COMPARATIVE EVALUATION OF THE ANTIBACTERIAL EFFICACY OF FOUR ROOT CANAL SYSTEM DISINFECTION METHODS AGAINST E. FAECALIS

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Erika Endriukaityte

5th Year, Group 13

COMPARATIVE EVALUATION OF THE

ANTIBACTERIAL EFFICACY OF FOUR ROOT CANAL

SYSTEM DISINFECTION METHODS AGAINST E.

FAECALIS

Master’s thesis

Eduardas Kelbauskas

PhD, Eduardas Kelbauskas

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY

FACULTY OF ODONTOLOGY ENDODONTICS

COMPARATIVE EVALUATION OF THE ANTIBACTERIAL EFFICACY OF FOUR ROOT CANAL SYSTEM DISINFECTION METHODS AGAINST E.FAECALIS

Master’s Thesis

This thesis was done

by student ……… Supervisor………...

(signature) (signature)

……….... ……….. (name surname, year, group) (degree, name surname)

………..20…. ………20…. (day/month) (day/month)

Kaunas, 2017

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EVALUATION TABLE OF THE MASTER’S THESIS

OF THE TYPE OF SYSTEMIC REVIEW OF SCIENTIFIC LITERATURE Evaluation: ... Reviewer: ...

(scientific degree. name and surname)

No. MT parts MT evaluation aspects

Compliance with MT requirements and evaluation Yes Partially No 1 Summary (0.5 point)

Is summary informative and in compliance with the

thesis content and requirements? 0.3 0.1 0

2 Are keywords in compliance with the thesis _essence? 0.2 0.1 0

3

Introduc-tion, aim and tasks (1 point)

Are the novelty, relevance and significance of the

work justified in the introduction of the thesis? 0.4 0.2 0 4 Are the problem, hypothesis, aim and tasks formed

clearly and properly? 0.4 0.2 0

5 Are the aim and tasks interrelated? 0.2 0.1 0

6 Selection criteria of the studies, search methods and strategy (3.4 points)

Is the protocol of systemic review present? 0.6 0.3 0 7

Were the eligibility criteria of articles for the selected protocol determined (e.g., year, language, publication condition, etc.)

0.4 0.2 0

8

Are all the information sources (databases with dates of coverage, contact with study authors to identify additional studies) described and is the last search day indicated?

0.2 0.1 0

9

Is the electronic search strategy described in such a way that it could be repeated (year of search, the last search day; keywords and their combinations; number of found and selected articles according to the combinations of keywords)?

0.4 0.1 0

10

Is the selection process of studies (screening, eligibility, included in systemic review or, if applicable, included in the meta-analysis) described?

0.4 0.2 0

11

Is the data extraction method from the articles (types of investigations, participants, interventions, analysed factors, indexes) described?

0.4 0.2 0

12

Are all the variables (for which data were sought and any assumptions and simplifications made)

listed and defined? 0.4 0.2 0

13

Are the methods, which were used to evaluate the risk of bias of individual studies and how this information is to be used in data synthesis, described?

0.2 0.1 0

14 Were the principal summary measures (risk ratio, _{difference in means) stated?} 0.4 0.2 0

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analysis of data (2.2 points)

the reasons in each stage of exclusion presented?

16

Are the characteristics of studies presented in the included articles, according to which the data were extracted (e.g., study size, follow-up period, type of respondents) presented?

0.6 0.3 0

17

Are the evaluations of beneficial or harmful outcomes for each study presented? (a) simple summary data for each intervention group; b) effect estimates and confidence intervals)

0.4 0.2 0

18 Are the extracted and systemized data from studies presented in the tables according to individual tasks?

0.6 0.3 0

19

Discussion (1.4 points)

Are the main findings summarized and is their

relevance indicated? 0.4 0.2 0

20 Are the limitations of the performed systemic _{review discussed?} 0.4 0.2 0 21 Does author present the interpretation of the _results? 0.4 0.2 0 22

Conclusions (0.5 points)

Do the conclusions reflect the topic, aim and tasks

of the Master’s thesis? 0.2 0.1 0

23 Are the conclusions based on the analysed material? 0.2 0.1 0

24 Are the conclusions clear and laconic? 0.1 0.1 0

25

References (1 point)

Is the references list formed according to the

requirements? 0.4 0.2 0

26

Are the links of the references to the text correct? Are the literature sources cited correctly and

precisely? 0.2 0.1 0

27 Is the scientific level of references suitable for

Master’s thesis? 0.2 0.1 0

28

Do the cited sources not older than 10 years old form at least 70% of sources, and the not older than 5 years – at least 40%?

0.2 0.1 0

Additional sections, which may increase the collected number of points

29 Annexes Do the presented annexes help to understand the _{analysed topic?} +0.2 +0.1 0

30 recommen-Practical dations

Are the practical recommendations suggested and

are they related to the received results? +0.4 +0.2 0

31 Were additional methods of data analysis and their results used and described (sensitivity analyses, meta-regression)?

+1 +0.5 0

32

Was meta-analysis applied? Are the selected statistical methods indicated? Are the results of each meta-analysis presented?

+2 +1 0

General requirements, non-compliance with which reduce the number of points

33

General

require-ments

Is the thesis volume sufficient (excluding annexes)?

15-20 pages (-2 points)

<15 pages (-5 points) 34 Is the thesis volume increased _{artificially?} -2 points -1 point

35 Does the thesis structure satisfy the

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Reviewing date: ...

*Remark: the amount of collected points may exceed 10 points.

Reviewer’s comments: ___________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ _________________________________________ ___________________________

Reviewer’s name and surname Reviewer’s signature

36 Is the thesis written in correct language,

scientifically, logically and laconically? -0.5 point -1 points 37 Are there any grammatical, style or _{computer literacy-related mistakes?} -2 points -1 points

38 Is text consistent, integral, and are the _{volumes of its structural parts balanced?} -0.2 point -0.5 points 39 Amount of plagiarism in the thesis. _{(not evaluated)}>20%

40

Is the content (names of sections and sub-sections and enumeration of pages) in compliance with the thesis structure and aims?

-0.2 point -0.5 points

41

Are the names of the thesis parts in compliance with the text? Are the titles of sections and sub-sections distinguished logically and correctly?

42 Are there explanations of the key terms _{and abbreviations (if needed)?} -0.2 point -0.5 points

43 Is the quality of the thesis typography (quality of printing, visual aids, binding) good?

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SUMMARY

Background:

Presence of resistant bacterial species such Enterococcus faecalis in the root canal has long been known to be a major contributor to persistent endodontic infections and post-treatment failure. Irrigation with sodium hypochlorite solution is widely considered as the “Gold standard” method of canal disinfection, but carries the disadvantages of host cell cytoxicity and unpleasant taste and odor, rationalizing the need for research into alternative methods.

Objectives:

To systematically review scientific Literature publications on four commonly used root canal disinfection methods in terms of their respective bactericidal efficacies against Enterococcus

faecalis whilst also evaluating their levels of cytotoxicity and dentinal tubule penetration depths.

