ENDODONTOLOGY Original Research - 4

Comparison of antimicrobial efficacy of biopure MTAD

and 2.5% sodium hypochlorite irrigation in infected root
canals following single visit endodontics - An in vivo study

SANDEEP SINGH *#
SUSHMA KRISHNA **##
SHASHI RASHMI ACHARYA ***#
P. SUGANDHI RAO****##
VASUDEV BALLAL*****#

ABSTRACT
Aims and Objectives: To effectively clean the root canal system a disinfecting agent must penetrate difficult to
reach areas and kill the microorganisms with minimal damage to the host tissues. The purpose of this in-vivo
investigation was to evaluate the antimicrobial efficacy of irrigating solutions 2.5% Sodium Hypochlorite and
BioPure MTAD in infected root canals following single visit endodontic procedure.
Materials and Methods: Twenty single canal anterior and premolar teeth that had pulpal or periapical pathology
were randomly divided in to two groups (n=10). Group I cases were irrigated with 2.5% Sodium Hypochlorite
only during biomechanical preparation , Group II cases were initially irrigated with 0.9% saline during the complete
biomechanical preparation, 5 minute final irrigation was done with BioPure MTAD. Microbiological samples for
culture were taken immediately after accessing the canal and following irrigation with test solutions.
Conclusion: The results of this study conclude that the use of 2.5% Sodium Hypochlorite resulted in significantly
greater reduction of anaerobic organisms when compared to BioPure MTAD.
Keywords: Irrigants, Sodium hypochlorite, BioPure MTAD, Antimicrobial efficacy

INTRODUCTION To determine the influence of infection at the

For more than 509 bacterial species found in
the oral cavity, factors such as nutrient availability,
low oxygen tension in the root canals with a
necrotic pulp and bacterial interactions ecologically
determine the criteria for the selection of the
microbiota in root canals. Selective growth
conditions lead to the predominance of facultative
and strict anaerobic microorganisms that survive
and multiply easily, cause resistant infections,
stimulate periapical bone resorption.
time of root canal therapy on the treatment outcome
of patients, Sjogren et al, 1 compared the success
rate of teeth with negative cultures at the time of
obturation to those that had positive bacterial
cultures before the same procedure. They reported
a significantly higher success rate when teeth were
free of bacteria (94% vs 68% ). The results agree
with the findings of Bystrom et al, 2
who also
showed improved clinical success rates after
effective disinfection of root canals.

* Post Graduate Student, ** Post Graduate Student, *** Professor and Head, **** Professor, ***** Reader, # Department of Conservative Dentistry and Endodontics,
Manipal College of Dental Sciences, Manipal. ## Department of Microbiology, KMC, Manipal

