EFFECT OF A SURFACTANT ON SODIUM HYPOCHLORITE …repository-tnmgrmu.ac.in/10841/1/240402319dinesh_kumar.pdf · effect of a surfactant on sodium hypochlorite combination in the elimination

EFFECT OF A SURFACTANT ON SODIUM

HYPOCHLORITE COMBINATION IN THE

ELIMINATION OF ENTEROCOCCUS FAECALIS IN

RETREATMENT PROCEDURE- AN INVITRO STUDY

Dissertation submitted to

THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY

In partial fulfillment for the Degree of

MASTER OF DENTAL SURGERY

BRANCH IV

CONSERVATIVE DENTISTRY AND ENDODONTICS

MAY2019

ACKNOWLEDGEMENT

I take this opportunity to sincerely thank my post graduate teacher and

my guide Dr.Shankar.P, M.D.S., Professor, Department of Conservative

Dentistry and Endodontics, Ragas Dental College and Hospital, for his

perseverance in motivating, guiding and supporting me throughout my study

period.

I extend my sincere thanks to Dr.R.Anil Kumar, M.D.S., Professor

andHead,Department of Conservative Dentistry and Endodontics, Ragas

Dental College and Hospital, for his encouragement, support and guidance all

throughout my study period.

My sincere thanks to Dr. R. Indira, M.D.S.,Professor and

formerHOD,Department of Conservative Dentistry and Endodontics, Ragas

DentalCollege and Hospital, who helped me with her guidance, support

andconstant encouragement throughout my study period.

My sincere thanks to Dr. S. Ramachandran, M.D.S.,

Professor&former Principal, Department of Conservative Dentistry and

Endodontics,Ragas Dental College and Hospital, who helped me with his

advice andimmense support throughout my post graduate curriculum.

I extend my sincere thanks to Dr.C.S.Karumaran, M.D.S., Professor,

for his constant encouragement throughout the completion of this work.

I extend my sincere thanks toDr.M. Rajesekaran, M.D.S., Professor,

for his constant encouragement throughout the completion of this work.

I extend my sincere thanks to Dr.B.Veni Ashok, M.D.S., Professor, for

his constant encouragement and support.

I would like to solemnly thank Dr. Shankar Narayan, M.D.S., Dr.S.M.

Venkatesan, M.D.S., Dr. M. Sabari M.D.S, Dr.Aravind, M.D.S.,

Dr.B.Venkatesh, M.D.S., Readers, for all the help and support during my

study period.

I would also like to thank Dr.Nirmala, M.D.S., Dr.Sudhakar,M.D.S.,

Senior lecturers, for their friendly guidance and support.

I also wish to thank the management of Ragas Dental College and

Hospital, Chennai for their help and support.

I sincerely thankmy seniorDr.R.Ashwin for his constant support and

encouragement throughout my study.

I remain ever grateful to all my seniors, batchmates, juniorsespecially

Dr.SaiSwathi.Randfriends for their support.

I would like to especially thank my father Mr.A.Thiruneela Prasad,

my mother Mrs.T.Selva Rani and my sister Ms.A.T.Anu Nanthini,for their

love, understanding, support and encouragement throughout these years

without which, I would not have never reached so far.

My sincere thanks to Mr.K.Thavamanifor his guidance and support in

DTP and Binding works.I extend my thanks to Dr.Bijivinfor his help in

statistical work and Mr.Balaji Msc. for his help in inoculation of bacteria and

bacterial colony counting.

Above all, I am thankful to God, who always guides me and has given

these wonderful people in my life.

CONTENTS

S. NO. INDEX PAGE.NO

1. INTRODUCTION 1

2. AIM AND OBJECTIVES 8

3. REVIEW OF LITERATURE 10

4. MATERIALS AND METHODS 33

5. RESULTS 40

6. DISCUSSION 42

7. SUMMARY 63

8. CONCLUSION 65

9. BIBLIOGRAPHY 67

10. ANNEXURES -

LIST OF TABLES

S.NO TITLE

Table 1 MEAN COLONY COUNT OF E.FAECALIS PRESENT AFTER THE

TREATMENT BY DIFFERENT TEST SOLUTIONS

Table 2 WILCOXON SIGN RANK TEST

LIST OF GRAPHS

S.NO TITLE

Graph 1

BEFORE IRRIGATION OF ALL GROUPS (G1)

Graph 2

BEFORE IRRIGATION OF NaOCl GROUP AND NaOCl/BAK

GROUP (G1)

Graph 3

AFTER IRRIGATION OF NaOCl GROUP AND NaOCl/BAK

GROUP (G2)

LIST OF FIGURES

S.NO. TITLE

FIGURE 1

TEETH SAMPLES

a) Positive control group

b) Negative control group

c) NaOCl group

d) BAK/NaOCl group

FIGURE 2

ARMAMENTARIUM FOR ROOT CANAL TREATMENT

FIGURE 3

FILES AND ENDOBLOC

FIGURE 4

XSMART PLUS ENDOMOTOR

FIGURE 5

ROOT CANAL TREATED AND SPECIMENS PLACED IN

MICROTUBES FOR INOCULATION

FIGURE 6

INOCULATION OF E.FAECALIS IN SPECIMENS AND

STORED FOR 21 DAYS

FIGURE 7

a) REMOVAL OF GUTTA PERCHA FROM ROOT CANAL

USING RETREATMENT FILE

b) IRRIGATION

c) PAPER POINT SAMPLING

FIGURE 8

BACTERIAL GROWTH

LIST OF ABBREVIATIONS

SL.NO ABBREVIATIONS DESCRIPTION

1 NaOCl Sodium hypochlorite

2 BAK Benzalkonium chloride

3 GP Gutta Percha

4 ESP Enterococaal Surface Proteins

5 PCR Polymerase Chain Reaction

6 SPSS Statistical Package for Social Sciences

software

7 CFU

Colony Forming Units

8

CMC

Critical Micellar Concentration

Introduction

Introduction

1

INTRODUCTION

Success of endodontics is generally attributed to the following basic

steps- mechanical shaping of the root canal, disinfection and three dimensional

obturation of root canal system. Among these steps chemomechanical

preparation of the root canal is the most vital. This is usually accomplished

using mechanical instrumentation and chemical irrigation and followed by

placement of an intracanal medicament in between treatment appointments 56

.

The anatomy of root canal system plays a significant role in success

and failure of root canal treatment. In addition to this natural factor, failures in

endodontic treatment may be attributed due to iatrogenic events such as

improper shaping, inadequate cleaning and deficiencies during obturation.

Re-infection of the root canal system can occur when the coronal seal

is lost after completion of root canal treatment. At times, root canal-treated

teeth may appear to be free of any disease, but yet may harbour

microorganisms in the root canal. Apparently balance exists between the

microorganisms lodged in the root canal, their environment, and on the host

response resulting in absence of disease. A change in this balance can result in

reinfection and disease. Leakage due to a break in the coronal seal may be one

of the major causes for reinfection. Occasionally even after the root canal is

well obturated, some microorganisms may invade the obturated canal and

Introduction

2

cause infection within few weeks or months. In such cases it may be necessary

to consider a retreatment of the endodontically treated tooth.

Endodontic retreatment varies in some aspects from primary

endodontic treatment. The main difference is the removal of the obturated

filling material from the filled root canals before reperforming adequate

chemomechanical preparation.

Microorganisms which thrive in such root canal treated teeth include

bacteria, yeasts and viruses. Anatomical factors and inadequate treatment may

cause the root canal space to acquire and harbour various species of bacteria

and fungi along with their toxins and by-products. 100 – 200 species of

bacteria are able to thrive in these inadequately treated root canals and can

produce secondary endodontic infection. Though it is difficult to attribute the

secondary infection to a single aetiological agent, the association of

enterococci, particularly enterococcus faecalis seems to be the main pathogen

capable of surviving and persisting within the space of the root canal treated

tooth.

Enterococcus faecalis is a facultative anaerobe, which means it is

capable of growing both in the presence or absence of oxygen. It may present

as single, in pairs or as short chains. It is also less dependent upon virulence

factors and can survive in harsh environmental conditions and resuscitate upon

returning to favourable conditions61

. It can undergo prolonged period of

nutrition deprivation and can bind to dentin and adequately invade the dentinal

Introduction

3

tubules. Previous animal studies, and pure cultures of various bacteria when

inoculated separately in the root canals have shown that E.faecalis unlike other

bacteria, are able to colonize the root canal and can survive without the

support of other bacteria. It can also remarkably resist commonly used

irrigants, medicaments like calcium hydroxide. Studies have shown that

prevalence of E.faecalis was in the range of 29-64 percent in previously root

filled teeth with apical periodontitis 38

.

E.faecalis has been often observed as a single infectious agent, but has

also been recovered from mixed colonies of bacteria within the root canal

system. E.faecalis is capable of forming a biofilm on its own on gutta percha,

and this biofilms seems to be thicker compared to the biofilms produced by

other organisms.

The Biofilm has been defined as a microbial community characterized

by cells that are attached to a substratum, are in a matrix of extracellular

polymeric substance (EPS).It offer their member cells several benefits, the

foremost of which is protection from killing by antimicrobial agents15

.

E. faecalis can develop into a biofilm under different growth

conditions such as aerobic, anaerobic, nutrient-rich, and nutrient-deprived

environment 37

.When tooth undergoes pulpal necrosis or inadequate root canal

treatment, with subsequently periradicular periodontitis and exudate may flow

in and out of the canal. This exchange of fluid provides proteins, glycoproteins

and other nutrients to the bacteria growing as a biofilm in the root canal. The

Introduction

4

complexities and variations in the root canal anatomy may provide a suitable

environment for the microorganisms to multiply and form a biofilm15 .

The major objective in root canal retreatment is to thoroughly disinfect

the entire root canal system. Irrigants play a central role during disinfection in

endodontic treatment and retreatment. During and after instrumentation, the

irrigants facilitate removal of microorganisms, tissue remnants, and dentin

chips from the root canal through a flushing mechanism. The goal is to reduce

or eliminate the bacteria from the reinfected root canal and also to remove the

hard and soft obturating materials completely from the obturated root canal.

Various irrigating solutions have been suggested to be used during this

retreatment procedure 57.

Sodium hypochlorite is the most commonly used endodontic irrigant

because of its antimicrobial property and tissue-dissolving activity55

. Sodium

hypochlorite is used in concentration ranging from 0.5% to 6% during

endodontic therapy as it demonstrates good antibacterial activity. However,

low concentrations of sodium hypochlorite are known to be inadequate in

completely eliminating the bacteria and other debris during retreatment. Hence

sodium hypochlorite at concentration of 6% was used in this study.

The major problem encountered during retreatment procedure is not only the

complete elimination of microorganisms but also the products from the

materials used during obturation. The sealers used during primary endodontic

treatment have the ability to adhere to the canal walls and also penetrate into

Introduction

5

the dentinal tubules .This requires the use of an irrigant which will facilitate

thorough debridement, penetrating even into the dentinal tubules.

Therefore the irrigants used must be in close contact with the dentin

walls and debris. This close contact depends on the wettability of the irrigant

and is correlated to the property of its surface tension.

The Surface tension is defined as “the force between molecules that

produces the tendency for the surface area of the liquid to decrease” 25

. This

force tends to limit the ability of the liquid to penetrate a capillary tube, like

the dentinal tubules. Hence the irrigants used in endodontic should have very

low surface tension25

.