Materials and methods:

A total of 33 literature sources published between 2012 and 2017 were systematically selected and reviewed. All sources were found on PubMed and Science Direct databases.

Results:

In terms of antibacterial efficacy against E. faecalis, 2.5%-5.25% proved to be the best

method of root canal disinfection, but fell short in dentin penetration abilities and cytotoxicity levels. Diode laser showed average antibacterial efficacy, but optimal dentin penetration depth. Passive ultrasonic irrigation did not prove to be more bactericidal than passive irrigation with NaOCl, but achieved slight improvement in dentinal penetration depth. Photoactivated disinfection proved favorable in terms of bactericidal efficacy, dentin penetration depth and cytotoxicity. CHX proved favorable only in terms of low cytotoxicity.

Conclusions: Irrigation with NaOCl proved itself justifiable in its mainstream use with impressive

results, however much evidence pointed to PAD as an acceptable alternative with impressive abilities across all three measurements.

Key words:

Enterococcus faecalis, canal disinfection, sodium hypochlorite irrigation, photoactivated disinfection, diode laser, passive ultrasonic irrigation

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INTRODUCTION

The main objective of all treatments of infected root canals is a thorough elimination of pathogenic microorganisms (1). The outcome of root canal treatment depends heavily on the efficacy of mechanical debridement and disinfecting irrigants (2). The aim of endodontic treatment is to achieve a root canal system free from damaging bacteria and other irritants, because the

presence of residual microorganisms after treatment may cause persistent inflammation. Insufficient root canal disinfection is possibly one of the main reasons for the occurrence of endodontic

treatment failure and periapical pathologies. Difficulties in achieving a thorough canal disinfection following mechanical debridement arise from the presence of a complex root canal system

anatomy. Presence of curved canals and accessory canals allow microorganisms to penetrate deep into the dentinal tubules where they are seldom reached by conventional irrigants (3), making it difficult if not impossible to entirely eliminate microorganisms from this system (4).

Enterococcus faecalis is a gram-positive anaerobic coccoid that has been reported in various studies

to be the predominant bacteria present in cases of endodontic failure. E. faecalis can adhere to host cells, alter the host-response, impair lymphocyte function and is able to persist in harsh

environmental conditions such as nutritional deprivation and high pH. E. faecalis is also capable of forming a dynamic structure known as a biofilm. This is composed of bacterial populations

surrounded by a polysaccharide polymetric matrix (5). A biofilm increases the bacterial resistance to antimicrobial agents by about 1000-1500 times when compared to the same bacterial species in their planktonic state. A biofilm also allows this microorganism to be substantially more resistant to destruction by phagocytes, antibodies and antimicrobials than species that are seldom biofilm forming (2). Furthermore, E. faecalis has a high virulence potential which can be attributed to its ability to invade dentinal tubules, compete with other microorganisms and persist in conditions of nutritional scarcity and high pH (6).

Presently, sodium hypochlorite is one of the most universally utilized irrigation solutions in endodontic dentistry. Its concentration can range from 0.5% to 5.25% and over. In an attempt to obtain optimal canal disinfection, there is some tendency by clinicians to use this solution at its highest concentration, which in effect brings about the issue of its cytotoxicity which increases with increasing concentration. There is some evidence that increasing the concentration past a certain point achieves only a statistically insignificant improvement in bactericidal efficacy whilst causing more harm to the host’s cells, necessitating the need for analysis in regard to this question. Aside from this, its other disadvantages include unpleasant smell, taste and corrosive potential (7). As

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technology progresses, alternative methods of canal disinfection are becoming more widely known and utilized in practice. However, an abundance of protocol varieties, contradicting data and differing opinions has led to a general lack of consensus as to which method of canal disinfection can most efficiently reduce the chance of endodontic post-treatment failure, the occurrence of which has persisted despite much scientific and technical progress. Four main methods, namely irrigating solution, diode laser, photoactivated disinfection and passive ultrasonic irrigation, will be considered.

This systematic review aims to gather results from multiple clinical and laboratory studies

conducted in the last 5 years and thereby draw conclusions on the method or methods with the most favorable attributes in terms of three specific parameters: 1) Method/s with low levels of

cytotoxicity 2) Method/s achieving highest dentin penetration depth and most importantly, 3) The resulting antibacterial efficacy of each method against Enterococcus faecalis.

Hypothesis: Conventional root canal irrigation using a needle and syringe and Sodium

Hypochlorite solution does not perform better than other root canal disinfection methods in terms of bactericidal efficacy against E. faecalis, dentin tubule penetration depth and cytotoxicity.

SELECTION CRITERIA OF THE ARTICLES. SEARCH METHODS AND

STRATEGY

Search strategy:

This systematic review utilized sources found on the database PubMed and also several literatures hand searched on Science Direct. To combine the chosen keywords in an efficient manner and generate the most suitable results, PubMed advanced search was used. To begin the search and selection, a combination of keywords was used by selecting either a “AND” or “OR” option of keyword combination. My chosen selection was: Enterococcus faecalis AND root canal disinfection AND sodium hypochlorite OR chlorhexidine irrigant OR root canal disinfection diode laser OR photoactivated disinfection OR photodynamic therapy OR passive ultrasonic irrigation OR root canal irrigation cytotoxicity OR root canal dentin penetration OR enterococcus faecalis biofilm. Some of the key variables of the search included various concentrations of irrigants and different types of light sources for PAD. The last date of search was 17/02/17.

Types of publications:

All the sources found written in the English language, with the exception of an article by Ivana Tolijan et al. (2016) which was translated into English from the Croatian language.

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Types of studies and population:

Types of studies used in the writing of this review included In vivo, In vitro, Ex vivo and controlled randomized trials. Studies were conducted on humans, extracted human or bovine teeth and studies on simulated root canal models were all included.

Inclusion criteria

- Articles written in or directly translated into the English Language

- Studies testing antimicrobial efficacies and/or penetration depths and cytotoxicity

Exclusion criteria

- Systematic and literature reviews - Publications older than five years

Data collection process

The use of these keywords generated an initial literature inclusion of 1416 reports/article. The generated sources were then further filtered in order to include only those that fit the criteria. The sources had to be full text and exclude systematic reviews, meta-analysis and literature reviews. The applied filters generated 710 sources (plus 3 additional hand searched articles), which were then screened for relativity based on titles and abstracts. Despite applied filters, a few manuscripts were generated which were excluded based on findings that they were not yet published and could therefore contain misinformation. Most of the generated sources were excluded due to it being generally unsuitable for the topic of research. A total of 62 articles were accepted based on their titles or abstracts and out of these, only 33 sources were deemed as eligible for use in this review.

Several studies such as (8) and (9) tested various disinfection protocols against biofilms composed of multiple bacterial strains. As the focus of this paper is elimination of E. faecalis only, these sources were only utilized for general information about disinfection protocols, but excluded from the main results and tables of evidence.