29
ENDODONTOLOGY
Orstavik and Haapasalo 3 showed the
importance of removal of the smear layer and the
presence of patent dentinal tubules for the
disinfecting effect of intracanal medications. In
addition to smear layer removal, an ideal root canal
irrigant should be able to disinfect the root canal
system, allow penetration of antimicrobial agents
present in the solution into the dentinal tubules and
to have sustained antibacterial effect after use. It is
important that it should not initiate any antigenic
reaction, and also it should be nontoxic and
noncarcinogenic.
Sodium hypochlorite has long been the irrigant
of choice for nonsurgical endodontic procedures.4
Sodium hypochlorite dissolves necrotic and vital
tissue, has antimicrobial activity, and aids as a
lubricant in the canal 4 6
It also has minimal
clinical toxicity when kept within the confines
of the canals. However, NaOCl is extremely toxic
to the periapical tissues if it is injected beyond the
apex of the tooth.7
A final flush with EDTA followed by NaOCl
seems to remove the smear layer; however, EDTA
is not antibacterial and has the ability to erode
dentin if the exposure time exceeds 1 minute. 8
The use of RC-Prep (Premier Dental Products,
Norristown, PA) is an accepted endodontic
protocol. 9 It can ease instrumentation by softening
dentin and wetting root canal walls. EDTA is a
chelating agent that helps remove the smear layer,
thus facilitating the entry of antibacterial irrigation
solutions and intracanal medicines into the dentinal
tubules. 10
Torabinejad et al. 11 investigated the effect of
a new root canal irrigant, BioPure MTAD, which is
30
SANDEEP SINGH, SUSHMA KRISHNA, SHASHI RASHMI ACHARYA, P. SUGANDHI RAO, VASUDEV BALLAL
a mixture of a tetracycline isomer, an acid, and a
detergent, on the removal of the smear layer from
the instrumented root canal walls. Their results
showed that BioPure MTAD is effective as a final
rinse to remove the smear layer with minimal
erosive changes on the surface dentin. In another
study, Shabahang et al. 12 investigated the ability of
BioPure MTAD to disinfect contaminated root canal
with whole saliva and compared its efficacy to that
of NaOCl. Based on their results, it seems that
BioPure MTAD is significantly more effective than
5.25% NaOCl in eradicating bacteria from infected
root canals. In addition, Beltz et al. 13
found that
the solubilizing effects of BioPure MTAD on pulp
and dentin were somewhat similar to those of
EDTA. The recommended regimen of BioPure
MTAD is initial irrigation of the canals by 1.3%
NaOCl during instrumentation and final 5 minute
irrigation with BioPure MTAD. But, this regimen
has always had doubts that the antimicrobial effect
which is achieved is of BioPure MTAD or Sodium
Hypochlorite.
The purpose of this study was to evaluate the
in vivo Antimicrobial efficacy of irrigating solutions
2.5% Sodium Hypochlorite and BioPure MTAD in
infected root canals following single visit
endodontic procedure.
MATERIALS AND METHODS
Screening
Health histories of all participating patients
with a complete written informed consents were
obtained. Patients who received antibiotic
treatment in the previous 3 months, or had any
systemic disease were excluded from the study.
Twenty anterior and premolar teeth with
Weines Type I root canal system that has pulpal
ENDODONTOLOGY COMPARISON OF ANTIMICROBIAL EFFICACY OF BIOPURE MTAD AND 2.5% SODIUM HYPOCHLORITE IRRIGATION IN INFECTED
ROOT CANALS FOLLOWING SINGLE VISIT ENDODONTICS. AN IN VIVO STUDY

or periapical pathology from patients aged 18 to the approximate length of the tooth. A pre-sterile
55 years were isolated. The periapical and pulpal paper point (DENTSPLY, MAILLEFFER)
pathology was assessed with the use of intra oral premoistened with sterile saline to provide a
periapical radiographs and pulp vitality tests. pooling effect for collection of bacteria was
introduced into the root canal and kept in the canal
The cases were randomly divided in to 2
for 1 minute and then dropped into a test tube
groups:- n=10
containing the culture medium. This constituted the
Group I:- 2.5% Sodium Hypochlorite was sample for the Pre irrigant culture (S1).
used as irrigation during biomechanical preparation
BIOMECHANICAL PREPARATION
with final application of RC-Prep paste in the root
All canals were prepared with rotary Protaper
canal.
files up to size F3.
Group II:- 0.9% saline was used as an
Group I :- Each instrument change was
irrigation during complete biomechanical
accompanied by irrigation with 2ml 2.5% Sodium
preparation followed by final irrigation with
Hypochlorite. RC prep was quoted on the last file
Biopure MTAD.
used and was kept in the canal for one minute
PREPARATION OF ACCESS CAVITY followed by 5ml final rinse of 2.5% Sodium
After rubber dam isolation, the tooth, clamp Hypochlorite solution.
and its surroundings were cleaned with 2.5%
Group II :- Each instrument change was
Sodium Hypochlorite solution for one minute. An
accompanied by irrigation with 2ml 0.9% saline.
initial opening was made 1 -2 mm into the dentin
After the completion of the instrumentation 5
with a sterile tapering fissure bur at high speed.
minute final rinse of 5ml BioPure MTAD was done.
The tooth was again swabbed with 2.5% Sodium
Hypochlorite solution for 10 seconds. Sodium After the instrumentation is completed in
Hypochlorite solution was deactivated with 5% Group I, the canal is flushed with 5% Sodium
Sodium thiosulfate. Thiosulfate to neutralize any residual effect of
NaOCl, whereas in Group II, following
Proceeding with the penetration of the pulp
biomechanical preparation canal was irrigated with
chamber, a sterile surgical length No 2 or No 4
0.9% Saline. All forms of irrigation was
tungsten carbide round bur was used at slow speed,
accomplished by using 30 gauge needle.
working the bur from the inside to the outside to
expose the entire pulp chamber. Root canal walls were gently filed and a
postinstrumentation sample was collected from the
COLLECTION OF SAMPLES
canal as aforementioned. This constituted the
Two sterile test tubes containing Robertsons
sample for the Post irrigant culture (S2).
Cooked Meat broth (RCM) as culture medium was
obtained for each case.