Sodium hypochlorite has been shown to have high surface tension as

compared to other irrigants, and is unable to reach or flow into the depth of the

dentinal tubules. Previous studies have shown that detergents like tween 80

and polypropylene glycol had been used to reduce the surface tension of

endodontic irrigants. Studies on irrigants with the addition of surfactants have

shown encouraging results with respect to depth of penetration of irrigant,

higher dentin permeability, improvement in cleaning and disinfection of canal

walls and better pulp tissue dissolution during primary endodontic treatment6.

However, there are not many studies done on the effect of the use of

surfactants with sodium hypochlorite in retreatment procedures.

Introduction

6

Benzalkonium chloride (BAK) is a surfactant detergent displaying a

high affinity to proteins in the cell membranes. The antibacterial potential of

BAK relies on the changes provoked on the ionic resistance of the cell

membranes. BAK is widely used in oral disinfectant mouthwashes.

It is a cationic detergent most commonly used in medicine. In

ophthalmology, it is the most common preservative to avoid contamination of

eye solutions. In dentistry, it is frequently used in dentin bonding agents,

orthodontic resins, and in commercial ethylenediaminetetraacetic acid

solution. It also may be used in recent commercial root canal irrigants whose

composition remains undisclosed.

BAK when combined with NaOCl can cause a greater reduction in the

bacterial load when compared with NaOCl used alone and allows a greater

diffusion of NaOCl in to the dentinal tubules. Combining BAK with NaOCl

reduced the contact angle of NaOCl by more than 50% (Bukiet et al) thereby

improving the wetting properties resulting in an overall 70-fold reduction in

biofilm accumulation 35

.

Bukiet et al6 stated Benzalkonium chloride may chemically react with

sodium hypochlorite. This chemical reaction alters the solvent ability of the

irrigant by modifying the free chlorine content. This may affect the

antibacterial and cytotoxic properties of mixture, leading to a loss of efficiency

and future complications.

Introduction

7

Hence, the purpose of this present invitro study was as follows:

1. To assess the effect of higher concentration of sodium hypochlorite (6%)

in removing bacteria E.faecalis during retreatment.

2. To see the efficacy of sodium hypochlorite (6%) when used with a surface

active agent BAK (0.008%) by estimating the colony forming units (CFU)

in retreatment cases.

Aim and Objectives

Aim and Objectives

8

AIM AND OBJECTIVES

AIM :

To analyse the in vitro effect of 0.008% benzalkonium chloride surfactant-

6% sodium hypochlorite combination in the elimination of E.faecalis during

retreatment procedure.

OBJECTIVES:

1. To determine a simple in vitro experiment for retreatment.

2. Is normal saline effective in eliminating the bacteria E.faecalis during

retreatment?

3. To determine the effective concentration of NaOCl during retreatment.

4. To check whether 6% NaOCl is effective in completely eliminating

bacteria E.faecalis during retreatment.

5. To check whether a surfactant needed is to be added to NaOCl 6% during

retreatment.

6. To determine whether addition of 0.008% BAK improves the surface

properties of 6% NaOCl.

7. To evaluate the efficacy of 0.008% BAK with 6% NaOCl in completely

eliminating E.faeclis during retreatment.

Aim and Objectives

9

8. To find a simple and effective method to check the bacterial status of the

root canal during treatment.

9. To enumerate the number of E.faecalis colonies formed (CFU) by using

the colony counter during retreatment.

Review of Literature

Review of literature

10

REVIEW OF LITERATURE

J. F. SIQUEIRA et al (1997)57

studied the effectiveness of 4.0%

sodium hypochlorite (NaOCl) used with three irrigation methods in the

elimination of Enterococcus faecalis from the root canal tested in vitro. Root

canals contaminated with E. faecalis were treated as follows: (i) irrigation with

2 mL of NaOCl solution and agitation with hand files; (ii) irrigation with 2 mL

of NaOCl solution and ultrasonic agitation; (iii) irrigation with NaOCl

alternated with hydrogen peroxide. The result showed there were no

statistically significant differences between the experimental groups.

However, NaOCl applied by the three methods tested, was significantly more

effective than the saline solution (control group) in disinfecting the root canal.

Tanriverdi F et al (1997)63

experimented an in vitro test model from

human teeth to comparatively examine antibacterial effectiveness of calcium

hydroxide, parachlorophenol (PCP) and camphorated parachlorophenol

(CPCP) against Enterococcus faecalis in infected root canals. The results

showed that the effectiveness of CPCP and PCP at one day was superior to the

effectiveness of Ca(OH)2. In the three-day group, CPCP was the most

effective, followed by Ca(OH)2.

Anders Molander et al (1998)44

made a study and examined the

microbiological status of 100 root-filled teeth with radiographically verified

apical periodontitis – the pathology (P) group – and of 20 teeth without signs

of periapical pathosis – the technical (T) group. In the P group 117 strains of


11

bacteria were recovered in 68 teeth. In most of the cases examined one or two

strains were found. Facultative anaerobic species predominated among these

isolates (69% of identified strains). Growth was classified as „sparse‟ or „very

sparse‟ in 53%, and as „heavy‟ or „very heavy‟ in 42%. Enterococci were the

most frequently isolated genera, showing „heavy‟ or „very heavy‟ growth in 25

out of 32 cases (78%). In 11 teeth of the T group no bacteria were recovered,

whilst the remaining nine yielded 13 microbial strains. Eight of these grew

„very sparsely‟. They concluded that the microflora of the obturated canal

differs from that found normally in the untreated necrotic dental pulp,

quantitatively as well as qualitatively. Nonsurgical retreatment strategies

should be reconsidered.

Axel Hartke et al (1998)31

experimented on the ability of

Enterococcus faecalis to metabolically adapt to an oligotrophic environment.

E. faecalis is able to survive for prolonged periods under conditions of

complete starvation established by incubation in tap water. During incubation

in this microcosm, cells developed a rippled cell surface with irregular shapes.

Analysis of protein synthesis by two-dimensional gel electrophoresis revealed

the enhanced synthesis of 51 proteins which were induced in the oligotrophic

environment. A comparison of these oligotrophy-inducible proteins with the

42 glucose starvation-induced polypeptides showed that 16 are common

between the two different starvation conditions. These proteins and the

corresponding genes seem to play a key role in the observed phenomena of


12

long-term survival and development of general stress resistance of starved

cultures of E. faecalis.

Goran Sundqvist et al (1998)57

conducted a study to determine what

microbial flora was present in teeth after failed root canal therapy and to

establish the outcome of conservative re-treatment. The microbial flora in

canals after failed endodontic therapy differed markedly from the flora in

untreated teeth. Infection at the time of root filling and size of the periapical

lesion were factors that had a negative influence on the prognosis. Finally he

suggested that three of four endodontic failures were successfully managed by

re-treatment.

B. P. F. A. Gomes et al (2001)26

experimented the effectiveness of

several concentrations of NaOCl (0.5%, 1%, 2.5%, 4% and 5.25%) and two

forms of chlorhexidine gluconate (gel and liquid) in three concentrations

(0.2%, 1% and 2%) in the elimination of E. faecalis.

Marcia Carneiro Valera et al (2001)68

evaluated the effect of 1%

sodium hypochlorite and five intracanals medications on Candida albicans

harvested inside root canals. This study reinforced the importance of

endodontic treatment in two sessions with the use of a long-term intracanal

medication to eliminate microorganisms present inside the root canals and also

highlights the presence of yeast, C. albicans, which may persist after

endodontic treatment.


13

H.H.Hancock et al (2001)34

study was to determine the composition

of the microbial flora present in teeth after the failure of root canal therapy in a

North American population. Fifty-four root-filled teeth with persistent

periapical radiolucencies were selected for retreatment. After removal of the

root-filling material, the canals were sampled with paper points, and by

reaming of the apical dentin. Both samples were grown under aerobic and

strict anaerobic conditions. Then the bacterial growth was analysed. The result

was the microbial flora was mainly of 1 to 2 strains of predominantly gram-

positive organisms. Enterococcus faecalis was the most commonly recovered

bacterial species. They concluded that, bacteria were cultivated in 34 of the 54

teeth examined in the study. E faecalis was identified in 30% of the teeth with

a positive culture.

Love.R.M. et al (2001)40

identified a possible mechanism that would

explain how E.faecalis could survive and grow within dentinal tubules and

reinfect an obturated root canal. It is postulated that a virulence factor of E.

faecalis in failed endodontically treated teeth may be related to the ability of E.

faecalis cells to maintain the capability to invade dentinal tubules and adhere

to collagen in the presence of human serum.

Peciuliene V et al (2001)48

made a study to determine the occurrence

and role of yeasts, enteric gram-negative rods and Enterococcus species in

root-filled teeth with chronic apical periodontitis, and the antimicrobial effect

of iodine potassium iodide (IKI) irrigation.. All third samples (after IKI)

except one were negative. They concluded the high prevalence of enteric


14

bacteria and yeasts in root-filled teeth with chronic apical periodontitis was

established. IKI improved the antimicrobial effect of the treatment.

Brenda Paula Figueiredo de Almeida Gomes et al (2002)27

made a

study to investigate the susceptibility of some microorganisms commonly

isolated from root canals to calcium hydroxide in combination with several

vehicles by the agar diffusion method. They conclude that, anaerobic Gram-

negative bacteria are more susceptible to calcium hydroxide pastes than

facultative Gram-positive microorganisms.

M. Evans et al (2002)19

conducted a study to clarify the mechanisms

that enable E. faecalis to survive the high pH of calcium hydroxide. E. faecalis

was resistant to calcium hydroxide at a pH of 11.1, but not pH 11.5. Pre-

treatment with calcium hydroxide pH 10.3 induced no tolerance to further

exposure at pH 11.5. No difference in cell survival was observed when protein

synthesis was blocked during stress induction, however, addition of a proton

pump inhibitor resulted in a dramatic reduction of cell viability of E. faecalis

in calcium hydroxide. They concluded that, survival of E. faecalis in calcium

hydroxide appears to be unrelated to stress induced protein synthesis, but a

functioning proton pump is critical for survival of E.faecalis at high pH.

Tanomaru Filho M et al (2002)20

evaluated the inflammatory

response to irrigating solutions injected into the peritoneal cavity of mice.

They concluded, the 0.5% sodium hypochlorite solution induced an

inflammatory response, however, the 2.0% chlorhexidine digluconate solution

did not induce a significant inflammatory response.


15

Christopher J. Kristich et al (2003)39

made an in vitro study that,

Enterococcus faecalis is a gram-positive opportunistic pathogen known to

form biofilms. The results demonstrated that in vitro biofilm formation occurs,

not only in the absence of esp, but also in the absence of the entire

pathogenicity island that harbors the esp coding sequence. Using scanning

electron microscopy to evaluate biofilms of E. faecalis OG1RF grown in the

fermentor system, biofilm development was observed to progress through

multiple stages, including attachment of individual cells to the substratum,

microcolony formation, and maturation into complex multilayered structures

apparently containing water channels. Microtiter plate biofilm analyses

indicated that biofilm formation or maintenance was modulated by

environmental conditions. Furthermore, their results demonstrated that

expression of a secreted metalloprotease, GelE, enhances biofilm formation by

E. faecalis.

C. E. Radcliffe et al (2004)50

experimented to determine the resistance

of microorganisms associated with refractory endodontic infections to sodium

hypochlorite used as a root canal irrigant. Using 0.5% NaOCl for 30 min

reduced cfu to zero for both strains tested. This compares with 10 min for

1.0%, 5 min for 2.5% and 2 min for 5.25% (P < 0.001). They finally

concluded the regression analysis for the dependent variable loge (count + 1)

with loge (time + 1) and concentration as explanatory variables gave rise to a

significant interaction between time and concentration (P < 0.001).