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SYSTEMIZATION AND ANALYSIS OF DATA

NCBI PubMed advanced search:

Search terms included: “enterococcus faecalis”, “root canal disinfection”, “sodium hypochlorite”, “chlorhexidine irrigant”, “ root canal disinfection diode laser”, “photoactivated

disinfection”, “root canal photodynamic therapy”, “passive ultrasonic irrigation”, “root canal irrigation cytotoxicity”, “root canal dentin penetration” and “enterococcus faecalis biofilm” Records identified through database searching:

(n =1416)

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eni

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Additional records identified through other sources (n =3 ) Records screened (n = 713)

Irrelevant records excluded (Irrelevant titles, systematic reviews etc.)

(n = 651)

Full-text articles assessed for eligibility

(n = 62)

Studies included in qualitative synthesis

(n = 33)

Full text articles excluded due to their unsuitability or inclusion criteria eg.

Multispecies biofilms, branded irrigation solutions, adjunctive use

reports. (n= 29)

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Enterococcus faecalis

There are a number of microorganisms that may be isolated from root canals in reported cases of endodontic failure, but due to the significant reports of the prevalence of Enterococcus faecalis, this paper will focus solely on this microorganism. Enterococcus faecalis is a gram-positive anaerobic bacterium frequently found in cases of post-treatment failure due to resistant root canal infection, with a prevalence reaching 90%. E. faecalis is nine times more frequently found in endodontically treated teeth than other primary infection causing bacteria (4). It has the ability to invade dentinal tubules rapidly and beyond a depth of 1000µm and is able to survive in the canal much longer than other microorganisms (10). Not only is this bacterium resistant to various antimicrobial agents, it has also shown varying degrees of resistance to basic and acidic environments. Its virulence can be attributed to particular anatomical structures such as cytoplasm, lytic enzyme and lipoteichoic acid. It is able to compete with other bacterial cells by expressing certain proteins (11).

The most commonly used passive irrigation solutions have a high pH which E. faecalis is able to resist due to the presence of an active protein pump in its cellular membrane (3). Its proliferation is undisturbed by temperature variations or solutions that are highly saline. It is even able to survive long periods of starvation in an obturated canal. Perhaps its most troublesome feature is that it has the ability to adhere to dentinal tubules and form intra- and extra-radicular biofilms that are not easily disturbed. They are composed of a complex three-dimensional shape consisting of an

extracellular polymeric matric in which microorganisms are embedded (12). These biofilms in fact are extremely difficult to eliminate from a root canal compared to the same species in its planktonic form. This can be attributed the overexpression or conversely a downregulation of certain genes in sessile vs. planktonic bacteria (13). An E. faecalis biofilm development undergoes three main phases: 1) Bacterial attachment and microcolony formation 2) Release of calcium and phosphate ions following dissolution of the dentin substrate 3) Calcification of biofilms as mineralization progresses. Following completion of all these stages, the biofilm becomes calcified, resulting in a persistent infection of the root canal.

E. faecalis is also able to alter its phenotype to increase resistance when exposed to a changing

physiologic or metabolic environment. Many of the commonly used irrigants have varying disinfecting efficacies, however they may have no effect on the deeper layers of root dentin.

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Passive irrigation

Irrigating solutions

A widely used and approved method of canal disinfection is using irrigating solutions. These are introduced into the canal with a syringe and 30-gauge needle inserted apically and streamed under high velocity. Favorable properties of irrigation solutions include low toxicity, efficient tissue dissolving capabilities and very high antibacterial properties with minimal damage to the host cells (7). This paper will focus on two of these solutions:

• Sodium Hypochlorite (NaOCl) • Chlorhexidine gluconate (CHX)

Sodium Hypochlorite

Sodium hypochlorite (NaOCl) is an alkaline canal irrigant used as a conventional disinfection material in conjunction with and following mechanical debridement. Its use as an irrigant is

reported to have begun as early as the 1920s (14). Presently, it is the most widely utilized irrigation method with a favorable broad spectrum antimicrobial activity and tissue dissolving capability. NaOCl may be used as a solution or as a gel, though studies have shown the solution form to be more effective and the gel form offers only the benefit of a lower risk of leaking through the root’s apex (7). It has a high pH that leads to interference of the integrity of bacterial cytoplasmic

membrane. It also causes changes in biosynthesis in cellular metabolism. NaOCl’s dissolving abilities are directly proportional to its concentration, which may vary from 0.5% to 6% (14). It is delivered into the canal via a 30-gauge syringe needle that is inserted apically and streamed at relatively high velocity. This type of delivery in somewhat in itself a limitation as the remainder of the root canal is irrigated at a lower velocity. The highest velocity is produced at and around the tip of the needle (15). Syringe irrigation also creates the possibility of air bubbles becoming trapped in the apical region of the root canal, making it nearly impossible to remove biofilm in that area (16).

Sodium hypochlorite also has a relatively high surface tension, meaning that it does not directly contact dentinal walls in areas of a more complicated anatomy. It may only remove microorganisms by contacting them directly (3), but according to some sources, NaOCl can only penetrate into the dentinal tubules by 130µm, which would mean that it would not be able to reach E. faecalis penetrating beyond this point. The accuracy of this data will be discussed at the end of this review. Along with this, NaOCl also has the disadvantage of unpleasant taste and odor. Its most noteworthy

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disadvantage though is its high cellular toxicity, which increases with increasing concentrations, whilst lowering its concentrations poses the problem of lowered bactericidal efficacy (13).

An additional problem with this irrigant is that its penetration into periapical tissues can induce an allergic reaction (7). These are some reasons for a current reduction in the desire to utilize it (4).

Currently, scientific literature contains many varying statements about the properties of this

solution, with a general lack of consensus as to which concentration is best to use and how effective this irrigant really is at preventing cases of post endodontic treatment failure. For example, a study by Rosen Eyal et al (2016) tested a 0.6% concentration of NaOCl and concluded that it was unsuccessful in reducing the biofilm mass on dentin disks, but was however most efficient at reducing the biofilm’s cells replication. The study also suggested that NaOCl induced a VMNC state in E. faecalis, meaning that when exposed to this irrigant, the bacterium becomes incapable of undergoing cell division whilst still continuing to be metabolically active (17). A study by Frough-Reyhani M. et al. (2016) concluded that NaOCl solution at a concentration of 1% is recommended specifically due to its low toxicity, however as it is not capable of eliminating mature biofilms, its use is not a proper choice. The same study found no significant difference in the antimicrobial efficacy of 2.5% and 5% NaOCl, concluding that it is sufficient to use the latter concentration for canal disinfection (2). Another study concluded that using 3% NaOCl did not completely eradicate

E. faecalis biofilm that was 21 days old (18).

Chlorhexidine gluconate

As an alternative to NaOCl, some studies have suggested the use of 2% chlorhexidine gluconate. CHX is a positively charged irrigant that acts on the positively charged bacterial cell wall via electrostatic interactions. As this process modifies the cell’s osmotic equilibrium, the result is cell death (19). Chlorhexidine’s possible advantage over NaOCl is its lower cytotoxicity and milder malodor, though there are contradicting findings on this matter. Another one of its favorable

properties is a higher viscosity, which ensures that the solution maintains contact with the root canal walls.

