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
Review of literature
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
Review of literature
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.
Review of literature
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
Review of literature
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.
Review of literature
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).
Review of literature
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.
Review of literature
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
conducted a study to compare the
efficacy of root canal irrigants against E. faecalis biofilms using a novel in
Review of literature
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.
Review of literature
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
Review of literature
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
Review of literature
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
Review of literature
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,
Review of literature
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
Review of literature
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
Review of literature
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
conducted a study to compare the
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
Review of literature
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.
Review of literature
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
Review of literature
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
Review of literature
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
Review of literature
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
Review of literature
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
Review of literature
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
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.)
Materials and Methods
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.
Materials and Methods
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,
Materials and Methods
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.
Materials and Methods
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.
Materials and Methods
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)
Materials and Methods
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.
Bibliography
Bibliography
BIBLIOGRAPHY
1. Abou-Rass M and Michael V. Piccinino.
The effectiveness of four clinical irrigation methods on the removal of
root canal debris.
Oral Surg. September, 1982.
2. Amelia I, E Herda and Y K Eriwati.
Sealing ability of zinc oxide eugenol and non-eugenol-based
temporary filling.
IOP Conf. Series: Journal of Physics: Conf. Series 1073 (2018)
062014.
3. Arias-Moliz MT, Carmen Marıa Ferrer-Luque, Miguel Espigares-
Garcıa and Pilar Baca.
Enterococcus faecalis Biofilms Eradication by Root Canal Irrigants.
Journal of Endodontics 2009;35:711–714
4. Arias-Moliz MT, M. Ruiz-Linares, G. Cassar, C.M. Ferrer-
Luque, P. Baca, R. Ordinola-Zapata.
The effect of benzalkonium chloride additions to AH Plus sealer.
Antimicrobial, physical and chemical properties.
Journal of Dentistry 2015.
67
Bibliography
5. Baron A, Kimberly Lindsey, Stephanie J. Sidow, Douglas
Dickinson, Augustine Chuang and James C. McPherson.
Effect of a Benzalkonium Chloride Surfactant–Sodium Hypochlorite
Combination on Elimination of Enterococcus faecalis.
Journal of Endodontics 2015.
6. Bukiet F, Guillaume Couderc, Jean Camps, Herve Tassery .
Wetting Properties and Critical Micellar Concentration of
Benzalkonium Chloride Mixed in Sodium Hypochlorite.
Journal of Endodontics 2012;38:1525–1529.
7. Cameron Jeffrey A.
The effect of a fluorocarbon surfactant on the surface tension of the
endodontic irrigant, sodium hypochlorite.
Australian Dental Journal 1986;31:5.
8. Carneiro Valera M, Juliana de Moraes Rego, and Antonio Olavo
Cardoso Jorge.
Effect of Sodium Hypochlorite and Five Intracanal Medications on
Candida albicans in Root Canals.
Journal of Endodontics Vol. 27, No. 6, June 2001.
9. Chavez de Paz LE, Dahlen G, Molander A, Moller, Bergenholtz
G. Bacteria recovered from teeth with apical periodontitis after
antimicrobial endodontic treatment.
International Endodontic Journal, 36, 500^508, 2003.
Bibliography
10. Chavez de Paz.
Redefining the Persistent Infection in Root Canals: Possible Role of
Biofilm Communities.
Journal of Endodontics 2007; 33:652– 66.
11. Claxton NS, Thomas J. Fellers, and Michael W. Davidson.
Laser Scanning Confocal Microscopy.
12. Clarkson PM, Michael B, Overell.
Environmental Reporting and its Relation to Corporate Environmental
Performance.
ABACUS, Vol. 47, No. 1, 2011
13. Clegg MS, F.J. Vertucci, Walker, M. Belanger and L.R. Britto.
The Effect of Exposure to Irrigant Solutions on Apical Dentin
Biofilms In Vitro.
Journal of Endodontics 2006; 32:434–437.
14. Deutschle T, U. Porkert, R. Reiter, T. Keck, H. Riechelmann.
In vitro genotoxicity and cytotoxicity of benzalkonium chloride.