16

Morgana Eli Vianna et al (2004)69

conducted a study to investigate in

vitro the antimicrobial activity of 0.2%, 1%, and 2% chlorhexidine gluconate

(CHX gel and CHX liquid), against endodontic pathogens and compare the

results with the ones achieved by 0.5%, 1%, 2.5%, 4%, and 5.25% sodium

hypochlorite (NaOCl).They resulted that, the timing required for 1.0% and

2.0% CHX liquid to eliminate all microorganisms was the same required for

5.25% NaOCl. The antimicrobial action is related to type, concentration, and

presentation form of the irrigants as well as the microbial susceptibility.

Charles H. Stuart et al (2005)61

stated that Enterococcus faecalis is a

microorganism commonly detected in asymptomatic, persistent endodontic

infections. Use of good aseptic technique, increased apical preparation sizes,

and inclusion of 2% chlorhexidine in combination with sodium hypochlorite

are currently the most effective methods to combat E. faecalis within the root

canal systems of teeth. In the changing face of dental care, continued research

on E. faecalis and its elimination from the dental apparatus may well define

the future of the endodontic specialty.

S. George et al (2005)23

made a study to evaluate the effect of

different growth conditions on the characteristics of E. faecalis biofilm on root

canal, and the penetration of E. faecalis into dentinal tubules. Finally, they

demonstrated distinct ultrastructural and physiochemical properties of the

biofilms formed and dentinal tubular penetration of E. faecalis under different

conditions.


17

Luciano Giardino et al (2006)25

conducted a study to compare the

surface tension of four common endodontic irrigants: Moltendo EDTA 17%,

Cetrexidin, Smear Clear, Sodium hypochlorite 5.25%, with the surface tension

of MTAD and Tetraclean. Freshly produced MilliQ water was used as a

reference. They concluded that, both new irrigants, MTAD and Tetraclean, are

capable of removing the smear layer. Thanks to their low surface tension,

increasing the intimate contact of irrigant solutions with the dentinal walls,

they may permit deeper penetration.

M.S. Clegg et al (2006)13

assessed in their study, the effectiveness of

different concentrations of sodium hypochlorite (NaOCl), 2% chlorhexidin

and BioPure MTAD. Intracanal contents were collected from 10 patients

diagnosed with chronic apical periodontitis. Finally, the results indicated that

6% NaOCl was the only irrigant capable of both rendering bacteria nonviable

and physically removing the biofilm.

Patricia Kho et al (2006)36

made a study to compare the antimicrobial

efficacy of irrigating with 1.3% NaOCl/ Biopure MTAD versus irrigation with

5.25% NaOCl/ 15% EDTA in the apical 5 mm of roots infected with

Enterococcus faecalis. Finally, they demonstrated that there is no difference in

antimicrobial efficacy for irrigation with 5.25% NaOCl/15% EDTA versus

irrigation with 1.3% NaOCl/Biopure MTAD in the apical 5 mm of roots

infected with E. faecalis.

Thomas R. Dunavant et al (2006)15


efficacy of root canal irrigants against E. faecalis biofilms using a novel in


18

vitro testing system. Biofilms grown in a flow cell system were submerged in

test irrigants for either 1 or 5 minutes. Post-hoc analysis showed a significant

difference between 1% and 6% NaOCl, and all other agents including Smear

Clear™, 2% chlorhexidine, REDTA, and Bio- Pure™ MTAD™ (P 0.05).

They concluded within the parameters of this study, both 1% NaOCl and 6%

NaOCl were more efficient in eliminating E. faecalis biofilm than the other

solutions tested.

Daniel P. Oliveira et al (2007)45

conducted a study to compare the in

vitro antimicrobial activity of 2% chlorhexidine gel against Enterococcus

faecalis with sodium hypochlorite in 2 different concentrations (1.5% and

5.25%). The 2% chlorhexidine gluconate gel and 5.25% sodium hypochlorite

were effective in eliminating E. faecalis even 7 days after the instrumentation;

Finally, the higher the concentration of sodium hypochlorite the better its

antimicrobial action.

Jason M. Duggan et al (2007)16

stated that Biofilms are complex

aggregations of microorganisms attached to a surface. The formation of

biofilms might facilitate certain survival and virulence characteristics under

some situations. This study tested the hypothesis that the ability of

Enterococcus faecalis to form biofilms is related to the source of the strains.

They finally showed, within the root canal and oral isolates there were no

significant associations between biofilm formation and the presence of the

virulence determinants asa, cylA, esp, and gelE.


19

Ronald Ordinola Zapata et al (2008)75

made a study to explore the

potential of confocal laser scanning microscopy (CLSM) for in situ

identification of live and dead Enterococcus faecalis in infected dentin. They

concluded that, CLSM analysis shows that the discrimination between viable

(green) and dead (red) bacteria in infected dentinal tubules could be observed

after staining with FDA/PI. Acridine orange was able to show metabolic

activity of the E. faecalis cells inside the dentinal tubules showed by its red

fluorescence. The viability of bacteria in infected dentin can be determined in

situ by CLSM. FDA/PI and acridine orange are useful for this technique.

Anne E. Williamson et al (2009)71

conducted study to create a

monoculture biofilm of a clinical isolate of Enterococcus faecalis and to

determine susceptibility against four antimicrobial irrigants. Biofilms were

subjected to 1-, 3-, and 5-minute exposures to one of the following irrigants:

6% sodium hypochlorite (NaOCl), 2% chlorhexidine gluconate (CHX) or one

of two new products, < 6% NaOCl with surface modifiers (Chlor-XTRA) or

2% CHX with surface modifiers (CHX-Plus™) (Vista Dental Products,

Racine, WI). Results indicated that 6% NaOCl and Chlor- EXTRA™ were

significantly superior against E. faecalis biolfilms compared to 2% CHX and

CHX-Plus™ at all time points except five minutes.

Maria Teresa Arias-Moliz et al (2009)3 They conducted a study to

evaluate the minimal biofilm eradication concentration (MBEC) of sodium

hypochlorite (NaOCl), chlorhexidine (CHX), EDTA, and citric and

phosphoric acids after 1, 5, and 10 minutes of exposure to biofilms of


20

Enterococcus faecalis. The biofilms grew in the MBEC high-throughput

device for 24 hours at 37˚C and were exposed to 10 serial two-fold dilutions of

each irrigating solution. NaOCl was the most effective agent, capable of

eradicating the biofilms after 1 minute at a concentration of 0.00625%.They

concluded that, CHX eradicated biofilm after 5 minutes at 2%. EDTA and

citric and phosphoric acid solutions were not effective against the biofilms at

any concentration or time tested.

Ling Zou et al (2010)76

Studies were to evaluate the effect of

concentration, time of exposure, and temperature on the penetration of NaOCl

into dentinal tubules. The result was the shortest penetration (77 mm) was

measured after incubation with 1% NaOCl for 2 minutes at room temperature.

The highest penetration (300 mm) was obtained with 6% NaOCl for 20

minutes at 45˚C. After the initial penetration during the first 2 minutes, the

depth of penetration doubled during the next 18 minutes of exposure.

Temperature had a modest effect within each group on the depth of

penetration and in most cases was not statistically significant (P > .05). Depth

of penetration increased with increasing hypochlorite concentration, but the

differences were small. Within each time group, depth of penetration with 1%

NaOCl was about 50%–80% of the values with the 6% solution. They

concluded that, temperature, time, and concentration all contribute to the

penetration of sodium hypochlorite into dentinal tubules.

Singamaneni Vijaykumar et al (2010)70

made an in vitro study

comparing the reduction of E. faecalis counts in root canals produced by


21

irrigation with distilled water, hydrogen peroxide, sodium hypochlorite,

chlorhexidine, and combinations of solutions After serial dilutions, samples

were plated onto Mitis salivarius agar, and the colony forming units were

counted. Results were analysed by Chi-square test, Fisher‟s exact test, and

one-way ANOVA followed by multiple range Tukey HSD test with the level

of significance set at P < 0.05.They concluded that, reduction of colony counts

in distilled water group was significantly lower than the mean reduction in all

the other groups. However, no other contrasts are statistically significant.

Combination of sodium hypochlorite and chlorhexidine showed the most

effective antimicrobial activity followed by sodium hypochlorite and hydrogen

peroxide together. Hydrogen peroxide was the least effective irrigant when

used alone.

Sonja Stojicic et al (2010)56

study was to evaluate and compare the

effects of concentration, temperature, and agitation on the tissue-dissolving

ability of sodium hypochlorite. In addition, a hypochlorite product with added

surface active agent was compared with conventional hypochlorite solutions.

They made a result, weight loss (dissolution) of the tissue increased almost

linearly with the concentration of sodium hypochlorite. Higher temperatures

and agitation considerably enhanced the efficacy of sodium hypochlorite. The

effect of agitation on tissue dissolution was greater than that of temperature;

continuous agitation resulted in the fastest tissue dissolution. Hypochlorite

with added surface active agent had the lowest contact angle on dentin and

was most effective in tissue dissolution in all experimental situations. They


22

concluded that, optimizing the concentration, temperature, flow, and surface

tension can improve the tissue-dissolving effectiveness of hypochlorite even

50-fold.

Juliane M. Guerreiro-Tanomaru et al (2011)30

made a study to

evaluate pH, available chlorine content, and antibacterial activity of

endodontic irrigants and their combinations. The pH and chlorine content of

sodium hypochlorite (NaOCl) were analyzed pure and in combination with

10% citric acid (CA) and apple vinegar (AV). The antibacterial effect of the

following solutions was measured by direct contact test against Enterococcus

faecalis: 2.5% NaOCl, 2.5% NaOCl 10% CA (7:3), 2.5% NaOCl AV (5:5),

10% CA, and AV. Sterile saline was used as control. The colony-forming

units were determined by serial decimal dilutions. The result was the

combination of 2.5% NaOCl with CA or AV lowered the pH and the chlorine

content. NaOCl, alone or in combination was able to eliminate E. faecalis in

30 seconds, and CA, after 10 minutes. AV promoted reduction (32.2%) after

10 minutes. Finally, they concluded that NaOCl with acidic solutions lowered

the pH and the chlorine content, but did not alter its antibacterial effect.

Palazzi F et al (2011)46

investigated the surface tension characteristics

of 5.25% sodium hypochlorite and three recently introduced sodium

hypochlorite solutions, which had been modified to reduce their surface

tension: Chlor-Xtra, Hypoclean A and Hypoclean B. They concluded that the

new 5.25% sodium hypochlorite solutions modified with surfactants,


23

Hypoclean A and Hypoclean B, had surface tension values that were

significantly lower (P < 0.01) than Chlor-Xtra and 5.25% NaOCl. Because of

their low surface tension and increased contact with dentinal walls, these new

irrigants have the potential to penetrate more readily into uninstrumented areas

of root canal system as well as allow a more rapid exchange with fresh

solution, enabling greater antimicrobial effectiveness and enhanced pulp tissue

dissolution ability.

David E. Jaramillo et al (2012)35

conducted a study that, Secondary

biofilm formation by oral bacteria after breakdown/fracture of temporary or

permanent restorations imposes a challenge to the outcome of root canal

treatment. This study focuses on benzalkonium chloride (BAK) coating on

dentin or polystyrene surfaces and its influence on the early adhesion and

biofilm formation by oral and root canal bacteria. Finally, they illustrated that

surface coating with a surfactant solution containing BAK does not cause cell

membrane damage but might interfere with cell mechanisms of adhesion.