Despite these favorable qualities, CHX is unable to remove the smear layer from the canal and in addition to this, is not able to eliminate necrotic tissue. These limitations may be detrimental to a successful disinfection. Also, CHX may cause toxicity or induce an inflammatory process. It has

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also been known to induce an allergic reaction in some cases, though CHX tends to be the disinfectant of choice for cases of open root apex and patients allergic to NaOCl (4) (10) .

One study concluded that 2% chlorhexidine had a higher antibacterial efficacy against E. faecalis than 2.5% NaOCl and noted than similar results were drawn in a 2009 study by Vianna et al., however both of these studies have some significant limitations as they used an in vitro method calculating zones of inhibition. This is a less accurate method of measurement than using a CFU (Colony forming units) count and also, conditions inside a root canal may contradict these results (13). CHX offers another favorable feature of substantivity (20) which allows a material to form an association with a substrate for a prolonged period of time. In this case, it means that unlike NaOCl, CHX has the ability to provide long-lasting antimicrobial effects and one study by D. E. Bottcher et

al. assessed how the CHX residual effect is influenced by an E. faecalis biofilm age and concluded

that CHX maintained its substantivity up to 30 days, with maximum effects presenting at the 7day mark. Substantivity was not influenced by the percentage of live cells in a biofilm (21). Findings of various studies testing the suitability of CHX as an irrigant in terms of its antibacterial capability, dentin penetration depth and cytotoxicity will be presented and discussed in this review.

Main outcome: 2.5%-5.25% NaOCl used with conventional needle irrigation showed very

favorable results in terms of antibacterial efficacy against E. faecalis, often achieving total elimination. Photoactivated disinfection also achieved either total or highly significant bacterial reduction.

1Antibacterial efficacy against E. faecalis

This section will focus on the ability of each disinfection method on reducing viable bacterial counts. The efficacy of achieving this is the prime objective of any utilized disinfection protocol. The outcomes of studies evaluating this efficacy are demonstrated in the outcome tables below.

1.1Colony Forming Unit (CFU)

A CFU count was done in the majority of these studies. As the name suggests, it aids in determining bactericidal efficacies by comparing the number of E. faecalis colonies before and after treatment or by drawing comparisons between control and intervention groups. This figure may be represented as a fraction, percentage or converted to log form when counting CFU/ml. A percentage format which was used in a study by Afkhami F et al. (2016) (5) for example would utilized the following equation:

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CFU’s (Before treatment) – CFU’s (After treatment) x 100 = %RCC CFU’s (Before treatment)

*RCC (Reduction in colony counts)

CFU is likely a much more reliable method of measurement than using more simplified methods like zone of inhibition measurement.

1.2Confocal Laser Scanning Analysis

This method of measurement uses one type of molecular probe such as the SYTT90 probe and this marks all bacteria in a specimen whilst the second propidium iodide probe labels only damaged bacteria as it penetrates only those bacterial cells that have a damaged plasmatic membrane. Once the bacteria have undergone this labeling, they are incubated and produce a green fluorescence if they are alive and a red fluorescence if they are dead. They are used using a CLSM microscope.

Outcomes: Antibacterial efficacies of NaOCl and CHX against E. faecalis Table 1. [Bactericidal effects of NaOCl and 2% CHX against E. faecalis] Author/ Year Level of evidence/ Study Design/ Participants/ Inclusion Criteria Control Groups and Intervention Outcome Measurements Results Frough-Reyhani, Ghasemi et al. (2016) Level IV 96 single rooted extracted human teeth Control group N=1 Intervention groups N=3 Control group: phosphate-buffered saline solution Intervention groups: 1% NaOCl, 2.5% NaOCl, 5% NaOCl

CFU NaOCl concentrations of 2.5% and 5% showed complete elimination of E. faecalis (CFU count = 0) Zand V. et al. (2015) Level IV 60 single rooted extracted human teeth Control group N=1 Intervention group N=3 Control group: No intervention Intervention groups: 1) 2.5% NaOCl 2) 5.25% NaOCl 3)

CFU Antibacterial effect of all groups significantly higher than control group (P<0.05).

No significant difference between 2.5% and 5.25%

NaOCl (P>0.05). Both totally eliminated E.

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Active irrigation

This paper will focus on three types of active root canal disinfection methods including: • Diode laser disinfection

• Photoactivated disinfection • Passive ultrasonic irrigation

2.5% NaOCl gel Daiana Elizabeth Bottcher et al. (2015) Level IV 123 extracted single rooted human teeth Control group N= 1 Intervention groups N= 1 Control group: Saline solution Intervention groups: 1) 2% CHX 2) Saline (Both groups subdivided into a time frame of 48h, 7 days and 30 days) CLSM After 48h, CHX significantly reduced percentage of viable cells compared with saline solution P=0.007. Difference remained after 7 days P=0.001.

After 30 days, viable cell count of CHX group became

similar to saline group P=0.623. No significant difference within groups when comparing time periods. There was a negative correlation (p= -0.435) between percentage of

live cells and amount of remaining CHX, showing CHX substantivity. Jerin Jose et al. (2016) Level IV E. faecalis inoculated into culture plates and spread into respective media using sterile spreader. 5 uniform wells prepared E. faecalis solution

was added to the wells. After 24h, zones of inhibition were recorded in centimeters. Control group N= 2 Intervention groups N= 3 Control group: (Positive controls) 1) 2.5% NaOCl 2) CHX Intervention groups: (Not included in this paper: QMiX, guava leaf, aloe vera extract) Zone of inhibition

CHX group showed a wider zone of inhibition than 2.5 % NaOCl and the difference was

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Diode laser

A laser is a device that converts various light frequencies into a chromatic radiation in the visible, infrared or ultraviolet regions. These waves are able to mobilize immense heat and power when they are focused at a close range (8). Laser application for endodontic use in microorganism

elimination is a relatively new method. A laser’s effectiveness against microorganisms is correlated with its dose. Diode laser is effective against E. faecalis by thermal modifications that damage bacterial cell walls. A root canal system has a complex anatomy that includes challenging areas such as fins, furcal canals and lateral canals. Conventional irrigants are unable to reach these areas allowing the E. faecalis biofilm to remain undisturbed (13).

Diode laser is able to penetrate these anatomic irregularities and has been suggested to improve the strength of root dentin by increasing the amount of cross-links in collagen fibres (5). E. Faecalis was found to reach dentinal tubule depths of >1000µm (Afkhami F et al. 2016). Therefore, an effective disinfectant must have the ability to penetrate the dentinal tubules up to or beyond this point to reduce chances or post-treatment failure. In one study by Vatkar N. et al (2016), diode laser was shown in a study to have the ability to reach dentinal tubule depths beyond 1000µm. The same study also suggested a possible explanation for this ability. The authors of the study proposed that once the laser light is emitted, it loses the characteristics of a concentrated beam. Instead, a “light fog’’ is created inside the dentin. The dentinal tubules along with the enamel prisms then propagate this light to the peripheral root dentine, which may then successfully reach and eliminate the e.

faecalis present there (10).