Toxicology in Vitro 20 (2006) 1472–1477
15. Dunavant TR, John D. Regan, Gerald N. Glickman, Eric S.
Solomon Comparative Evaluation of Endodontic Irrigants against
Enterococcus faecalis Biofilms.
Journal of Endodontics 2006; 32:527–531.
Bibliography
16. Duggan J.M and Christine M. Sedgley.
Biofilm Formation of Oral and Endodontic Enterococcus faecalis.
Journal of Endodontics 2007; 33:815– 818.
17. Du Tianfeng , Zhejun Wang, Ya Shen, Jingzhi Ma, Yingguang
Caoand Markus Haapasalo, Dr Odont.
Combined Antibacterial Effect of Sodium Hypochlorite and Root
Canal Sealers against Enterococcus faecalis Biofilms in Dentin
Canals.
J Endod 2015;-:1–5.
18. Engstrom, B.
The significance of enterococci in root canal treatment.
Odontol Revy 15, 87–106.1964.
19. Evans M, Davies JK, Sundqvist G, Figdor D
Mechanisms involved in the resistance of Enterococcus faecalis to
calcium hydroxide.
International Endodontic Journal, 35, 221–228, 2002.
20. Filho TM, Leonardo MR, Silva LAB, Anibal FF, Faccioli LH.
Inflammatory response to different endodontic irrigating solutions.
International Endodontic Journal, 35, 735^739, 2002.
21. Figdor D, Davies JK, Sundqvist G.
Starvation survival, growth and recovery of Enterococcus faecalis in
human serum. Oral microbial immunol 2003: 18: 234-239.
Bibliography
22. Frough-Reyhani M, Negin Ghasemi, Mohammadhosien Soroush-
Barhaghi, Mahsa Amini, Yousefreza Gholizadeh.
Antimicrobial efficacy of different concentration of sodium
hypochlorite on the biofilm of Enterococcus faecalis at different
stages of development.
Journal of Clinical Experimental Dentistry 2016.
23. George. S, A. Kishen, and K. P. Song.
The Role of Environmental Changes on Monospecies Biofilm
Formation on Root Canal Wall by Enterococcus faecalis.
American Association of Endodontists 2005.
24. Gilbert P and L.E. Moore.
Cationic antiseptics: diversity of action under a common epithet.
Journal of Applied Microbiology 2005, 99, 703–715
25. Giardino L, Emanuele Ambu, Carlo Becce, Lia Rimondini and
Marco Morra.
Surface Tension Comparison of Four Common Root Canal Irrigants
and Two New Irrigants Containing Antibiotic.
J Endod 2006;32:1091–1093
Bibliography
26. Gomes BPFA, Ferraz CCR, Vianna ME, Berber VB, Teixeira FB,
Souza-Filho FJ.
In vitro antimicrobial activity of several concentrations of sodium
hypochlorite and chlorhexidine gluconate in the elimination of
Enterococcus faecalis.
International Endodontic Journal,34, 424–428, 2001.
27. Gomes BPFA, Caio Cezar Randi Ferraz, Morgana Eli Viannai,
Pedro Luiz Rosalen.
In Vitro Antimicrobial Activity of Calcium Hydroxide Pastes and
their Vehicles Against Selected Microorganisms.
Braz Dent J (2002) 13(3): 155-161
28. Gomes BPFA, Pinheiro ET, Gade-NetoCR.
Microbiological examination of infected dental root canals.
Oral Microbiology Immunology 2004.
29. Grossman’s Endodontic Practice. 13th
edition
30. Guerreiro-Tanomaru J.M, Renata D. Morgental, Danilo L.
Flumignan, Fabricia Gasparini .
Evaluation of pH, available chlorine content, and antibacterial
activity of endodontic irrigants and their combinations against
Enterococcus faecalis. Volume 112, Number 1 2011.
31. Hartke A, Jean-Christope Giard, Jean Marie Laplace.
Bibliography
Survival of nterococcus faecalis in an Oligotrophic Microcosm:
Changes in Morphology, Development of General Stress Resistance,
and Analysis of Protein Synthesis.
Journal of Applied and Environmental Microbiology 1998 Nov;
64(11): 4238–4245.