Investigations into the clinical utility of BAK as an antibiofilm medication are

warranted.

Frederic Bukiet et al (2012)6 made a study to (1) assess the effect of

the addition of benzalkonium chloride to sodium hypochlorite on its wetting

properties, contact angle, and surface energy; (2) determine the critical

micellar concentration of benzalkonium chloride in sodium hypochlorite; and

(3) investigate the influence of addition of benzalkonium chloride on the free

chlorine level, cytotoxicity, and antiseptic properties of the mixture. The result


24

was the contact angle (P < .001) as well as the surface energy (P < .001)

significantly decreased with increasing benzalkonium chloride concentrations.

The critical micellar concentration of benzalkonium chloride in sodium

hypochlorite was 0.008%. At this concentration, the addition of benzalkonium

chloride had no effect on the free chlorine content, cytotoxicity, or

antibacterial efficiency of the mixture. They concluded that, the addition of

benzalkonium chloride to sodium hypochlorite at the critical micellar

concentration reduced the contact angle by 51.2% and the surface energy by

53.4%, without affecting the free chloride content, cytotoxicity, or

antibacterial properties of the mixture.

Qian-Qian Wang et al (2012)72

investigated the prevalence of

Enterococcus faecalis in saliva and filled root canals of patients requiring

endodontic retreatment for apical periodontitis. Patients with apical

periodontitis who were referred for endodontic retreatment were examined.

The type and quality of the restoration, symptoms, quality of obturation were

recorded. Finally, they concluded, E. faecalis is more common in root canals

of teeth with apical periodontitis than in saliva. The prevalence of E. faecalis

in root canals is associated with the presence of E. faecalis in saliva.

Zhejun Wang et al (2012)73

study was to compare the antibacterial

effects of different disinfecting solutions on young and old E.faecalis biofilms

in dentin canals using a novel dentin infection model and confocal laser

scanning microscopy (CLSM). The bacteria were introduced into the dentinal

tubules by centrifugation. After 1 day and 3 weeks of incubation, 40 infected


25

dentin specimens were subjected to 1 and 3 minutes of exposure to

disinfecting solutions, which included 2% sodium hypochlorite (NaOCl), 6%

NaOCl, 2% chlorhexidine (CHX) (Sigma Chemical Co, St Louis, MO), and

QMiX (Dentsply Tulsa Dental, Tulsa, OK). The proportions of dead and live

bacteria inside the dentinal tubules after exposure to these disinfectants were

assessed by CLSM using a LIVE/DEAD bacterial viability stain. Finally the

study showed that mature E. faecalis biofilms in dentin canals are more

resistant to disinfecting solutions than young biofilms. Six percent NaOCl and

QMiX had stronger antibacterial effects against young and old E. faecalis

biofilms in dentin than 2% NaOCl and 2% CHX.

Kenny T. Tran et al (2013)66


efficacy of the pulverization and sterile paper point techniques for sampling

root canals using 5.25% NaOCl/17% EDTA and 1.3% NaOCl/MTAD as

irrigation regimens. They ended that, the pulverization technique was more

efficacious in detecting viable bacteria. Furthermore, this technique showed

that 1.3% NaOCl/MTAD regimen was more effective in disinfecting root

canals.

Manikandan R et al (2013)41

aimed to evaluate the alkaline tolerance

ability of Enterococcus faecalis; one of the most commonly isolated bacterium

from failed root canal treatments. E. faecalis was grown in Brain heart

Infusion (BHI) broth and maintained at different alkaline conditions (pH); its

ability to form biofilm in polystyrene plates was assayed by O‟Toole method.

Intracanal irrigants sodium hypochlorite (NaOCl), chlorhexidine digluconate


26

(CHX) and MTAD were used to evaluate the suppression of E. faecalis

biofilm. The data obtained was statistically analysed by Anova and

Kolmogorov-Smirnov test. E. faecalis was able to survive and form biofilm at

all tested pH range (7.3-12.3). 2% NaOCl and 1% CHX were highly effective

in suppressing E. faecalis biofilm whereas MTAD had limited activity.

Rahul Halkai et al (2013)32

conducted a study to know the ability of

Enterococcus faecalis invasion into root dentin. Forty single rooted human

intact teeth were selected, after access opening and canal debridement, all the

samples were subjected for gamma sterilization to ensure complete absence of

microorganisms, then exposed to Enterococcus faecalis broth, broth is placed

with the help of micro pipette into root canal and also at the same time apical

1/3 of tooth were immersed into broth for 8 weeks, biomechanical preparation,

obturation and coronal sealing done using GIC followed by examination under

confocal laser scanning microscope after splitting the teeth samples into two

halves buccolingually. The results shows invasion of Enterococcus faecalis up

to 160 μm deep in to root dentin, and they concluded that, penetration and

survival of Enterococcus faecalis deep into dentin in extreme conditions may

be the possible reason for persisting infection after root canal treatment.

Zhejun Wang et al (2013)74

examined the effect of the smear layer on

the antibacterial effect of different disinfecting solutions in infected dentinal

tubules. Cells of Enterococcus faecalis were forced into dentinal tubules

according to a previously established protocol. After a 3-week incubation

period of infected dentin blocks, a uniform smear layer was produced.


27

Confocal laser scanning microscopy and viability staining were used to

analyse the proportions of dead and live bacteria inside the dentin. They

concluded, the smear layer reduces the effectiveness of disinfecting agents

against E. faecalis in infected dentin. Solutions containing 6% NaOCl and/ or

QMiX showed the highest antibacterial activity.

Halkai R et al (2014)33

made a study to address the cause of persistent

infection of root cementum by Enterococcus faecalis. E.faecalis broth was

placed in the root canal and apical one-third of the tooth was immersed in the

broth for 8 weeks with alternate day refreshment followed by biomechanical

preparation, obturation and coronal seal. The samples were observed under a

confocal microscope after splitting the teeth into two halves. They made a

result an E.faecalis penetrated 160 μm deep into the root cementum in group

III samples and only showed adhesion in group II samples. Penetration and

survival of E. faecalis deep inside the cementum in extreme conditions could

be the reason for persistent infection.

Baron et al (2015)5 investigated to determine the effect of a sodium

hypochlorite–surfactant combination on the removal of Enterococcus faecalis

from infected teeth. They concluded that the addition of BAK to NaOCl

significantly reduced the number of remaining bacteria within the canal after

irrigation compared with NaOCl.

M.T. Arias-Moliz et al (2015)4 determined the antimicrobial and

antibiofilm activities and physicochemical properties of AH Plus sealer mixed


28

with different concentrations of benzalkonium chloride. AH Plus was tested

alone and mixed with1%, 2% and 3% of BC. Microhardness and contact angle

tests were also performed. They concluded that the addition of 2% or higher

concentrations BC to AH Plus showed antimicrobial and antibiofilm activities

without affecting the properties specified in ANSI/ADA standards. However,

additives to the root canal sealer altered other physical and chemical properties

that are not commonly found in the literature to evaluate filling materials.

Rina Verma et al (2015)52

made a study that, Management of

abscessed primary teeth often present endodontic failure owing to questioned

efficiency of dressings or obturating pastes to eliminate Enterococcus faecalis,

a resistant bacterium, residing in depth of dentinal tubules. The present study

evaluates the antimicrobial efficacy of two antibacterial and two obturating

pastes in dentinal tubules of primary teeth infected with Enterococcus faecalis

using viability stain and confocal laser scanning microscope. They finally

concluded that, all medicaments were effective against E. faecalis in dentine

of primary teeth and their efficacy increased with longer contact with

1%CHX+CH being most effective at day 15. Inclusion of 1% CHX in

dressings or obturating pastes might minimize the endodontic relapse and

maximize the tooth retention in functional state in pediatric dentistry.

Tianfeng Du et al (2015)17

presented a study and it aimed to evaluate

the antibacterial effect of the combined use of sodium hypochlorite (NaOCl)

and root canal sealers on Enterococcus faecalis biofilms using a dentin

infection model. The result was the combined use of NaOCl and sealers (30


29

and 60 days) killed significantly more bacteria than NaOCl or sealers alone

(P < .05). NaOCl + MTA Fillapex were the most effective antibacterial

combination by killing 83% bacteria in dentin tubules in 60 days. Thirty and

60 days of exposure to the sealers resulted in significantly more dead bacteria

in dentin biofilms than 7- day exposures (P < .05). Finally, they concluded the

placement of root canal sealer after NaOCl treatment enhanced antibacterial

effects against E. faecalis in the dentinal tubules. Little additional effect was

obtained after 30 days of exposure to sealers.

Tuhina Banerjee et al (2015)67

made a study to determine the

prevalence of various virulence factors phenotypically and genotypically in

enterococci and study their association with multidrug resistance. A total of

310 enterococcal isolates were studied, comprising 155 E.faecium and 155 E.

faecalis. Antimicrobial susceptibility testing was done by disc diffusion and

agar dilution method. However, increase in resistance was associated with

significant decrease in expression or acquisition of virulence genes. Further,

acquisition of vancomycin resistance was the significant factor responsible for

the loss of virulence traits. Though it is presumed that increased drug

resistance correlates with increased virulence, acquisition of vancomycin

resistance might be responsible for reduced expression of virulence traits to

meet the “biological cost” relating to VRE.

Mohammad Frough-Reyhani et al (2016)22

investigated the

antimicrobial efficacy of 1%, 2.5 % and 5% sodium hypochlorite in

eliminating E. faecalis biofilms at different stages of development. Dentin chip


30

suspensions were used for colony forming unit (CFU) counting to estimate

remaining E.faecalis counts. Statistical comparison of the means was carried

out with Kruskal-Wallis test, and pair-wise comparisons were made by Mann-

Whitney U test, at a significance level of P<0.05. The results showed that

2.5% and 5% NaOCl completely eliminated E. faecalis biofilms in three stages

of biofilm development, whereas 1% NaOCl resulted in 85.73%, 81.88% and

78.62% decreases in bacterial counts in 4-, 6- and 10-week-old biofilms,

respectively, which was significantly more than those with PBS (p<0.05).They

concluded, the bacteria in mature and old biofilms were more resistant to 1%

NaOCl than were the bacteria in young biofilms. Overall survival rate and

residual bacteria increased with biofilm aging.

Jun Zou et al (2016)78

made a study, to find that after phagocytosis by

macrophages, enterococci-containing vacuoles resist acidification, and E.

faecalis is resistant to low pH. Ultrastructural examination of the enterococci

containing vacuole by transmission electron microscopy revealed a single

membrane envelope, with no evidence of the classical double membraned

autophagosomes. Western blot analysis further confirmed that E. faecalis

could trigger inhibition of the production of LC3-II during infection. By

employing cells transfected with RFP-LC3 plasmid and infected with GFP-

labelled E. faecalis, we also observed that E. faecalis was not delivered into

autophagosomes during macrophage infection.

