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
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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
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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
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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
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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,
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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
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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
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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.