32. Halkai R, Mithra N. Hegde & Kiran Halkai.
Enterococcus faecalis cause for persisting infection a confocal
analysis. Nitte University Journal of Health Science Vol. 3, No.4,
December 2013, ISSN 2249-7110.
33. Halkai R, Mithra N. Hegde & Kiran Halkai.
Evaluation of the presence of Enterococcus Faecalis in root
cementum: A confocal laser scanning microscope analysis.
Journal of Conservative Dentistry 2014 Volume: 17.
34. Hancock H.H III, Asgeir Sigurdsson, Martin Trope and Julian
Moiseiwitsch.
Bacteria isolated after unsuccessful endodontic treatment in a North
American population.
Oral Surgery Oral Medicine Oral Pathology Vol. 91 No. 5 May 2001.
35. Jaramillo D.E, Alberto Arriola, Kamran Safavi Luis E. Chavez de
Paz Decreased.
Bacterial Adherence and Biofilm Growth on Surfaces Coated with a
Solution of Benzalkonium Chloride. Journal of Endodontics 2012;
38:821–825.
Bibliography
36. Kho P and J. Craig Baumgartner.
A Comparison of the Antimicrobial Efficacy of NaOCl/Biopure
MTAD versus NaOCl/EDTA against Enterococcus faecalis.
Journal of Endodontics 2006; 32:652– 655.
37. Kishen A, S. George,R. Kumar.
Enterococcus faecalis-mediated biomineralized biofilm formation on
root canal dentine in vitro.
Inc. J Biomed Mater Res 77A:406–415, 2006.
38. Kowalski William J, Edward L. Kasper, John F. Hatton, Barbara
E. Murray.
Enterococcus faecalis Adhesin, Ace, Mediates Attachment to
Particulate Dentin.
Journal of Endodontics 2006; 32:634–637.
39. Kristich CJ, Yung-Hua Li, Dennis G. Cvitkovitch, and Gary M.
Dunny.
Esp-Independent Biofilm Formation by Enterococcus faecalis
Journal of Bacteriology, Jan. 2004, p. 154–163.
40. Love RM.
Enterococcus faecalis – a mechanism for its role in endodontic failure.
International Endodontic Journal, 34, 399–405, 2001.
41. Manikandan, Mithra N Hegde, Veena shetty A, Geethashri A.
Comparative evaluation of biofilm formation ability of E.faecalis in
Bibliography
alkaline conditions and its susceptibility to endodontic irrigant
regimens – An In vitro microbiological study.
IOSR Journal of Dental and Medical Sciences (IOSR-JDMS) Volume
4, Issue 2 (Jan. - Feb. 2013), PP 49-52.e-ISSN: 2279-0853, p-ISSN:
2279-0861.
42. McNaught AD, Wilkinson A (1997)
IUPAC. Compendium of Chemical Terminology, 2nd edn. (The
‘‘Gold Book’’).
Oxford:Blackwell Scientific Publications, XML on-line corrected
version: http://goldbook.iupac.org (2006-) created by M. Nic, J. Jirat,
B. Kosata; updates compiled by A. Jenkins. ISBN 0-9678550-9-8. doi:
10.1351/goldbook.
43. Menezes MM, Valera MC, Jorge AOC, Camargo CHR
Invitro evaluation of the effectiveness of irrigants and intracanal
medicaments on microorganisms within root canal.
Journal of Endodontics 37,311-311, 2004.
44. Molander A, C. Reit, G. Dahlen & T.Kvist.
Microbiological status of root-filled teeth with apical periodontitis.
International Endodontic Journal (1998) 31, 1–7.
45. Oliveira DP, Joao V. B. Barbizam, Martin Trope and Fabricio B.
Teixeira, Chapel Hill, NC.
In vitro antibacterial efficacy of endodontic irrigants against
Enterococcus faecalis.
Bibliography
Endodontology Vol. 103 No. 5 May 2007.
46. Palazzi F, Morra M, Mohammadi Z, Grandini S, Giardino L.
Comparison of the surface tension of 5.25% sodium hypochlorite
solution with three new sodium hypochlorite-based endodontic
irrigants. International Endodontic Journal, 45,129–135, 2012.
