms PCA resulting in

reduction of Clo. Even though small amount of CHX exists in Smear OFF group the combination

of Cl- and Clo could be seen, suggesting that the small amount of CHX is concentrated on the

surface. Some profiling was performed using a combined mono/cluster source. No significant

changes were observed for CHX implying a thick layer whereas the amount of Cl- was quickly

reduced for Smear OFF group implying a few monolayers coverage. These results are consistent

with calcium and phosphate ions from the substrate being observed in the Smear OFF treated

samples and not in CHX treated samples. The fact that no precipitate was formed in the Smear

OFF group can be because of various reasons. Smear OFF is composed of CHX, EDTA, a

surfactant acidic polymer and water. The surfactant forms a tertiary structure with CHX which

39

protects the CHX from hypochlorite anion (OCl-) attack and eliminates the formation of a

precipitate. Moreover, the layer that the detergent formed could have been the one analyzed from

TOF – SIMS. However, lack of detection of chlorine in TOF-SIMS could also be attributed to the

possible small amount of CHX in the irrigant.

40

CONCLUSIONS & FUTURE DIRECTION:

- Within the limitations of this in vitro study utilizing dentin blocks, TOF-SIMS and XPS

analyses have shown that precipitate, resulting from NaOCl being mixed with CHX, may

form on the root canal dentin surface. These results, confirm previous study from our

group using TOF-SIMS solely to examine that.

- TOF-SIMS and XPS have indicated this precipitate to contain PCA, and to occlude the

tubules on the dentin of the root canal surface when dentin blocks are irrigated with 6%

NaOCl, followed by 17% EDTA, 6% NaOCl and 2% CHX.

- It has been noted that no precipitatation occured and no PCA was detected on root canal

dentin surface with TOF-SIMS and XPS analyses when dentin is irrigated with 6%

NaOCl, followed by final rinse with Smear OFF.

- The results of the present in vitro study suggest that Smear OFF can be safely applied after

NaOCl, as a final irrigant, without the need of an extra step of saline rinse. However,

assessment of its antibacterial properties is a research question that is prudent to be

examined in future studies.

41

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APPENDICES Appendix 1:

Letter of ethics approval from Health Sciences Research Ethics Board (REB), University

Toronto

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Appendix 2: Molecular structures

Figure A): Molecular structure of Chlorhexidine (CHX)

Figure B): Molecular structure of Ethylenediaminetetraacetic acid (EDTA)

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Appendix 3: Pictures

Picture 1: Sequence to create the dentin blocks

Picture 2: Dentin blocks in resin

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Picture 3: Dentin polishing with glass knife with Leica EM UC6/FC6 Ultracryomicrotome

Picture 4: Brown precipitate after irrigation with NaOCl followed by CHX in plastic cup & tooth

Picture 5: Minor change of color in a yellowish hue after combination of NaOCl and Smear OFF

in plastic cup & tooth

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Picture 6: Samples being analyzed in TOF - SIMS

Picture 7: Leica EM UC6/FC6 Ultracryomicrotome

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Picture 8: TOF- SIMS- ION – TOF GmbH

Picture 9: ThermoFisher Scientific Escalab 250Xi

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Appendix 4: Figures

Figures with the high mass resolutions and high spatial resolution from TOF-SIMS and survey

spectra, carbon and chlorine fits from XPS analyses

Figure 1: Sample 4: Positive ions – TOF-SIMS selected spectra.

Note the position of the peaks of the breakdown products of CHX (153u, 170u, 195u) and the PCA

(127u)

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Figure 2: Sample 4: Negative ions – TOF- SIMS selected spectra

Note the relative abundance of Cl- (35 and 37u) and the absence of PO-2 (63u) and PO-3 (79u)

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Figure 3: Sample 4: Positive ions –TOF-SIMS selected spectra-Images

Note the irregular precipitate occluding the dentinal tubules

63

Figure 4: Sample 4: Negative ions- TOF-SIMS selected spectra - Images

Note the irregular precipitate occluding the dentinal tubules

64

Figure 5: Sample 10: Positive ions TOF-SIMS selected spectra- Images

Note the absence of CHX breakdown (153u, 170u, 195u) and PCA(127u) peaks

65

Figure 6: Sample 10: Negative ions, TOF-SIMS selected spectra

Note the peaks PO-2 (63u) and PO3- (79u)

66

Figure 7: Sample 10 – Positive ions, TOF-SIMS selected spectra -Images

Note the open and patent dentinal tubules

67

Figure 8: Sample 10- Negative ions, TOF-SIMS selected spectra -Images

Note the open and patent dentinal tubules

68

Figure 9: Sample 8 - Positive ions, TOF-SIMS selected spectra

Note the absence of CHX breakdown (153u, 170u, 195u) and PCA(127u) peaks

69

Figure 10: Sample 8 - Negative Ions, TOF-SIMS selected spectra

Note the peaks PO-2 (63u) and PO3- (79u)

70

Figure 11: Sample 8: Positive ions, TOF-SIMS selected spectra, Images

Note the open and patent dentinal tubules

71

Figure 12: Sample 8- Negative ions, TOF-SIMS selected spectra, Images

Note the open and patent dentinal tubules

72

Figure 13: Sample 1- XPS analysis - Survey spectra CHX group

Figure 14: Sample 2 XPS analysis Survey spectra, CHX group

73

Figure 15: Survey spectra CHX- Sample 3 & Relative atomic percentages for all three samples

Figure 16: Sample 1: XPS analysis- Survey Spectra, Smear OFF group

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Figure 17: Sample 2- XPS analysis- Survey Spectra – Smear OFF group

Figure 18: Sample 3- XPS analysis, Survey Spectra – Smear OFF group

75

Figure 19: Sample 1: Carbon (C1s) fit – Smear OFF group

76

Figure 20: Sample 2: XPS analysis, Carbon (C1s) fit – Smear OFF group

77

Figure 21: Sample 3: XPS analysis, Carbon (C1s) fit – Smear OFF group