A preoperative radiograph was used to assess

31
ENDODONTOLOGY SANDEEP SINGH, SUSHMA KRISHNA, SHASHI RASHMI ACHARYA, P. SUGANDHI RAO, VASUDEV BALLAL

MICROBIOLOGICAL PROCESSING
FOR THE GROWTH OF ANAEROBIC
MICROORGANISMS
Isolation and identification of anaerobes
z Transported samples were plated on
neomycin blood agar and sheep blood agar by semi
quantitative technique . Each agar plate was divided
in to 4 quadrants and using a calibrations loop of
6mm diameter holding 0.01ml of the transport
media. After streaking, plates were incubated for
37 degrees for 72 hours in anaerobic jar (dynox). RESULTS
Anaerobiasis were maintained through out and Statistical analysis was done using Mann-
ATCC strain of Bacteroides fragilis 28285 were to Whitney U Test. 2.5% Sodium Hypochlorite
be tested grown with every jar holder. The number resulted in significantly greater reduction of
of viable organisms were counted and expressed anaerobic organisms when compared to BioPure
as colony forming units. MTAD (p < 0.005). All the pretreatment samples
showed anaerobic growth which was >105 colony
Group I (2.5% NaOCl) forming units. In Group I ( 2.5% Sodium
Hypochlorite) 9 out of 10 samples did not show
any post treatment growth, where as in Group II
(BioPure MTAD) only 3 out of 10 samples did not
show any post treatment growth.

DISCUSSION
It is believed that thorough debridement before
sealing the root canal system is the key to long-
term success of endodontic therapy.
Chemomechanical preparation is one of the most
important phases of endodontic treatment The best
Group II ( BioPure MTAD)
known and most commonly used of these
chemicals is Sodium hypochlorite (NaOCI) Its
established clinical efficacy results from its ability
to dissolve necrotic tissue and organic remnants
and its antimicrobial activity. 14

A culture dependent approach was used in

the present investigation. It is one of the most
reliable methods of detecting viable bacteria,

