Comparison of the Antibacterial Efficiency of MTAD, NaOCl, CHX Against Enterococcus Faecalis

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ENDODONTOLOGYENDODONTOLOGYENDODONTOLOGYENDODONTOLOGYENDODONTOLOGY

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Comparison of the antibacterial efficiency of MTAD,2.5% sodium hypochlorite and 2%chlorhexidine againstenterococcus faecalis - An ex vivo study.

SRIKUMAR G. P. V. *#K. RAVI VARMA **#K. HARISH KUMAR SHETTY ***#VIDYA ****##

* Post Graduate Student, ** Professor and HOD, *** Professor, **** Reader, # Department of Conservative Dentistry & Endodontics, ## Department of Microbiology,Yenepoya Dental College, Deralakatte, Mangalore, Karnataka

ABSTRACTObjectives: To evaluate the antibacterial efficiency of MTAD, 2.5%NaOCl and 2%Chlorhexidine when used asroot canal irrigants against enterococcus faecalis.

Materials and methods: 70 extracted human mandibular single rooted premolar teeth were selected and the teethwere decoronated. An innoculum of 10µlit of 24hrs pure culture suspension of enterococcus faecalis was insertedinto root canals of all the teeth. After incubation for 48hrs at 37°c of temperature all the teeth were randomlydivided into 7 groups of 10 teeth in each group. The teeth were instrumented in step back technique and canalswere enlarged till size 40. During the process of instrumentation respective root canal irrigants were used as perthe groups. In Group A- only MTAD was used, Group B-only 2.5%NaOCl was used, Group C-MTAD followedby the use of 2.5%NaOCl, Group D-2.5%NaOCl followed by the use of MTAD, Group E-MTAD followed bythe use of 2%Chlorhexidine,Group F-2.5%NaOCl followed by 2%Chlorhexidine, Group G-only saline was used.Results were statistically analysed using One way ANOVA and LSD Post Hoc test.

Results: There was a statistically significant difference in the antibacterial efficiency between all the groups exceptbetween group A with group E and group F.

Conclusion: Group C where MTAD was used as initial rinse followed by the use of 2.5%NaOCl as final rinse asroot canal irrigants showed the maximum antibacterial efficiency against enterococcus faecalis followed byGroup D, Group A, Group E, Group F, Group B, Group G.

Key words: Irrigants, MTAD, Sodium hypochlorite, Chlorhexidine, Enterococcus faecalis.

Abbreviations: MTAD - Mixture Tetracycline Citric Acid and Detergent. NaOCl – Sodium hypochlorite. CHX –Chlorhexidine.

INTRODUCTION:The success of root canal therapy is dependent

on efficient cleaning and shaping of the canals with

suitable irrigants and complete obturation of the

canal system. Microbial infection is the most

common reason for root canal therapy1.

Sodium hypochlorite, EDTA and

Chlorhexidine were used as irrigants in regular

endodontic practice, but they do not satisfy all the

properties of an ideal root canal irrigant.

The recent introduction of MTAD( Mixture

Original Research - 6

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Tetracycline Citric Acid and Detergent)

commercialized as Biopure MTAD( Dentsply Tulsa

Dental, Tulsa,OK), an endodontic irrigant, which

represents an innovative approach in simultaneous

removal of endodontic smear layer and complete

disinfection of root canals. It has been shown to be

clinically effective and biocompatible, with

potential antibacterial substantivity.2

MATERIALS AND METHODSMTAD, 2.5%NaOCL, 2%Chlorhexidine,

Sterile physiological saline, sterile distilled water,

Enterococcus faecalis were the materials used in

this study.

PREPARATION OF SPECIMENS70 extracted human mandibular single rooted

premolar teeth, non carious with matured, closed

apices were selected. Each teeth were radiographed

to confirm for the presence of a single canal and

only those teeth with single canal were selected.

All the teeth were decoronated with a diamond

disc using a low speed straight hand piece, so as to

standardize roots of all the teeth approximately

of the same length. Working lengths for all the teeth

were determined using a size 10 K-file (Dentsply/

Maillefer,Tulsa,Okla) and radiographs were taken

and recorded.

Then all the teeth were sterilized in an

autoclave at a temperature of 121°c under 15 lbs

of pressure for 20 minutes.

The bacterial strain used in the present study

was E.faecalis(ATCC 29212). It was grown in Brain

Heart Infusion(BHI) broth for 24 hrs at 36.5°c. An

innoculum of 10 µlit, 24hrs pure culture suspension

of E.faecalis was suspended into each canal of all

the teeth by using a sterile micropipette inside the

laminar air flow under fully aseptic conditions.