Igor Tsesis et al (2017)65

investigated in a study to evaluate

Enterococcus faecalis colonization at the apical part of root canals following


31

root-end resection and filling using confocal laser scanning microscopy

(CLSM). The result was fluorescence-stained areas were larger in the bucco-

lingual directions compared with the mesio-distal directions (p < 0.05). The

mean and maximal depths of bacterial colonization into the dentinal tubules

were 755 and 1643 μm, respectively, with no differences between the root-end

filling materials (p > 0.05). However, more live bacteria were found in the

MTA group in comparison to IRM and Biodentine groups (p < 0.05).They

concluded that, CLSM can be used to histologically demonstrate bacterial

root-end colonization following root-end filling. The results from this study

offer new insights into the interaction of E.faecalis with host cells and may

provide a new approach to treatment of enterococcal infections.

V. Nair et al (2017)58

conducted a study to detect in vitro adherence of

Enterococcus faecalis to the infected dentinal tubules of human extracted teeth

using confocal laser scanning microscope. Samples were washed, thoroughly

sectioned and examined by confocal laser scanning microscopy. The result

was, E. faecalis was able to invade the dentinal tubules to a depth of 1–400 μm

and adhere to 1–200 μm depth. Finally, they conclude adherence of E. faecalis

as evaluated by confocal laser scanning microscope was highest at the depth of

1–100 μm which may have an impact on the shaping and cleaning procedures

on the root canal.

Stefanie Nio (2017)62

conducted a study to compare the killing

effectiveness of two different sodium hypochlorite concentrations (2 % vs.

5.25 %) at two different temperatures (20 °C vs. 60 °C) against two strains of


32

E.faecalis biofilm at different ages of maturation (3 days vs. 3 weeks), in a

previously described dentin block model. The result was the killing efficacy of

E.faecalis in dentin tubules was affected by an increase in temperature of the

NaOCl solution. The concentration of the NaOCl solution and exposure time

to the irrigant played a role in the killing efficacy of NaOCl. Overall, 5.25 %

NaOCl demonstrated a greater effect on the killing efficacy on E. faecalis,

except when NaOCl is used at 60 °C with an exposure time of 10 minutes.

There was no significant difference (p>0.5) between the two different strains

of E. faecalis or between the 3-day and 3-week old biofilms in their sensitivity

to the bactericidal effect of NaOCl. He concluded bacterial killing by NaOCl

is enhanced by an increase in temperature and concentration.

Materials and Methods


33

MATERIALS AND METHODS

ARMAMENTARIUM AND MATERIALS

Sixty four mandibular single rooted premolar

Straight hand piece (NSK Japan)

K –files no 10, 15 and 20 ( MANI,INC )

2.5 ml syringe (Dispovan)

ProTaper Universal (DentsplyMaillefer, Ballaigues, Switzerland)

6% Gutta Percha (Dentsply)

Normal Saline (Eurolife healthcare Pvt. Ltd)

Zinc Oxide powder and Eugenol liquid (Dental Products of India)

Endodonticmotor (X-smartTMplus, Dentsply Tulsa Dental,Tulsa,OK)

ProTraper Universal Retreatment files (DentsplyMaillefer, Ballaigues,

Switzerland)

Brain-heart infusion (BHI) broth (Becton Dickinson and Company,

Sparks, MD)

E.faecalis (ATCC 29212)

6% Sodium hypochloride irrigating solution (Prime Dental Products

P Ltd)

0.008% Benzalkonium Chloride (Dermocare Laboratories Pvt.Ltd.)


34

SAMPLE COLLECTION

Human mandibular premolars extracted for various reasons unrelated

to the study were collected for the study. All teeth were subjected to root canal

treatment. The negative control samples were filled with sterile brain-heart

infusion (BHI) broth and the other 3 groups were stored in E.faecalis broth for

21 days and was incubated at 37˚C and 95% humidity.

INCLUSION CRITERIA

Mandibular premolars with fully formed apex having one separate

straight canal.

EXCLUSION CRITERIA

Teeth with root caries, open/immature apices, calcifications, external

resorption, dilacerations, anastomosis between canals and C-shaped canals

were excluded.

SAMPLE SIZE

Sixty four human mandibular premolars were selected from the pool of

collected samples which met the inclusion and exclusion criteria.


35

Study Groups:

The teeth were divided into four groups, (n=16)

Three experimental groups were inoculated with E. faecalis and

cultured for 21 days:

Group A: Positive control group, no irrigation (n= 16)

Group B: Negative control group received medium only and no inoculate

(n=16)

Group C: NaOCl group, irrigated with 5 mL 6% NaOCl (n=16)

Group D: BAK/NaOCL group, irrigated with 5 mL 0.008%

benzalkonium chloride (BAK) and 6% NaOCl (n=16)

Paper point sampling of the canals was obtained before irrigation (C1)

for all 4 groups and for 2 groups after irrigation (C2) to determine remaining

colony forming units.

PREPARATION OF THE SPECIMENS

This study was approved by the Institutional Review Board. Extracted

human mandibular premolars were stored at 3% hydrogen peroxide to retard

bacterial growth. Sixty-four teeth were radiographed in a mesiodistal direction

to screen for the presence of a single canal, resulting in a total sample size of

N = 64. The initial step, access cavity preparation was made for all teeth,


36

working length determined using a #10K file. The canals were instrumented

using ProTaper Universal rotary files and irrigated with normal saline. The

tooth was obturated with 6% gutta percha using zinc oxide eugenol sealer. The

canal was Irrigated with 6% NaOCl and replenished after each file by using a

30-gauge side-vented needle. The apical foramen was sealed by using Glass

ionomer cement, and the root surface was sealed with varnish. Teeth were

placed individually in 1.5mL microtubes and steam autoclaved at 121˚C for 30

minutes. The teeth were divided into four groups: positive control (group A),

negative control (group B), NaOCl 6% (group C), BAK 0.008% + NaOCl 6%

(group D) with 16 specimens in each group. The specimens were infected by

using a 24-hour pure culture suspension of E. faecalis cultivated in Brain

Heart Infusion agar (BHI) and then inoculated with 15 mL of 1 x10⁸ colony-

forming units (CFU)/mL suspension of E. faecalis (determined by serial

dilution and plating). Teeth were incubated at 37˚C and 95% humidity for 21

days. The negative control group was not inoculated and were filled with

sterile brain-heart infusion (BHI) broth (Becton Dickinson and Company,

Sparks, MD).

After 21 days, the tooth from all the four groups was taken from the

medium and re-treatment procedure was done using ProTraper Universal

Retreatment files (DentsplyMaillefer, Ballaigues, Switzerland) .Normal saline

was used as an irrigant during re-treatment procedure. After complete removal

of 6% gutta percha, the specimens were subjected to bacterial viability.


37

After re-treatment procedure, the teeth were placed into the medium.

A sterile paper point was used at working length to sample the canals from all

4 groups (G1). After paper point sampling, the specimens in the two tested

groups and positive control group were irrigated with 5 mL of the respective

irrigant as described. All irrigants were delivered at a flow rate of 0.04 mL/s

using a side-vented 30-gauge needle. A second post-irrigation paper point

sample (G2) was obtained from each canal in the NaOCl and NaOCl/BAK

groups as described earlier. The positive controls received no irrigation

whereas the specimens in negative control group were irrigated with normal

saline.

The paper points were placed individually in 1.5-mL microtubes

containing 0.5 mL BHI broth. One hundred microliters of medium was plated

on BHI agar in triplicate, and the remainder was stored at 4˚C. ll plates we e

ncu ated at 3 C and 5 um d t for 24 hours, after which the colonies

were counted by using a colony counter.

The number of colony forming units (CFU) was calculated and

subjected to statistical analysis using Wilcoxon sign test.


38

FLOWCHART ILLUSTRATING THE METHODOLOGY OF THE STUDY

Access cavity preparation was made for all teeth, then

the working length determination was made by using a

#15 file.

Sixty four human mandibular premolars were selected from

the pool of collected samples which met the inclusion and

exclusion criteria.

The teeth were obturated with 6% gutta percha using zinc

oxide eugenol as sealer and teeth were divided into four

experimental groups- Group A,B,C,D

The canals were instrumented by using ProTaper

Universal rotary files and irrigated with normal saline

All the specimens were then inoculated with 15 mL of 1

x10⁸colony-forming units (CFU)/mL suspension of E.faecalis

(ATCC29212)

After 21 days the number of bacteria present within the canal of

each specimen was counted (CFU/ml)


39

Group A

Positive

control, No

irrigation

(n=16)

Group C

NaOCl

group,

6%NaOCl

(n=16)

Group B

Negative

control,

irrigation

with 0.9N/n

normal saline

(n=16)

Group D

BAK/NaOCl

group, 0.008%

BAK and 6%

NaOCl

(n=16)

Determination of Bacterial Viability

After irrigation, a sterile paper point was used at working length to sample

the canals from all the specimens and the paper point is placed in BHI

broth.

The obturated teeth were subjected to retreatment using Pro Taper

retreatment files (torque) and GP with sealer was removed from all the

canals.

Specimens in different groups subjected to irrigation protocol.

The colony forming is determined by the colony counter and it is counted by

CFU/ml=total number of colony counted divided by dilution factors for 24-48 hours

Figures

Figures

ARMAMENTARIUM

Figure 1: TEETH SAMPLES

a) Positive Control group (Group A)

b) Negative Control group (Group B)

Figures

c) NaOCl group (Group C)

d) BAK/NaOCl group (Group D)

Figures

FIGURE 2: ARMAMENTARIUM FOR ROOT CANAL TREATMENT

FIGURE 3: FILES AND ENDOBLOC

Figures

FIGURE 4: X SMART PLUS ENDOMOTOR

Figures

FIGURE 5: ROOT CANAL TREATED AND SPECIMENS PLACED IN

MICROTUBES FOR INOCULATION

FIGURE 6: INOCULATION OF E.FAECALIS IN SPECIMENS AND

STORED FOR 21 DAYS

Figures

FIGURE 7: a) REMOVAL OF GUTTA PERCHA FROM ROOT

CANAL USING RETREATMENT FILE

Figures

b) IRRIGATION

c) PAPER POINT SAMPLING

Figures

FIGURE 8: BACTERIAL GROWTH

A B

C D

(A) Positive control group (B) Negative control group (C) 6% NaOCl group

(D) 0.008% BAK/ 6% NaOCl group.

Results

Results

40

RESULTS

This in vitro study was designed to analyse the effect of 0.008%

benzalkonium chloride surfactant- 6% sodium hypochlorite combination in the

elimination of E.faecalis during irrigation in retreatment procedure

The experimental groups were divided into four groups:

A. Positive control group (No irrigation)

B. Negative control group (Normal saline)

C. NaOCl group (6%)

D. BAK/NaOCl group (0.008%/6%)

The primary endodontic treatment was done for all specimens and

inoculation of E.faecalis for 21 days and retreatment was done. The paper

point samples were taken for all the groups and placed in BHI broth to count

the remaining colony forming units (CFU) and statistically analyzed.

Results

41

STATISTICAL ANALYSIS

The data collected were compiled using MS-Office Excel and was

subjected to Statistical analysis using IBM corp. SPSS (Statistical package for

social sciences) Statistics for windows, version 20.0 (Armonk, NY) Statistical

significance was set at P < 0.05. Descriptive and inferential statistics using

Wilcoxon Sign Rank Test were used to analyse the data. Normality of the data

was assessed.

Table 1 and Graph 1&2 illustrated the result of the present study and

it showed that the pre irrigation groups (G1) presented with a mean of

3.54x10⁴ CFU/mL for group A (positive control group), 1.22x101CFU/mL

for

group B (negative control group), 3.25x10⁴CFU/mL for group C1 (6% NaOCl

group), 3.32x10⁴ CFU/mL for group D1 (0.008% BAK/6% NaOCl ).