47. Palazzi F, Andrea Blasi, Zahed Mohammadi, Massimo Del
Fabbro, Carlos Estrela.
Penetration of Sodium Hypochlorite Modified with Surfactants into
Root Canal Dentin.
Brazilian Dental Journal (2016) 27(2): 208-216
48. Peciuliene V, Reynaud AH, Balciuniene I, Haapasalo M.
Isolation of yeasts and enteric bacteria in root-filled teeth with chronic
apical periodontitis.
International Endodontic Journal, 34, 429–434, 2001.
49. Pinheiro ET, Gomes BPFA, Ferraz CCRl.
Evaluation of root canal microorganisms isolated from teeth with
endodontic failure and their antimicrobial susceptibility.
Oral microbiology immunology 2003: 18: 100-103.
50. Radcliffe CE, Potouridou L, Qureshi R, Habahbeh N, Qualtrough
A, Worthington H, Drucker DB.
Antimicrobial activity of varying concentrations of sodium
hypochlorite on the endodontic microorganisms Actinomyces israelii,
A. naeslundii, Candida albicans and Enterococcus faecalis.
Bibliography
International Endodontic Journal, 37, 438–446, 2004.
51. Rocas I.N, Jose F. Siqueira Jr and Ka tia R. N. Santos
Association of Enterococcus faecalis With Different Forms of
Periradicular Diseases.
Journal of Endondontics. Vol. 30, No. 5, May 2004
52. Rina Verma, Sharma DS, Pathak AK.
Antibacterial Efficacy of Pastes against E Faecalis in Primary Root
Dentin: A Confocal Microscope Study.
The Journal of Clinical Pediatric Dentistry Volume 39, Number
3/2015.
53. Sathorn C, P. Parashos & H. Messer.
Antibacterial efficacy of calcium hydroxide intracanal dressing: a
systematic review and meta-analysis.
International Endodontic Journal, 40, 2–10, 2007
54. Sedgley CM, Lennan SL, Appelbe OK.
Survival of Enterococcus faecalis in root canals ex vivo.
International Endodontic Journal, 38, 735–742, 2005.
55. Spencer H.R, V. Ike and P. A. Brenna. Review:
the use of sodium hypochlorite in endodontics — potential
complications and their management.
British Dental Journal. Volume 202 No. 9 May 12 2007.
Bibliography
56. Sonja Stojicic, Slavoljub Zivkovic, Wei Qian, Hui Zhang and
Markus Haapasalo.
Tissue Dissolution by Sodium Hypochlorite: Effect of Concentration,
Temperature, Agitation, and Surfactant.
Journal of Endodontics 2010; 36:1558–1562.
57. Sundqvist G, David Figdor, Sten Persson.
Microbiologic analysis of teeth with failed endodontic treatment and
the outcome of conservative re-treatment.
Oral Surgery Oral Medicine Oral Pathology Volume 85, umber 1
1998.
58. Siddarth Nair.V, Moksha Nayak, MK Ramya .
Detection of adherence of Enterococcus faecalis in infected dentin of
extracted human teeth using confocal laser scanning microscope: An
In vitro Study.
J Pharm Bioall Sci [serial online] 2017 [cited 2019 Jan 27];9,
59. Siqueira J.F, A. G. Machado, R .M. Silveira, H. P. Lopes &
M.Deuzeda.
Evaluation of the effectiveness of sodium hypochlorite used with three
irrigation methods in the elimination of Enterococcus faecalis from
the root canal, in vitro.
International Endodontic Journal (1997) 30, 279–282.
Bibliography
60. Siqueira & Rôças.
Polymerase Chain Reaction–Based Analysis Of Microorganisms
Associated With Failed Endodontic Treatment.
Oral Surg Oral Med Oral Pathol Oral Radio Endod. 2004 Jan;
97(1):85-‐94.
61. Stuart CH, Scott A. Schwartz,Thomas J. Beeson and Christopher
B. Owatz.
Enterococcus faecalis: Its Role in Root Canal Treatment Failure and
Current Concepts in Retreatment.
Journal of Endodontics 2005; 32:93–98.
62. Stefanie Nio.
The effectiveness of heated sodium hypochlorite on Enterococcus
faecalis in infected dentinal tubules 2017.