An in vitro study published in the Iranian Endodontic Journal by Sohrabi K. et al. however, showed that despite its ability to penetrate deeper into dentinal tubules, the diode laser showed a lower bacterial reduction than 5.25% NaOCl (96.56% and 99.87% respectively) when calculating colony-forming units (CFU). Another study by Beltes C. et al (2017) concluded that the use of a diode laser alone without a photoactivator achieved a relatively insignificant bacterial load reduction of 70%, whilst 2% CHX, 2.5% NaOCl and NaOCl combined with photoactivated disinfection achieved a total elimination of the bacteria.

Photoactivated disinfection

At its initial inception, photoactivated disinfection was aimed at treating pre-malignant diseases or various oral pathologies including tumors. Nowadays however, its use as a canal disinfection

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noteworthy advantage. The heat generated from lasers is capable of charring dentin, melting

cementum, causing root ankylosis or resorption and possibly lead to periapical necrosis (15). These dangers can be avoided by first sensitizing the bacteria to the laser heat with the use of a

photosensitizer (PS). A photosensitizer or photoactive agent works together with a light source (laser or LED) to release singlet oxygen and free radicals.

When exposed to light of a suitable wavelength, the PS such as a nontoxic photoactive dye, becomes activated. The suitability of the wavelength coincides with the peak absorption of the particular PS in use (4). These wavelengths range from 625nm-805nm (9). When activation occurs in the presence of oxygen, energy is transferred from the activated PS to the available oxygen, giving rise to singlet oxygen, which is a highly reactive oxygen species (ROS), or free radicals. These have the ability to destroy microorganisms by causing damage to some of their essential cellular components such as membrane lipids, proteins and nucleic acids. This process is known as antimicrobial photodynamic therapy (aPDT).

PSs having a cationic charge have the ability to bind to and penetrate bacterial cells at a rapid pace. This property consequently allows PSs to maintain a high degree of selectivity for killing

microorganisms rather than the host’s mammalian cells. This is an advantage over using a NaOCl solution, which may potentially be injurious to the host (15). Another advantage of this method is that bacteria are highly unlikely to develop a resistance to this cytotoxic action in comparison to using antibiotics, which is more than likely to be the reason behind the increase in multi-resistant bacteria (7). PAD is not only effective on bacteria but also kills viruses, fungi and protozoa (23). PAD produces a very low risk of temperature increase in hard and soft tissues as it can make use of low-energy lasers, which results in clinically insignificant heat generation (less than 0.5°C) (24). It is considered to be minimally invasive and may be a great adjuvant to conventional treatment in an effort to eliminate bacterial load (1)(4). In addition to killing bacteria, PAD also has the ability to detoxify endotoxins like lipopolysaccharides. Polysaccharides can trigger the production of pro-inflammatory cytokines (25). PADs effects are immediate and have the ability to reach complex areas like pits over the root and furcations, which leads to a reduced chance of bacteremia in patients who are immunocompromised.

A study by I. Bago et al. 2012 showed that PAD using a toluidine blue as a PS and a 660nm diode laser, achieved better bacterial load reduction than both NaOCl 2.5% and high-power diode laser (15). Another study by Samiei M. et al 2016 contradicted these findings, concluding that 2.5%

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NaOCl achieved better results than PAD, though a possible explanation for this may be the use of a different PS or lower energy laser (24).

A study by Zand V et al. (2014) study found that PDT and 2.5% NaOCl did not differ in their bactericidal efficacies against 4, 6 and 8-week old E. faecalis biofilm and both achieved its complete elimination. The study also mentioned that use of both MB and TB achieved complete inhibition of bacteria, and that TB was equally as effective as MB when used at a lower

concentration. In addition, TB had a greater ability to form dimers, which are essential in the ability of the dye to cause bactericidal photodamage (25). Lopez-Jimnez L et al. (2015) stated that PDT was effective and caused severe morphological changes to the bacterial cells, whereas untreated E.

faecalis remained intact with its coccoid cells unaltered. Use of TBO and MB alone barely affected

the bacteria at all. Light therapy on its own affected the biofilm morphology slightly (12). Another interesting factor to take into account is that in order for PAD to obtain a close to ideal effect, it must be in an oxygen rich environment. In an anoxic or low-oxygen environment such as that inside a root canal, the output of ROS outweighs the input and ultimately decreases PADs antibacterial potential (26). This suggests that there is a need for further modifications for this method (27).

Outcomes: Antibacterial efficacy of PAD against E. faecalis Table 2. [Bactericidal effects of PAD against E. faecalis] Author/

Year Level of evidence/Study

Design/Participants/ Inclusion Criteria

Intervention and

Control Groups Outcome Measurements Results

Asnaashari M. et al. (2016) Level I- RCT* 20 patients Inclusion criteria: 1) previously treated molar 2) Visible PA lesion in radiograph 3) no clinical symptoms of pain or swelling 4) No existing systemic disease Control group: N=2 Intervention groups: N=2 Control group: Pre-treatment group Intervention groups: 1st sample taken following removal of gutta-percha and conventional irrigation. Samples not containing E. faecalis excluded. 10 randomly selected patients treated with PDT. 2nd sample tested in laboratory.

CFU Number of colonies was significantly decreased after treatment (p=0.01<0.05)

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Mohan D et al. (2016) Level IV 53 single rooted extracted human teeth Control group N=1 Intervention group N=3 (5) Control group: No intervention Intervention groups: 1) CET (2.5% NaOCl, 17% EDTA) 2a) PAD 4min 2b) PAD 2min 3a)CET+PAD 4min 3b) CET+PAD 2min

CFU Highest percentage of cell death -Group 3a. CET group got a higher % cell death than PAD at both irradiation times. Differences between surviving fraction and cell death between groups 2a and 2b were statistically significant p=0.005 Sunil Bumb S. et al. (2014) Level IV 20 single rooted extracted human teeth Inclusion criteria: Non carious teeth, single rooted teeth Exclusion criteria: Carious, deciduous teeth, dilacerated roots, multi-rooted teeth. Control group N=1 Intervention group N=1 Control group: No intervention Intervention group: 1) PAD

CFU %CFU reduction in

control group was 60.5x10^6, PAD group was 2.0x10^6, which means a reduction of 96.7%.

*CFU: Colony forming unit

*RCT: Randomized controlled trial *CET: Conventional endodontic treatment

The two main types of light sources used in PAD are low energy diode lasers and a light emitting diode (LED). An LED light source has the advantage of being most cost-effective than a laser as they utilize less energy and is known to have a lower thermal productivity and lower tissue injury potential. One study compared the bactericidal efficacies of these two light sources and concluded that PAD using a toluidine blue PS achieved better results when using LED (630nm) as a light source (9). Results of two studies comparing the use of these two light sources is demonstrated in the table below. As both studies came to different conclusions, more research into the subject should be carried out in order for PAD to be used to its full potential.