Table 1 and Graph 1&3 shows the results of the post irrigation group

(G2) 6% NaOCl group (C1) showed reduction in the bacterial load or colony

forming units from 3.27x10⁴ to 0.89x10² CFU/mL. In group (D1) 0.008%

BAK/6%NaOCl showed initial growth of 3.33x10⁴ CFU/mL and on irrigation

during retreatment reduced the bacterial load to almost 0.5x10¹ CFU/mL.

Addition of 0.008% BAK completely eliminated the entire population of

E.faecalis from the retreated canal. The significant P value for 6% NaOCl and

0.008% BAK/6% NaOCl were respectively (.000 and .001).

Tables & Graphs

Photographs

TABLES AND GRAPHS

Mean colony count of E.faecalis present after the treatment by different test

solutions.

DESCRIPTIVE STATISTICS

Mean Std. Deviation

Positive (Group A) 35443.7500 1186.01223

Negative (Group B) 122.0000 40.98130

Before NaOCl (Group C1) 32712.5000 287.22813

Before NaOCl/BAK(Group D1) 33262.5000 404.76331

After NaOCl (Group C2) 89.1125 42.61286

After NaOCl/BAK (Group D2) .5467 1.14009

WILCOXON SIGN RANK TEST

GROUP D2 Std. Deviation P value

NaOCl 89.1125 42.61286 .000

NaOCl and BAK .5467 1.14009 .001

Adj.Sig= p value

Photographs

GRAPH 1: BEFORE IRRIGATION OF ALL GROUPS (G1)

0

5000

10000

15000

20000

25000

30000

35000

40000

MEAN

Positive Negative Bef NaOCl Bef BAL/NaOCl

Photographs

GRAPH 2: BEFORE IRRIGATION OF NAOCL GROUP AND

BAK/NAOCL GROUP (G1)

GRAPH 3: AFTER IRRIGATION OF NaOCl group and BAK/NaOCl group

(G2)

0

5000

10000

15000

20000

25000

30000

35000

NAOCL NAOCLBAK

BEFORE

Discussion

Discussion

42

DISCUSSION

Success in endodontic treatment is dependent on the complete

elimination of bacteria and their by-products from the root canal system and

also in preventing reinfection. According to Ingle 60% of endodontic failures

are due to incomplete obturation of the root canals while incomplete

disinfection and biomechanical preparation contribute to the rest. Mechanical

shaping of the root canal augmented with the potential irrigation plays a role

in disinfecting the root canal system completely.

Shaping and cleaning is performed mechanically by instruments which

remove both the infected and non-infected root canal tissue within the root

canal along with some dentin from the walls of the root canal and by chemical

disinfection, removing the bacteria and their products from within the root

canal space. Chemical disinfection using various irrigants helps in removing

the microorganisms and their by-products from within the root canal space29

.

Primary endodontic infection is polymicrobial and is mainly made up

of obligate anaerobes and small proportion of facultative anaerobes. The

obligate anaerobes can be easily eliminated completely to a great proportion

by mechanical instrumentation and chemical irrigation, whereas the facultative

anaerobes cannot be eliminated completely as a small percentage of them

survive the mechanical and chemical cleansing and lodge themselves in the

root canal niche and contribute to persistence of infection in endodontically

treated teeth 9.

Discussion

43

Enterococcus faecalis, a gram-positive facultative anaerobe is one of

the species of bacteria frequently present from previously root canal treated

teeth which can produce secondary infections. Several studies have reported

that E.faecalis susceptibility to irrigant solution is low27, 69

and are also not

eliminated by intracanal medicaments such as calcium hydroxide68, 27, 43

.

Therefore E.faecalis is capable of persisting within the root canal even after

instrumentation, intramedication, and also after obturation, thereby causing

post treatment disease.

Studies have shown that E.faecalis is the predominantly isolated

species of bacteria obtained from the root canal in retreated teeth with post

treatment apical periodontitis. Engstrom B18

investigated the occurrence of

enterococci in 223 teeth. There was bacterial growth in 134 samples and

enterococci in 20 cases (15%). Molander44

reported that bacteria were found

in 68 percentages of teeth which were subjected to retreatment following

obturation. E.faecalis was found to be the most frequent species with 47

percentages of the teeth tested showed positive culture. Sundqvist et al57

did a

study on retreatment of 54 teeth with post treatment disease. They found

microbial growth in 24 teeth (45%), out of which E.faecalis was found in 9

teeth (38%) and was the most frequently isolated bacterium, on each occasion

it was isolated in pure culture.

Hancock et al34

was able to obtain microbial growth from the root

canals of 33 teeth (61%) in a study for retreatment in 54 root filled teeth

showing post treatment disease. They found E.faecalis in 10 teeth out of the 33

Discussion

44

teeth. Of this, 6 teeth showed a pure culture of E.faecalis. Peciuliene et al48

did a study in 40 root filled teeth with asymptomatic apical periodontitis and

detected microbial growth in 33 teeth (83%) and could isolate E.faecalis in 21

teeth (64%). E.faecalis was the only isolated bacteria in 11 teeth, and was

isolated together with other bacteria or yeast in remaining 10 teeth. However

E.faecalis was found to be the dominance species in 8 teeth out of this10 teeth.

It can be inferred that in root canal treated teeth, the localization of the

bacteria within the root canal greatly depends on the space available after

obturation. The root canal sealers and obturating material may restrict the

possibility of the microorganisms present to interact with the periapical tissues

through the apical foramen. Hermetic sealing of the root canal space may also

prevent reoccurrence of infection and retreatment.

Siqueira and Rocas60

analysed microorganisms using PCR associated

with post treatment. Root canal samples were taken from 22 root filled teeth

with persistent disease and were subjected to endodontic retreatment.

E.faecalis was the most prevalent species detected in 77% of the teeth .Most of

the microorganisms were isolated from the apical third of the root canals filled

shorter than 2 mm from the apex. Pinheiro et al48

from their study inferred

that it is primarily the anaerobic bacteria that are associated with acute

symptoms in teeth with posttreatment disease, and not E.faecalis or other

enterococci. However Siqueira et al59

found E.faecalis are more commonly

present in symptom free teeth than in teeth with acute symptoms. E.faecalis is

the dominant species in endodontically treated teeth with periapical

Discussion

45

periodontitis and resisted many treatment protocols. There is no substantial

evidence to state that it is responsible for severe acute infections.

E.faecalis has been confirmed as most commonly found

microorganism in persistent secondary infection51,60

and also have been

identified in asymptomatic cases, posttreatment44

. The reason why E.faecalis

is isolated in cases both with and without disease needs to be clarified but may

be related to its virulence capability. Several of this virulence factors like

cytolysin and proteolytic enzymes, adhesion like enterococci surface protein,

and capsular and cellular polysaccharides, have been studied. Among such

virulence factors, gelatinase and enterococcal surface proteins (ESP) have

greater potential to colonize and produce disease 39, 54

. Another virulence

factor, gelatinase is capable of enhancing biofilm formation using E.faecalis

alone39

.

The virulence traits of E.faecalis are cell surface-associated protein,

namely, enterococcal surface proteins (ESP)67

. ESP is a cell wall-associated

protein that enriches the persistence of E.faecalis. The high prevalence of ESP

within oral isolates suggests that this surface protein may be a potential

virulence trait that participates in colonization of different niches of the oral

cavity. It stimulates biofilm production and helps the organism to adhere to

epithelium through mucin or uroplakin 78

.

E.faecalis is capable of invading the dentinal tubules of the root canal

system and can survive prolonged periods of adverse conditions such as

Discussion

46

starvation21, 54

and can also resist the high pH of calcium hydroxide

medication 19, 27

.

The collagen binding protein (Ace) and serine protease (Spr) are the

primary substrate for specific binding of E.faecalis to dentin and root canal

wall. Enterococcus faecalis is the most unique species from oral infections

including marginal periodontitis, infected root canals, and periradicular

abscesses, it has the ability to invade dentinal tubules and strongly get adhered

to collagen, which is abundantly present in root dentin and cementum 58

.

Sodium hypochlorite is the most commonly used irrigating solution

and it has a cytotoxic effect when injected into the periapical tissues20

. There

is no universally accepted concentration of sodium hypochlorite for use as an

endodontic irrigant55

, its optimal concentration, which ranges from 0.5% to

6%. Sodium hypochlorite brings its anti-microbial activity by direct contact

with the microorganisms. There is no universally accepted concentration of

sodium hypochlorite for use as an endodontic irrigant.

Sodium hypochlorite due to its high surface tension has less ability to

penetrate into dentine and therefore its antibacterial effectiveness within

dentinal tubules is reduced25

. The penetration of NaOCl into dentinal tubules

has been reported to be around 300 µm with a 6% solution for 20 min at

450C

77.

Surface tension can be reduced by adding chemicals known as

surfactants71, 46

. Surfactant molecules are identified by a hydrophobic portion,

organic/oil soluble or water insoluble, and a hydrophilic region, water soluble.

Discussion

47

Each molecule may contain a positively charged group (cationic class), a

negatively charged one (anionic class), both (amphoteric class) or no electric

charge (nonionic class) 42

.

BAK is an amphoteric surfactant that contains amphipathic molecules,

such as quaternary nitrogen associated with a hydrophobic substituent, that are

capable of reducing surface tension and increasing the surface area of

hydrophobic, water-insoluble growth substrates 35

.

Adding a surfactant to the root canal irrigant theoretically permits a

better spreading of the irrigant in minute spaces that are inaccessible to the

endodontic instruments6. Addition of surfactants to irrigants produce an

improvement in irrigants penetration depth, better permeability into dentin,

thereby providing better cleansing and disinfection of root canal walls and also

better dissolution of the pulp tissue 6.

Benzalkonium chloride (BAK) can chemically react with sodium

hypochlorite. This chemical reaction modifies the free chlorine content of

NaOCl and therefore can alter the solvent property of the irrigant. This effect

may also lead to a loss of the efficiency of sodium hypochlorite. To prevent

this loss, a homogeneous mixing of BAK in NaOCl solution was maintained

with a magnetic stirrer to prevent BAK adsorption at the solution/air interface.

Reducing the surface tension and the contact angle of a root canal irrigant by

51.2% by adding 0.008% BAK seems to be a promising technique 6.

Discussion

48

The purpose of this study was to assess the effect of the addition of

BAK to NaOCl, on the antibacterial activity in the root canal during

retreatment.

For this study, human mandibular first premolars without any fractures

or carious lesions, extracted for orthodontic treatment were used. Radiographs

of these teeth were taken and 64 teeth showing single straight or mildly curved

(less than 20 degrees) canal were selected and used for this study. The teeth

were thoroughly cleaned, and access was gained into the root canal and

working length determined using 15 size K file, till the file exited the apical

foramen. The canals were shaped with protaper universal rotary files and

cleaned with 2.5% sodium hypochlorite solution and finally rinsed with

normal saline. The canals were then dried and obturated using a single 6%

gutta percha cone and zinc oxide eugenol as sealer. Single cone technique was

used as it produced space within the root canal for the bacteria to colonize and

multiply after inoculation. Zinc oxide eugenol was the sealer of choice as it is

commonly used and has good penetrability in to the dentinal tubules

preventing growth of microorganisms2. After completion of obturation, the

access cavity coronally and the apical end of the root canal were sealed with

type II Glass Ionomer Cement and left for 24 hours. This was done to prevent

contamination of the root canal till inoculation and for the purpose of

retreatment procedure.