32
ENDODONTOLOGY COMPARISON OF ANTIMICROBIAL EFFICACY OF BIOPURE MTAD AND 2.5% SODIUM HYPOCHLORITE IRRIGATION IN INFECTED
particularly when samples are taken immediately
after antimicrobial treatment where viability may
not be ascertained by most of the culture
independent methods. 15
However, it is worth pointing out that a
negative culture does not imply sterility. Indeed, a
negative culture usually means that cultivable
bacterial populations were reduced to levels below
the detection ability of culture dependent methods
& that these levels can be compatible to
periradicular healing in most of the cases.
All root canals in this study harbored bacteria
before treatment, confirming the strong correlation
between bacteria and apical pathology. Sodium
Hypochlorite gold standard of irrigation of root
canals was used in the comparison. It has a broad
spectrum antimicrobial activity. 16
The main advantage of NaOCl is ability to
dissolve necrotic tissues. The combined use of
various preparations of EDTA and NaOCl is
recommended for smear layer removal and has
been shown to be more effective than NaOCl alone.
17
In the present study RC-Prep was used after
instrumentation to aid in smear layer removal.
BioPure MTAD a new irrigant which has 3%
Doxycycline 150mg/5ml, 4.25 % citric acid ; 0.5%
polysorbate 80 detergent. Torabinejad et al reported
that BioPure MTAD irrigation regime has superior
antibacterial efficacy when compared to Sodium
Hypochlorite against E Faecalis. 18
In the present study, Group II (BioPure MTAD)
involved initial irrigation with saline during the
instrumentation so as to determine the sole efficacy
of BioPure MTAD as a 5 minute final rinse irrigant
in reducing the microbial load.
33
ROOT CANALS FOLLOWING SINGLE VISIT ENDODONTICS. AN IN VIVO STUDY
Using the direct method, a study made by
measuring zones of inhibition on agar plates has
shown that BioPure MTAD was as effective as
5.25% NaOCl in eradicating bacteria. 19
It is known that bacteria organized as biofilms
have been found in inaccessible areas of necrotic
pulp space and on root surfaces and cemental
lacunae.20,21,22 In the present in vivo study the
inferior result outcome of BioPure MTAD can be
attributed to its ineffectiveness in the presence of
biofilm.
Dunavant et al,23 have shown that only NaOCl
is highly effective against the whole bacteria
population organized in biofilm and that its activity
is strictly correlated to its concentration. The
percentage of bacteria killed by BioPure MTAD in
the same study was only 16.08%. These findings
could be correlated with the bacteriostatic activity
of doxycycline, according to Torabinejad et al,24
the action against bacteria of these irrigants should
be caused by the doxycycline present in the
mixture, which is a bacteriostatic antibiotic and it
cannot kill bacteria.
Kho and Baumgartner, 25 compared the action
of 1.3% NaOCl/BioPure MTAD versus 5.25%
NaOCl/17% EDTA in the apical 5mm of teeth
infected with E faecalis. After irrigation, the root
canal apexes were resected and pulverized to
expose E faecalis in dentinal tubules. In this study,
there were no differences in antimicrobial efficacy
for irrigation with 5.25% NaOCl/17% EDTA versus
1.3% NaOCl/BioPure MTAD in 5-mm apical
dentinal tubules infected with E faecalis.
In another study, Clegg et al. 26
reported that
only 6% and 3% NaOCl were capable of disrupting
ENDODONTOLOGY
and removing the biofilm; 1.6% NaOCl/BioPure
MTAD was capable of disrupting but not
eliminating bacteria completely.
Johal et al,27 found no growth of E Faecalis in
root canals irrigated with 5.25% NaOCl/15%
EDTA, while 50% of the canals irrigated with 1.3%
NaOCl/Biopure MTAD demonstrated the growth
of E. Faecalis. The difference was statistically
significant. According to a study by Luciano
Giardino et al, 28 BioPure MTAD, shows a better
action, but the goal of the total disappearance of
the biofilm is reached only after 30 to 60 minutes
of irrigation. This is too long for a clinical use of
this irrigation regime.
BioPure MTAD has only 45% solubilizing
effect on organic tissue like pulp where as 5.25%
NaOCl has more than 90% effective in dissolving
organic tissue such as pulp. 29 This fact would have
been also responsible for inferior results shown by
BioPure MTAD.
Noteworthy, inactivation of Biopure MTAD
was not accomplished before post treatment
sample was obtained from the root canal. As till
date there is no agent capable of inactivating all its
content.30 But, it is known that reactive oxygen
species release from sunflower roots inactivates
tetracycline antibiotics via their conversion to
quinone products but these products are neither
marketed nor manufactured. 31
The previous reported success of Biopure
MTAD lies in the fact that the initial irrigation was
done with Sodium Hypochlorite and BioPure
MTAD might not be achieving much on its own
inside the root canal.
To conclude, in the present in vivo study, 2.5%
34
SANDEEP SINGH, SUSHMA KRISHNA, SHASHI RASHMI ACHARYA, P. SUGANDHI RAO, VASUDEV BALLAL
Sodium Hypochlorite irrigation was proven
superior to sole irrigation regime of BioPure MTAD.
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