Table 3. [Bactericidal effects of PAD using LED vs DIODE against E. faecalis]

Author/ Year Level of evidence/Study Design/Participants/ Control Groups and intervention groups Outcome Measurements Results

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Several sources of literature used in the writing of this paper investigated multiple methods of root canal disinfection against E. faecalis. For ease of interpretation, the outcomes of all these studies in represented in Table 4 below:

Outcomes: Antibacterial efficacies of NaOCl, CHX, DL and PAD against E. faecalis

Table 4. [Bactericidal effects of NaOCl, CHX, DL and PAD against E. faecalis Author/ Year Disinfect ion protocol s included Level of evidence/ Study design/Participant s/ Inclusion criteria Control groups and Intervention groups Outcome measurements Results Vahid Zand et al. (2014) NaOCl • Level IV 120 single rooted extracted human teeth

All methods tested

Control group: PBS Intervention groups: 1) CFU 2.5% NaOCl and PAD achieved 100% bacterial killing. There CHX Inclusion Criteria L. Lopez-Jimenez et al. (2015) Level IV

24-well culture plates with E. faecalis Control group N= 3 Intervention groups N= 3 Control group: Biofilms treated with 1) No treatment 2) Laser only (30s) 3) LED only (30s) Intervention groups: Biofilms treated with 1) TB in dark 1min 2) TB in dark 1min + LED 30s 3) MB in dark 1min 4) MB in dark 1 min+ diode laser 30s CSLM imaging P values less than 0.05 considered statistically significant. Biofilms treated with PS only achieved almost no effect. Light therapy alone caused slight changes in the biofilms. TB+LED achieved 79% lethality, MB+DL achieved 95% lethality. P>0.05 (Not SS) Asnaashari M et al. (2015) Level IV 56 single-rooted extracted human teeth Control N=2

Intervention groups N=2

Control group: -ve control- No bacteria +ve control- E.faecalis injected, no intervention Intervention groups:1)PAD using diode laser 2)PAD using LED lamp

CFU CFU’s in LED

group were significantly lower than in diode laser group p=0.021

Both groups had significantly lower CFU than control group p<0.01. No bacterial growth in –ve control group.

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DL against 4-, 6-, 8- old E. faecalis biofilms Control group N=1 Intervention group N=3 PAD 2) 1% NaOCl 10min 3) 2.5% NaOCl 10min was no statistical significance P=1.000 PAD • Sohrabi K. et al (2015) NaOCl • Level IV 18 extracted single rooted human teeth Control group N=1 Intervention group N= 2 Control group: (-ve control) Intervention groups: 1) 5.25% NaOCl (5min) 2) 980-nm diode laser (25sec) CFU NaOCl resulted in 99.87% removal of bacteria and its bactericidal effect was statistically significantly more than that of the diode laser (P<0.05) CHX DL • PAD Vatkar N et al. (2016) NaOCl • Level IV 60 dentine blocks Control group N=1 Intervention group N=5 Control group: No intervention Intervention groups: 1) Saline solution 2) 5.25% NaOCl 3) CHX 4) NI 5) Diode laser ZDB

CLSM Control group showed bacteria penetrating to a depth of 965.45-1175.78 µm. Diode laser group achieved total E. faecalis elimination. CHX • DL • PAD Afkhami F et al. (2016) NaOCl • Level IV 65 single rooted extracted human teeth Control group N=1 Intervention group N=4 Control group: 2.5% NaOCl Intervention groups: 1) Diode laser, 2) NI 3) PAD 4) NI CFU Significant reductions in all groups (P<0.05) NaOCl got 94.61% reduction compared PAD’s 68.47%. DL alone got the best result of 97.41%. CHX

DL •

PAD •

Bago I. et

al (2012) NaOCl • Level IV 120 single extracted rooted human teeth Control group N=1 Control group: Saline solution Intervention CFU PAD significantly more effective than DL and CHX DL •

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PAD • Intervention group N=5 groups: 1) Diode laser 2) PAD 3) NI 4) 2.5% NaOCl 5)NI NaOCl in reducing CFUs (P<0.05) Higher power DL not significantly different to NaOCl irrigation (P>0.05) Samiei M. et al. (2015) NaOCl • Level IV 60 single rooted extracted human teeth Control group N=1 Intervention group N=3 Control group: No intervention Intervention groups: 1)PAD 2) 2% CHX 3) 2.5% NaOCl CFU Bacterial growth inhibited in all groups significantly (P<0.05). 2.5% NaOCl was significantly better than PAD and CHX. P<0.001 and P=0.007) CHX • DL PAD • Beltes C, Sakklas H et al. (2017) NaOCl • Level IV E. faecalis suspension in a sterile Eppendorf tube Control group N=2 Intervention groups N=7 Control groups: Positive control- (ICG-, Light-) Negative control- (No bacterial suspension) Intervention groups: 1) PAD in medium energy fluence, 2) PAD in high energy fluence, 3) PS only 4) Laser only 5) 2.5% NaOCl 6) 2.5% NaOCl+PAD 7) 2.0% CHX CFU Gr.1 and 2: significant reduction in bacterial load (99.99% disinfection. P<0.01) , Gr. 3 and 4:no significant reduction in bacterial load, Gr. 5,6 and 7: total elimination of planktonic bacteria CHX • DL • PAD •

*A “•” symbol denotes that the corresponding method of root canal disinfection has been tested in the respective study.

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Passive ultrasonic irrigation

Contrary to what the name suggests, passive ultrasonic irrigation is an active form of canal disinfection. It is sometimes referred to as ultrasonically activated irrigation, but generally carries the former name due to the fact that it is used “passively” i.e. without intent to remove dentin or shape the canals. However, PUI is an active disinfection method that works in unison with irrigating solutions. In this technique, a canal irrigant obtains a transfer of energy from an oscillating file. This energy transfer leads to be creation of ultrasonic waves and a type of acoustic screaming of the irrigant, which aids in canal cleansing (18). Acoustic streaming creates shear stress on the bacterial cells which helps destroy them. Along with this, PUI creates cavitation, which leads to a collapse of gas bubbles and hence a creation of a pressure vacuum effect, which aids in bacterial destruction and root canal cleansing (28).

It is generally agreed that the effectiveness of a root canal irrigant is dependent upon its direct contact with the internal root surface. This is hindered due to the roots anatomical complexities. Passive ultrasonic irrigation (PUI) is a technique in which a canal irrigant obtains a transfer of energy from an oscillating file. This energy transfer leads to the creation of ultrasonic waves and a type of acoustic screaming of the irrigant, which aids in canal cleansing (18). The PUI has been reported to be efficient in the removal of intracanal smear layer and debris and to facilitate the disruption of endodontic biofilms. The efficacy of ultrasonic irrigation is related with the establishment of a hydrodynamic environment in canals that are well-shaped and filled with an irrigant (15). As previously established, the cytotoxicity and other drawbacks of NaOCl increases with increasing concentration. Therefore, aside from hoping that PUI cleanses the canal more efficiently, the idea behind utilizing it is based upon the belief that it will do so using NaOCl or other irrigation solutions at a lower concentration and therefore reducing the damage potential of these solutions.