63. Tanriverdi F, Esener T, Erganis O.
An in vitro test model for investigation of disinfection of dentinal
tubules infected with Enterococcus faecalis.
Brazilian Dental Journal 1997.
64. Tasman F, Zafer C. Çehreli, Canan Ogan and Ilker Etikan.
Surface Tension of Root Canal Irrigants.
Journal of Endodontics Vol. 26, No. 10, October 2000
Bibliography
65. Tsesis I & Shlomo Elbahary & Nuphar Blau Venezia & Eyal
Rosen Bacterial colonization in the apical part of extracted human
teeth following root-end resection and filling: a confocal laser
scanning microscopy study.
Journal of Oral Clinical Investigation 2017.
66. Tran K.T, Mahmoud Torabinejad, DMD, Shahrokh Shabahang,
Bonnie Retamozo
Comparison of Efficacy of Pulverization and Sterile Paper Point
Techniques for Sampling Root Canals.
Journal of Endodontics 2013; 39:1057–1059.
67. Tuhina Banerjee and Shampa Anupurba.
Prevalence of Virulence Factors and Drug Resistance in Clinical
Isolates of Enterococci: A Study from North India.
Journal of Pathogens Volume 2015, Article ID 692612
68. Valera M.C, Juliana de Moraes Rego, and Antonio Olavo Cardoso
Jorge.
Effect of Sodium Hypochlorite and Five Intracanal Medications on
Candida albicans in Root Canals.
Journal of endodontics Vol. 27, No. 6, June 2001
69. Vianna M.E, Brenda P. F. A. Gomes, Vanessa Bellocchio Berber,
Alexandre Augusto Zaia, Caio Cezar Randi Ferraz, and Francisco
Jose´ de Souza-Filho.
Bibliography
In vitro evaluation of the antimicrobial activity of chlorhexidine and
sodium hypochlorite.
Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004; 97:79-84.
70. Vijaykumar S, Madiraju GunaShekhar, Sura Himagiri.
In vitro effectiveness of different endodontic irrigants on the
reduction of Enterococcus faecalis in root canals.
J Clin Exp Dent. 2010; 2(4):e169-72.
71. Williamson A.E, Jared W. Cardon and David R. Drake.
Antimicrobial Susceptibility of Monoculture Biofilms of a Clinical
Isolate of Enterococcus faecalis.
Journal of Endodontics 2009;35:95–97.
72. Wang Qian-Qian, Cheng-Fei Zhang, Chun-Hung Chu and Xiao-
Fei Zhu.
Prevalence of Enterococcus faecalis in saliva and filled root canals of
teeth associated with apical periodontitis.
International Journal of Oral Science (2012) 4, 19–23.
73. Wang Z, Ya Shen, Jingzhi Ma and Markus Haapasalo.
A New Noninvasive Model to Study the Effectiveness of Dentin
Disinfection by Using Confocal Laser Scanning Microscopy.
Journal of Endodontics 2012;38:948–953
74. Wang Z, Ya Shen and Markus Haapasalo.
Effect of Smear Layer against Disinfection Protocols on Enterococcus
faecalis–infected Dentin.
Bibliography
Journal of Endodontics 2013; 39:1395–1400.
75. Zapata R.O, Clovis M. Bramante, Ivaldo Gomes de Moraes,
Norberti Bernardineli.
Confocal Laser Scanning Microscopy Is Appropriate to Detect
Viability of Enterococcus faecalis in Infected Dentin.
Journal of Endodontics 2008; 34:1198 –120.
76. Zou L, Ya Shen, Wei Li and Markus Haapasalo.
Enterococcus faecalis Biofilms Eradication by Root Canal Irrigants.
Journal of Endodontics2009; 35:711–714.
77. Zou L, Ya Shen, Wei Li and Markus Haapasalo.
Penetration of Sodium Hypochlorite into Dentin.
Journal of Endodontics 2010; 36:793–796.
78. Zou J and Nathan Shankar.
The opportunistic pathogen Enterococcus faecalis resists phagosome
acidification and autophagy to promote intracellular survival in
macrophages.
Cellular Microbiology (2016) 18(6), 831–843.
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