After obturation, the teeth were placed individually in 1.5mL

microtubes (Axygen AXY) for 24 hours under aseptic conditions. The 64

Discussion

49

samples were then done randomly divided into four groups of N=16- Group A

(positive control), Group B (negative control), Groups C1 and D1 (testing

groups). Glass Ionomer Cement was removed from all the specimens

coronally. Out of the 4 groups, the negative control group (group B) received

only the medium and no inoculate. Group B samples were filled with sterile

brain heart infusion (BHI) broth (Becton Dickinson and Company, Sparks,

MD). The other three groups of specimens ( N=48) were infected by using a

24 hour pure culture suspension of E.faecalis (ATCC 29212) cultivated in BHI

and then inoculated with 15µL of 1x10⁸ colony- forming units (CFU)/mL

suspension of E.faecalis determined by serial dilution and plating. All the

specimens were then incubated at 37˚C and at 95% humidity for 21days.The

BHI was removed from the canal by gentle aspiration and replenished at the

end of 4th

day, till 21 days.

After 21 days the medium was removed from all the specimens and re-

treatment procedure was done using ProTraper Universal Retreatment files

(DentsplyMaillefer, Ballaigues, Switzerland). Normal saline was used as an

irrigant during re-treatment procedure. After complete removal of 6% gutta

percha, a sterile paper point was used at working length to sample the canals

from all 4 groups.

The paper points were placed individually in 1.5mL microtubes

containing 0.5 mL of BHI broth and were vortexed at highest speed for 3

consecutive intervals of 15 secs each. 100µLs of the medium was plated on

BHI agar in triplicate the reminder was stored at 4˚C.

Discussion

50

After paper point sampling, the specimens in the two tested groups and

positive control group were irrigated with 5 mL of the respective irrigant as

described. The negative control group specimens (Group B) were irrigated

with normal saline and the specimens in group C1 were irrigated with 6%

NaOCl solution, and specimens in group D1 were irrigated with 0.008% BAK

solution and 6% NaOCl solution. All irrigants were delivered at a flow rate of

0.04 mL/s using a side-vented 30-gauge needle. A second post-irrigation paper

point sample (C2 and D2) was obtained from each canal in the NaOCl and

NaOCl/BAK groups respectively. The positive controls received no irrigation

and were only sampled before irrigation. This second paper point samples

were placed similarly in 1.5m L microtubes and were plated as described

earlier.

All the plates (both pre irrigation and post irrigation) were incubated at

37˚C and 95% humidity for 24 hours, a colony counter (TRINITY V3) was

used to enumerate the number of colonies formed (CCU). The results were

recorded and tabulated, and the data collected were compiled using MS-Office

Excel and was subjected to Statistical analysis using IBM corp. SPSS

(Statistical package for social sciences).

From the table, following obturation and before retreatment the

negative control group (group B) showed minimal bacterial growth with mean

of 1.22x10² CFU/mL, inoculation of bacteria into the other three groups

produce growth with the mean of 3.54x10⁴, 3.25x10⁴, 3.32x10⁴ in the positive

control group (group A), NaOCl group (group C1) and BAK/NaOCl (group

Discussion

51

D1) respectively on comparison of the above 3 groups. It was seen that there

was no significant difference in the values between these groups (Kruskal

Wallis test). But on comparison with the negative group there was significant

difference between the values. The negative control group produced minimal

growth of bacteria (E.faecalis) probably due to presence of the bacteria from

the BHI broth, colonizing and multiplying within the available root canal

space after obturation. The other three groups showed high colony counts

probably due to the inoculation of the bacterial E.faecalis. This shows that

available space within the root canal permits minimal growth of bacteria from

BHI broth as compared to inoculation.

From biological perceptive it can be considered that the root canal is a

highly control zone with limited number of niches. Growth of bacteria in such

niches is related to the environmental factors available in the space such as

oxygen and nutrient availability. After root canal treatment, other factors such

as pH and short or long term effects of antibacterial medicament supply, type

of sealer used become the limiting factors for bacterial growth 9.

Earlier studies have shown that E.faecalis is able to survive in such

controlled environments, especially after root canal treatment and depends on

the ability of this microorganism to adapt to the existing condition.

Bacterial survival especially E.faecalis after root canal treatment will

depend not only on its robustness, but also how effectively it adapts to the

newer limiting factors within the root canal and its corresponding niches.

Discussion

52

Following retreatment and irrigation in retreated root canal with

sodium hypochlorite (Group C2) reduced the bacterial load or colony forming

units from 3.27x10⁴ to 0.89x10² CFU/mL.

Among the various root canal irrigants being used sodium hypochlorite

is the most effective irrigant and due to its ability to dissolve the tissue along

with its proteolytic action and due to its bactericidal effect on microorganisms

and bacterial biofilms, it is a powerful disinfectant. Most common

concentrations of sodium hypochlorite used in endodontics as a root canal

irrigant are in the range of 0.5-5.25% 22

. However research has shown that

irrigation with 5 % NaOCl during endodontic instrumentation has shown that

nearly one third to one half of the root canal remains contaminated22

.

E.faecalis has the ability to form a biofilm in monoculture as it can

adapt to harsh environmental conditions. Overtime this biofilm increases in

growth and becomes calcified. As a result it becomes more difficult to remove

this matured and mineralized biofilm with the use of routine irrigants

including 5.25% NaOCl5. Retreatment involves not only removing the

microorganisms from the treated root canal but also the obturated material and

the sealer which would have penetrated the dentinal tubules. Hence, in this

invitro study a higher concentration of NaOCl at 6% was used to remove the

set cement from the dentinal walls.

Removal of the sealer along with the obturated material followed by

irrigation with NaOCl 6%, probably nearly eliminated completely the

inoculated E.faecalis from the root canal. Studies by Zou et al 76

have showed

Discussion

53

that an irrigation time of 20 mins was necessary with 6% NaOCl for a

maximum penetration of up to 300µm in dentinal tubules. In the present study,

irrigation was done only for 5 minutes and with only 5 ml of irrigant which

probably resulted in complete elimination of E.faecalis.

During primary endodontic treatment, use of irrigants allows its

penetration into the narrow long dentinal tubules either by capillary force or

by diffusion into the dentin. However during retreatment the irrigants are

unable to permeate into the dentinal tubules due to the penetrability and setting

of the sealer used, which prevent complete elimination of the microorganisms

lodged.

Use of surface acting agent added to NaOCl could improve their

wettability on the dentin surface in the root canal thereby resulting in better

adaptation to dentin and penetration in to the dentinal tubules resulting in

improved bacterial clearance46

. Cameron7 showed that addition of surface

acting agent to NaOCl enhances its ability to dissolve organic material.

Clarkson et al12

reported that addition of surfactants added to NaOCl dissolve

porcine pulp in shorter time than regular NaOCl. Various agents like ethanol,

polysorbate 80, chlorhexidine, fluorad 99, EDTA, benzalkoniumchloride have

all been tried as surface acting agent. Available evidence suggests that surface

active agent improve the penetration of NaOCl in the main root canal.

However recent studies show that addition of surfactants results in better

lubrication, debris removal and efficient antimicrobial action when used along

Discussion

54

with NaOCl. Hence in the present study benzalkonium chloride at 0.008% was

used as a surface active agent prior to irrigation with 6% NaOCl5, 6,35,36,47

.

From the results it can be seen that addition of 0.008% benzalkonium

chloride as an irrigant when used for 5 minutes prior to use of 6%NaOCl

(Group D2) completely eliminated the bacteria from the dentinal walls of the

root canal during retreatment.

Benzalkonium chloride is a surfactant and is capable of improving the

wetting properties. Jaramillo et al35

have found that BAK is capable of

producing an overall 70-fold reduction in biofilm accumulation. BAK when

combined with NaOCl is capable of reducing a bacterial load more than

sodium hypochlorite alone as it helps NaOCl to diffuse further in to the

dentinal tubules.

BAK may chemically react with NaOCl which can modify the free

chlorine content which can alter the solubility of the irrigant. This may lead to

the loss of efficiency as it may affect the antibacterial and cytotoxic properties

of the mixture.

In the present study, inoculation of E.faecalis into the specimens before

retreatment produced growth of 3.33x10⁴ CFU/mL and on irrigation during

retreatment reduced the bacterial load to almost 0.5x10¹ CFU/mL. Addition of

0.008% BAK completely eliminated the entire population of E.faecalis from

the retreated canal.

The major disadvantages of sodium hypochlorite are its high surface

tension which may affect its wettability and prevent NaOCl to penetrate into

Discussion

55

the depth of dentinal tubules. The wettability of a solution depends on its

surface tension. Surface tension is defined as the force between molecules

tending to reduce the surface area of the liquid64

. To decrease the surface

tension of a liquid surfactants or surface active agents can be added,

commonly referred to as detergents. Different detergents added to

antimicrobial solutions improve their bactericidal efficacy1.

To achieve this optimal wettability, the surface energy of the substrate

(i.e. dentin) must be as high as possible and the surface tension of the liquid

contacted (i.e. irrigant) with the substrate must be as low as possible. Surface

tension due to intramolecular attraction/repulsion prevents the spreading of a

solution over the surface. When this intramolecular attraction is destroyed the

surface tension decreases 7. Therefore a low surface tension would increase the

penetration of irrigants into the root canal system, lateral canals and dentinal

tubules and thereby increase their contact with the dentin walls 46

.

Surface tension can be reduced while using heat or adding chemicals

known as surfactants. Surface active agents may consist of monomers,

polymers or complex mixtures which contain an active portion within the

inactive base. Surfactant molecules are characterized by a hydrophobic portion

which can be organic/oil soluble or water insoluble and have a hydrophilic

region which is water soluble. Each molecule may also contain a positively

charged group (cationic), a negatively charged group (anionic), both the

groups (amphoteric) or low electric charge (non-ionic). Non-ionic agents do

not ionize but contain water soluble polar groups and/or have hydrogen

Discussion

56

bonding capabilities which may provide interactions with water molecules,

improving 42

.

The mode of action of BAK against bacterial cells may involve the

cytoplasmic membrane and outer cell membrane of the bacteria involving the

lipid bilayer. This may cause a generalized and progressive leakage of

cytoplasmic materials to the environment that will result in bacterial death.

Previous reports suggest that the hydrophobic changes and electrostatic

repulsion of BAK surfactant prevent biofilm formation. The repulsive action

might be associated to the positively charged quaternary nitrogen which reacts

with the head groups of acidic phospholipids within the cell membrane of the

bacteria. This interaction may decrease the adhesive potential of bacterial

adhesions 24.

The results of the study done by Jaramillo et al35

suggest that the

surface coating BAK solution has a very high biofilm reducing capacity. On

continuous exposure to BAK it reduced the microorganisms ability to persist

and adapt to the given concentrations of BAK. Therefore it can be concluded

that bacterial adhesion depends on the conditioning film properties rather than

on the surface itself. This study also showed that the biofilm reducing

potential of BAK seems to be related in preventing the biofilm formation at its

initial stages.

The primary function of an endodontic sealer is to fill all the irregular

spaces within the root canal system including dentinal tubules. Bacteria

present inside dentinal tubules can survive after biomechanical preparation.

Discussion

57

Earlier studies have shown that adhesion of antimicrobial agents to root canal

sealers can result in improving their antibacterial effect and thereby improving

the disinfection of the root canal system 4.