A study by Toljan I et al. (2016) tested PUI with 3% NaOCl as an irrigant but generated

disappointing results. The methods shortcomings however may have been due to the short time of irrigation (20s) (18). A study by Guerreiro-Tanomaru M J et al. (2015) compared the antibacterial efficacy of conventional needle irrigation (CNI) used with 1% NaOCl and PUI combined with the same irrigant and concluded that there was no statistical difference between the antibacterial efficacy of the two methods and neither method was able to completely eradicate E. Faecalis from the root canal system. The same result was achieved when substituting NaOCl with a saline solution. In this experiment, the total irrigation time was 2 minutes (28). Although in this

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experiment the irrigation time was higher than the former study, the concentration of NaOCl used was quite low. Nevertheless, since PUI combined with NaOCl and NaOCl used conventionally with mechanical instrumentation drew no significant differences, it can be said that it was the method of irrigation rather than the time or concentration that had the most significant effect.

A study by Pladisai P et al. (2016) supports these results as their study found conventional

irrigation by mechanical instrumentation (MI) and NaOCl 2.5% to be the most effective method of root canal disinfection when compared with PUI + NaOCl 2.5%. However, their study only tested these methods on large root canals, creating a possibility of bias (29). A study by Paula de Almeida A. et al. supports these results and also found that the antibacterial efficacy of 2.5% NaOCl with or without PUI drew no significant difference. Another study by Saifalarab A et al. (2016) found PUI+ NaOCl 2.5% to be more effective than manual agitation of NaOCl and suggested that this was due to the higher velocity of NaOCl flow as this is what aids in the biofilm removal.

Overall outcome: It was found that only one examined study supported the use of PUI as a more

efficient method of irrigation in terms of its bactericidal efficacy. The majority of data indicated that conventional irrigation with a syringe during endodontic treatment has the most desired outcome.

Table 5. [Bactericidal effects of PUI against E. faecalis] Author/

Year Level of evidence/ Study design/Participants/Inclusion criteria Control groups and Intervention groups Outcome measurements Results Tolojan I et al. (2016) Level IV

48 single rooted extracted human teeth Control group N=2 Intervention groups N=3 Control group: +ve control infected with E. faecalis, treater with 0.85% saline solution, – ve control- sterile broth Intervention groups: 1) 3% NaOCl 2) NI 3)PUI CFU Antimicrobial efficacy of syringe irrigation

and PUI using 3% NaOCl were similar (p=0.049) Maria Guerreiro-Tanomaru J et al. Level IV

75 single rooted extracted human teeth Control group N=1 Control group: No irrigation Intervention CFU No statistical difference between PUI/ NaOCl and CNI/

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(2015) Intervention groups N=4 groups: 1)PUI/Saline(SS) 2)PUI/1% NaOCl 3)CNI/saline 4)CNI/1%NaOCl NaOCl (p>0.05)PUI with 1% NaOCl was unable to completely eradicate E. faecalis Pladisai P et al. (2016) Level IV

50 single rooted extracted human teeth Control group N=1 Intervention groups N=4 Control group: No irrigation Intervention groups: 1) MI/ NaOCl 2) 2.5% NaOCl 3) PUI/ NaOCl 4) Saline CFU Gr. 1,2,3,4 achieved bacterial reductions of 99.99%, 96.83%, 99.22% and 60.93% respectively. Gr.1resulted in significantly less intracanal bacteria than Gr.2&3 Paula de Almeida A et al. (2014) Level IV

60 single rooted extracted bovine teeth Control group N=1 Intervention groups N=5 Control group: No irrigation Intervention groups:1)No treatment 2)Distilled water 3)2.5% NaOCl 4)NI 5) NaOCl/PUI 6)NI CFU No statistical difference in NaOCl irrigation with or without PUI (P<0.05). No group achieved complete decontamination. Saifalarab A. Mohmmed Level IV

45 identical root canal models manufactured using 3D printer

Control group N=1 Intervention groups N=4 Control group: No irrigation Intervention groups:1) 2.5% NaOCl delivered with a precision pump and left stagnant 2) 2.5% NaOCl delivered in the same way and agitated with

gutta- percha 3) NI 4) 2.5% NaOCl/PUI CLSM The active irrigation groups (manual, ultrasonic) exhibited less residual biofilm on the model surface than the passive irrigation and untreated groups. The difference was statistically significant. P<0.05

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Aliu Xevdet et al. (2014)

Level IV

78 single rooted extracted human teeth Control group N=1 Intervention groups N=5 Control group: No irrigation Intervention groups: 1) PAD for 1min 2) PAD for 3min 3) PAD for 5min 4) 2.5% NaOCl

5sec 5)

2.4%NaOCl/PUI 10sec

CFU PUI achieved

total elimination and its effect was statistically significant compared to the other methods (p<0.001) PAD 5min showed statistically relevant difference compared to 1min irradiation (p=0.003), but not with 3min

irradiation (p=709).

*CNI: conventional needle irrigation

2Dentin penetration

There is a limited number of studies looking into E. faecalis is known to be able to penetrate the dentinal tubules 1000 µm (20), therefore a desired feature of a disinfection method is that it must be able to penetrate dentin beyond this point and kill all bacteria present there. The outcomes of studies evaluating penetration depth are presented in the table X below:

Overall outcome: Diode laser was the only method that demonstrated the ability to penetrate the

total dentinal tubule depth. PAD showed an ability to penetrate to a substantial depth as well.

Table 6. [Dentin penetration depths] Author/ Year Level of evidence/Study Design/Participants/ Inclusion Criteria Control Groups and intervention groups Outcome measurements Results Vadhana S et al. (2015) Level IV 20 single-rooted extracted human teeth. CHX mixed with Rhodamine B dye. Control group: Conventional syringe irrigation Intervention group: PUI CLSM Mean penetration depths of CHX in coronal middle and

apical thirds: Group 1:138 µm, 80

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Intervention group N=1 Group 2: 209µm, 138µm and 72µm. The difference was statistically significant at all three

levels (p<0.01, p<0.001, p<0.001) Sunil Bumb S. et al. (2014) Level IV 20 single rooted extracted human teeth Inclusion criteria: Non- carious teeth,

single rooted teeth Exclusion criteria: Carious, deciduous teeth, dilacerated roots, multi-rooted teeth. Control group N=1 Intervention group N=1 Control group: No intervention Intervention group: PDT group

CLSM Bacteria in the control group had a penetration depth of 980µm. PDT treatment lead to bacterial death at 890-900 µm Vatkar N et

al. (2016) 60 dentine blocks Level IV Control group N=1 Intervention group N=5 Control group: No intervention Intervention groups: 1) Saline solution 2) 5.25% NaOCl 3) CHX 4) NI 5) Diode laser ZDB

CLSM Control group showed bacteria penetrating to a depth of 965.45-1175.78 µm. NaOCl killed bacteria in the 88.45-110.43 µm range. CHX: 109.89–

194.14 µm. Diode laser group achieved

total elimination. Yanhuang Wang and Xiaojing Huang Level IV 27 bovine root canals

Control group N=1 Intervention groups N=2 Control group: PUI with 2.5% NaOCl Intervention groups: 1) PAD 2) PUI +PAD

SEM E. faecalis biofilm contained intact cells at 600 µm after PAD.

3Cytotoxicity of root canal disinfection methods

Cytotoxicity of root canal irrigants is of vital importance since irrigating solutions on their own or in conjunction with PUI come in close contact with the host tissues (30). A minimal toxic effect on periapical tissues in a desired property of any root canal disinfection method.

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Overall outcome: The majority of studies concluded that 2% CHX was less cytotoxic than NaOCl

of various concentrations. Photoactivated disinfection showed only slight cytotoxicity that was much more favorable than that of NaOCl.

Table 7. [Cytotoxicity using different root canal disinfection methods] Author/ Year Level of evidence/Study Design/Participants/ Inclusion Criteria Control Groups and intervention groups Outcome measurements Results Narges Farhad Mollashahi et al. (2016) Level IV Stem cells isolated

from immature, impacted third molar and transferred to 24-well plates. Control group N=1 Intervention groups N=6 Control group: No intervention Intervention groups: 1-3) NI 4) CHX 5) 5.25% NaOCl 6) Sterile saline

MTT assay Difference between control and saline group and the study groups was statistically significant (P<0.0001). Cytotoxicity of CHX was statistically significantly lower than that of NaOCl (P<0.05) Evren OK et al. (2015) Level IV Human PDL fibroblast viability tested Control group N=0 Intervention groups: N=3 Intervention groups: 1) NI 2)NI 3) CHX 4) 5.25% NaOCl WST-1 assay After 1h, 5.25% NaOCl was more cytotoxic than CHX, but at 24h and 72h, their cytotoxic effects were similar. Dominika Bajrami et al. (2014)* Level IV Rat PDL fibroblasts Control N= 1 Intervention groups N=3 Control group: No intervention Intervention groups: 1) 3% NaOCl 2) NI 3) CHX WST-1 assay (Non-cytotoxic (>90% cell viability); slightly cytotoxic (60–90%); moderately cytotoxic (30– 59%); and strongly cytotoxic (<30%). At 100 µl/mL dilution: After 1h and 24h, CHX was less cytotoxic than NaOCl, though still considered highly cytotoxic. At 48h, NaOCl was less cytotoxic than CHX P=0.0022. At 0.1 µl/mL dilution: NaOCl least (moderately) cytotoxic, CHX highly cytotoxic at all time points. T. Vouzara

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(2015)* fibroblasts Control group N=1 Intervention group N=3 Intervention groups: 1) CHX 2) NaOCl 3) NI (Both groups tested at concentrations of 0.05%, 0.025%, 0.01%, 0.005% and 0.0025%) NaOCl at all concentrations. The difference was statistically significant (P<0.05) Homes-Filho J et al. (2016) Level IV Mouse fibroblasts Control group N=1 Intervention groups N=3 Control group: Culture medium Intervention groups: 1) Saline 2) 5% NaOCl 3) PDT MTT 0 – non-cytotoxic (less inhibition than 25%); 1 – slightly cytotoxic (inhibition between 25% and 50%); 2 – moderately cytotoxic (inhibition between 50% and 75%) and 3 – strongly cytotoxic (greater than 75% inhibition) Control, saline solution and PDT inhibited cell viability by <25%. 5% NaOCl- >75% (most cytotoxic, p<0.05)

* Solutions used in the study were diluted due in order to give more realistic results as cultured cells used in these studies are much more susceptible to cytotoxic damage than periapical cells.

3.1 MTT assay

The MTT (Mosmann’s Tetrazolium Toxicity) assay method of measurement was first introduced in 1980 by Mosmann. It is a method used for assessing the cytotoxicity of biological materials and works by evaluating how capable viable cells are in converting water-soluble tetrazolium salts into formazan crystals which are insoluble. Viable cells carry out this conversion by utilizing

mitochondrial dehydrogenase enzyme activity. In short, the MTT assay assesses a dental materials cytotoxicity based on changes in viable cell numbers, cell morphology and metabolism (31).

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3.2 WST-1 test

In the WST-1 test, tetrazolium dye is converted into yellow-orange formazan crystals by

dehydrogenases inside the mitochondria of living cells. If cells were damaged or non-living, there would be insufficient energy to grow or perform a metabolic function such as this conversion. Once the cells have broken down the dye, the optical density of the resultant formazan product can then be measured and this will represent both cellular respiration and metabolic rates. In this way, the optical density is proportional to cell viability and hence, WST-1 provides a good model for

cytotoxicity (30). The test works similarly to MTT, but is quicker than the MTT test which requires 52-75h to complete (32).

% Viability of cells = OD test compound × 100 OD OD Control

3.3 SRB assay

SRB (Sulforhodamine B) is another method of measuring cytotoxicity. It works via fluorescent colorimetry. SRB binds to the basic amino acids of cell proteins and thus measures the protein content of cells in a cell culture. A study that used SRB assay in their investigation claimed that it was a more sensitive measurement of cytotoxicity (33).

Risk of bias

In this review, the risk of bias at outcome level was estimated and represented in graph form for ease of interpretation. A risk of bias assessment allows one to establish the reliability of conclusions made. As most of the evaluated studies included in this review were laboratory tests, the lowest risk of bias was seen in the “Incomplete outcome data” category. Both “Blinding of outcome data” and “Performance bias” were subjectively judged as having a relatively high risk of bias since the compared disinfection methods would be very difficult to hide from the laboratory researcher. The judgements of each individual risk are subjective to interpretation, particularly when judging possibility of bias at the reporting stage. Therefore, risk of bias does not necessarily factually indicate presence of bias and the sources utilized in the writing of this review came from esteemed scientific journals and are likely to be reliable sources of data.

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Table 7. Risk of bias graph

DISCUSSION

An ideal method of root canal disinfection should have the properties of maximal antimicrobial efficacy whilst causing the least possible damage to the host via cytotoxicity or otherwise (31). A thorough root canal disinfection should be given utmost importance in order to provide quality endodontic treatment. In this systematic review, root canal irrigation using two main irrigating solutions, passive ultrasonic irrigation, diode laser and photoactivated disinfection was compared using data from scientific literature and conclusions were drawn regarding certain properties of all these disinfection methods in terms of their ability to penetrate dentinal tubules, cytotoxicity and a main focus on bactericidal efficacy against enterococcus faecalis. It was hypothesized that

alternative methods to needle and syringe irrigation with sodium hypochlorite solution can provide better outcomes. Despite a lot of data being available on the subject matter of this paper, lack of consensus amongst clinicians remains ubiquitous. Conventional irrigation using a needle and syringe to stream irrigating solutions such as sodium hypochlorite is extremely widespread due to the general agreement that this particular protocol has favorable outcomes. It is also a very

convenient and cheap method or root canal disinfection and is additionally not technique sensitive. One important aspect to consider in regards to this form of irrigation is that an irrigating solution may only reach 1mm deeper than the depth of the tip of the needle, which is often inserted only to the middle third of the canal, leaving the apical anatomy untouched (20). NaOCl was also said to be highly cytotoxic and have poor dentin penetration depth abilities (7) and the evidence in the tables of outcome in this paper agree with this statement, though found NaOCl’s antibacterial efficacy to be very favorable in the majority of studies.

COMPARATIVE EVALUATION OF THE ANTIBACTERIAL EFFICACY OF FOUR ROOT CANAL SYSTEM DISINFECTION METHODS AGAINST E. FAECALIS

Erika Endriukaityte