In this study done by Arias-Moliz et al4 was found that bacteria are

able to grow on the surface of sealers like AH Plus and about 57% of these

bacteria were alive. This probably could be the reason for the bacterial growth

seen in the present study before retreatment (3.37x10⁴CFU/mL). In the

previous study the adhesion of BAK to the sealer confirmed antibacterial

properties and was found to be concentration dependent. The bacterial bio

volume with 3% BAK group was 33 times lower in comparison to the sealer

AH Plus without BAK. This indicates that fewer bacteria were allowed to

grow on the sealer surface as their concentration increased, showing that BAK

has a good antibiofilm activity

Previous studies done on the microhardness of dentin have shown a

reduction when BAK is added to sealers. AH Plus containing 2% BAK and

AH plus containing 3% BAK produced a reduction in microhardness close to

50% and 75% respectively as compared with AH Plus4. Since higher

concentrations can have an influence on microhardness, a low concentration of

0.008% was preferred in this present study.

Further, surface tension of any liquid decreases according to the

surfactant concentration. More the concentration of surfactants leads to a

saturation and is called critical micellar concentration (CMC). Above this

CMC, the addition of surfactant alone keeps the surface tension relatively

Discussion

58

constant and contributes to the formation of micelles in the liquid. The best

wetting properties are achieved in this concentration.

A study by Deutschle et al14

reported that micelle formation was

considered the possible cause of the sudden termination of BAK activity at

higher concentration. Study by Bukiet et al6

confirmed that the contact angle

and the surface energy significantly decreased with increasing BAK

concentration.

The addition of BAK to NaOCl at the CMC reduced the contact angle

by 51% and the surface energy by 53% without affecting the free chloride

content, cytotoxicity or antimicrobial properties of the mixture. The CMC of

BAK in NaOCl was found to be 0.008% and hence used in this study.

Chloramines are derivatives of ammonia where 1, 2, 3 hydrogen atoms

are substituted by chlorine atoms6. BAK is a quaternary ammonium salt and

will react with NaOCl to produce chloramines. So when BAK is combined

with NaOCl for irrigation, this could lead to chlorine content after mixing.

This could result in loss of solvent activity on organic compounds and may

also increase the cytotoxicity of the mixture. Therefore it was mandatory to

assess the chlorine content of the mixture NaOCl + BAK at the CMC. The

study showed that BAK at the CMC level did not modify the chlorine content

of 2.4% NaOCl solution. This may be due to the very small BAK

concentration that is needed to reach the CMC. Further addition of this

concentration of BAK did not alter the action of the mixture in cytotoxic and

antibacterial studies. No significant chemical reaction occurred at 0.008%

Discussion

59

concentration of BAK with NaOCl at the CMC, and no harmful effect occurs

from the mixture of these compounds.

The antibacterial effectiveness of NaOCl is highly dependent on its

concentration, temperature, volume, refreshment rate and contact time into the

root canals and dentinal tubules especially during retreatment. After

endodontic treatment microorganisms especially E.faecalis may persists in

areas such as isthmus, ramification and lateral canals, as these areas are

difficult to clean completely. Further, penetration of E.faecalis into dentinal

tubules during obturation may provide them protection from the antimicrobial

actions of irrigants used during retreatment. Incomplete action of irrigation

may result in failure of retreatment. Hence, presence of surfactants added to

irrigant may play a role on efficient cleansing during retreatment.

According to Wang et al74

, the addition of surfactants did not

significantly improve the depth of penetration of 6% NaOCl solution. They

also suggested that higher concentrations of NaOCl without any surfactant

produced maximal killing within a time frame of 1-3 minutes for disinfection

on surface. Because penetration can occur either by capillary forces or by

diffusion/flow, low surface tension could limit the penetration of modified

NaOCl solutions into narrow and long dentinal tubules71

. Moreover Wang et

al74

reported better antibacterial performance by NaOCl solutions containing a

surfactant which was effective in all layers and not just in deeper areas of

dentin. The concentration of NaOCl provided a lesser contribution to

penetration in solutions with surfactants. The depth of penetration decreased

Discussion

60

with increasing concentration beyond CMC level. Differences were not

statistically significant at all times and temperatures for 1% versus 2% NaOCl

and 5.25% versus 6% NaOCl. However the bactericidal action varied or

increased with respect to the depth of penetration into the dentinal tubules, the

action being more prominent for 3% than 0.5% NaOCl74

. Zou et al77

investigated on the penetration depth of various concentration of NaOCl

solution at different times of exposure and temperature by observing bleaching

action on stained dentin and found penetration depth extended up to 77µm for

1% NaOCl and up to 300µm for 6% NaOCl with no reference to time.

Appropriate contact time for root canal irrigants to achieve complete

debridement and disinfection in clinical situation is unclear60

. Longer exposure

time results in deeper penetration of NaOCl with lowered surface tension and

regular concentrations. According to Zou et al77

speed of penetration for

NaOCl modified with a surfactant, decreased sharply with respect to time.

Addition of surfactants also increases the penetration depth with increasing

temperature. As temperature increases the surface tension values of regular

NaOCl and NaOCl with surfactants may show a decrease. Predictable increase

in the temperature of irrigants may have an effect on the success rate of

endodontic irrigation. The temperature and increase in concentration provides

a lesser contribution to the penetration of NaOCl into root canal dentin, the

exposure time significantly improves the penetration depth of NaOCl when

surfactants are added.

Discussion

61

In the present study, irrigation with 0.008% BAK followed by

6%NaOCl solution for a period of 5 minutes completely eliminated the

bacteria from the root canal in all the specimens of Group D2, during

retreatment. Since this study was done in laboratory conditions (in vitro), a

contact time of 5 minutes completely disinfected the root canals. However, in

clinical situations such long contact time is not recommended and hence use of

a surfactant like 0.008% BAK even with a lower concentration of

NaOCl, in a shorter time (1-2 minutes) may be effective in cleansing the

retreated root canal.

Antibiofilm or surfactant coatings can alter the root

canal surface properties and interfere with bacterial adhesion. In the present

study a surface coating with a solution of BAK reduced the formation of

growth by E.faecalis as compared to minimal growth with NaOCl.

One limitation of present study was use of a single bacterium

E.faecalis which made the study straight forward. However, various

microorganisms are able to invade root canals and form a complex biofilm.

Since, in most of the root canal retreatment cases or with persistent infections

E.faecalis is the commonest species isolated and on its stability to form a

biofilm even in single culture it was chosen for this study. Previous studies

(Chevaz de paz et al)10

, (Sathorn et al)53

have well documented that

E.faecalis is found primarily in teeth with previous endodontic treatment.

In the present study, the paper point sampling method was used for

colony counting which is a limitation of this study. The paper point sampling

Discussion

62

method is a less sensitive method for monitoring treatment strategies within

the canal.

Previous studies have reported decrease in bacterial load but not

complete elimination when using surfactants with current irrigation

techniques. All these studies were done during primary endodontic treatment.

Since there are not many studies on the effect of irrigants and surfactants

during irrigation in retreatment cases, the present study was undertaken.

In conclusion, the results of the present study demonstrate that the

addition of the surfactant 0.008% BAK to 6% NaOCl (or even lower

concentration) significantly eliminated the bacteria E.faecalis in infected root

canals during retreatment. Other methods to activate root canal irrigants and

improve their efficiency by enhancing bacterial clearance like passive

ultrasonic techniques, sonic activation, laser and photodynamic activation,

manual agitation, use of Endovac etc needs to be studied in vivo during

retreatment for successful outcomes in retreatodontics.

Summary

Summary

63

SUMMARY

The present in vitro study was done to analyse the effect of 0.008%

benzalkonium chloride surfactant- 6% sodium hypochlorite combination in the

elimination of E.faecalis during irrigation in retreatment procedure. A total

number of 64 extracted human mandibular premolars were employed in this

study and divided into 4 major groups: A positive control (group A), negative

control (group B), NaOCl 6% (group C), BAK 0.008% + NaOCl 6% (group

D) with 16 specimens in each group. The specimens were infected by using a

24-hour pure culture suspension of E. faecalis cultivated in Brain Heart

Infusion agar (BHI) and then inoculated with 15 mL of 1 x10⁸ colony-forming

units (CFU)/mL suspension of E. faecalis (determined by serial dilution and

plating) after primary endodontic treatment. Teeth were incubated at 37˚C and

95% humidity for 21 days. The negative control group was not inoculated and

were filled with sterile brain-heart infusion (BHI) broth (Becton Dickinson

and Company, Sparks, MD). After 21 days, the tooth from all the four groups

was taken from the medium and re-treatment procedure was done using

ProTraper Universal Retreatment files (Dentsply Maillefer, Ballaigues,

Switzerland). After complete removal of gutta percha, the teeth were again

placed into the medium. The paper point sampling was done in all 4 groups

before irrigation (G1). A second post-irrigation paper point sample (G2) was

obtained from each canal in the NaOCl (C2) and NaOCl/BAK (D2) groups.

The positive control group (A) received no irrigation and were only sampled

Summary

64

before irrigation. The specimens in negative control group (B) were irrigated

with normal saline during retreatment. After paper point sampling, the teeth

from group A, C and D were placed into the medium and the teeth from

negative control group were again placed into BHI broth. The paper point

samples of pre and post irrigation was to determine the remaining colony

forming units and statistically analysed.

Conclusion

Conclusion

65

CONCLUSION

Within the limitations of the present study the following conclusion

were made

1. E.faecalis is the commonest bacteria found in persistent endodontic

infections and can be lodged in the walls of the dentinal tubules even

as a single bacterial agent following obturation. Use of a monoculture

organism E.faecalis provided a straight forward in vitro experimental

design for retreatment.

2. Sodium hypochlorite (NaOCl) can be used in various concentrations

(0.5 % -5.25%) as a root canal irrigant during primary endodontic

treatment. However, an increased concentration (6%) has been found

to be effective in retreatment cases.

3. NaOCl (6%) does not eliminate the bacteria E.faecalis completely from

the dentinal walls of the root canal during retreatment (Group C2).

4. Addition of surface active agent to NaOCl, reduces the surface tension

and improves its wettability.

5. Addition of 0.008% Benzalkonium chloride (BAK) to NaOCl (6%)

seems to be a promising technique, to reduce the surface tension of

NaOCl (6%) and improve its penetrability into the dentinal tubules.

Conclusion

66

6. Irrigation of the root canal with 0.008% BAK followed by 6% NaOCl

was effective in completely removing all the bacteria (E.faecalis) from

the root canal walls during retreatment (Group D2).

7. Irrigation of the root canal with normal saline (Group B) was

ineffective in eliminating the bacteria from the root canal walls during

retreatment.

8. The paper point sampling method is found to be very effective to

confirm the bacterial status of the root canal both during primary

endodontic treatment and retreatment.

9. Incubating the teeth for 3 weeks with E.faecalis provided adequate

colony forming units (CFU) during the investigation.

Further investigations should be performed under dynamic conditions

to confirm the beneficial effects of the use of surfactants with irrigants, before

transposing these results to clinical use.

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Textbook References:

Ingle's Endodontics 6th Edition.

Cohen's Pathways of the Pulp11th Edition.

Weine’s Endodontic Therapy 6th

Edition.

Torabinejad’s Endodontics 5th Edition Principles and Practice.

Annexures

Annexures

ANNEXURE –I

Annexures

ANNEXURE –II

Documents

EFFECT OF A SURFACTANT ON SODIUM HYPOCHLORITE …repository-tnmgrmu.ac.in/10841/1/240402319dinesh_kumar.pdf · effect of a surfactant on sodium hypochlorite combination in the elimination