Later each tooth was transferred carefully into

a test tube containing 4ml of sterile physiological

saline and incubated for 48hrs at temperature of

37°c. After incubation, the teeth were removed

from the test tubes and were rinsed thoroughly with

sterile physiological saline.

All the teeth were randomly divided into 7

groups of 10 teeth in each group followed by

mounting of all the teeth in polyvinyl siloxane

impression material (Reprosil Heavy body,

Dentsply/Caulk) till the cervical line.

GROUP A: Here only MTAD was used as

root canal irrigant.

As per the working lengths determined, Root

canals of all the teeth were instrumented with a

step back technique and the canals were enlarged

using K-file (Denstply/Maillefer,Tulsa,Okla) upto

size 40, And during the process of instrumentation,

canals were irrigated with 2ml of MTAD per tooth.

Following completion of instrumentation, a final

rinse of each root canal was carried out by using

3ml of MTAD for 5 minutes.

Then each canal was irrigated with 4ml of

sterile distilled water followed by drying the canals

with sterile paper points. After accomplishing the

removal of excess moisture from the canal, a dry

sterile standardized paper point of size 40 was kept

in the canal to the full working length for 10

seconds.

Later the paper point was taken out and

transferred into a test tube of 2ml SPS( Sterile

Physiological Saline) and was vortexed for 1 min.

Then 1:10 serial dilutions were obtained from

SRIKUMAR G. P. V., K. RAVI VARMA , K. HARISH KUMAR SHETTY, VIDYA

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each sample(test tube) and bacterial culture was

carried out using a standard Loop technique for

semiquantitative culture on 5% sheep blood agar

and the inoculated plates were incubated at 37°c

for 48hrs under aerobic conditions.

This entire procedure was carried out in

complete aseptic conditions. Finally bacterial

colonies were counted using a colony counter.

GROUP B: Here 5ml of 2.5% sodium

hypochlorite alone was used as root canal irrigant

per canal. 2ml of 2.5%NaOCl during the process

of instrumentation and 3ml as final rinse. The entire

procedure was carried out just similiar to that of in

Group A, except that instead of MTAD, 2.5%

NaOCl was used as root canal irrigant.

GROUP C: The same procedure was carried

out here also as described for Group A, except that

here during the process of instrumentation 2ml of

MTAD per canal was used as irrigant and following

the completion of instrumentation 3ml of MTAD

was used as initial rinse for 2 min.

Then the canal was thoroughly irrigated with

4ml of sterile distilled water for 2 min, then final

rinse was carried out using 5ml of 2.5% NaOCl

for 5 min.

GROUP D: The same procedure was carried

out as described for Group A, except that 2ml of

2.5% NaOCl was used as irrigant during the process

of instrumentation followed by use of 5ml of 2.5%

NaOCl as initial rinse for 2 min. Then the canal

was thoroughly irrigated with 4ml of sterile distilled

water for 2min and then 5ml of MTAD was used as

a final rinse for 5 min.

GROUP E: The same procedure was carried


MTAD was used as irrigant during the process of

instrumentation followed by the use of 3ml of

MTAD as an initial rinse for 2 min. Then the canal

was thoroughly irrigated with 4ml of sterile distilled

water for 2min followed by the use of 5ml of 2%

chlorhexidine as a final rinse for 5 min.

GROUP F: The same procedure was carried


2.5% NaOCl was used as irrrgant during the process

of instrumentation followed by the use of 3ml of

2.5% NaOCl as an initial rinse for 2 min.

The canal was thoroughly irrigated with 4ml

of sterile distilled water for 2 min, and 5ml of 2%

chlorhexidine was used as a final rinse for 5 min.

GROUP G: The same procedure was carried


sterile physiological saline was used as an irrigant

during the process of instrumentation followed by

the use of 5ml of saline for 5 min as a final rinse.

RESULTSOne plate per group was selected randomly

by lottery method for counting the Colony forming

units (C.F.U)

The results were tabulated and statistically

analysed.

Mean and standard deviation were estimated

for all the groups and One way ANOVA shows

that there is significant difference between the root

canal irrigants. To find exactly which root canal

irrigant differs from the other, LSD (Least Significant

Difference) Post Hoc test was done and the table is

listed in Page No:21 and Barr diagram in Page

No:22.

COMPARISON OF THE ANTIBACTERIAL EFFICIENCY OF MTAD,2.5% SODIUM HYPOCHLORITE AND 2%CHLORHEXIDINE AGAINST ENTEROCOCCUS FAECALIS-AN EXVIVO STUDY.

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The results indicates that group C (MTAD as

initial rinse followed by the use of 2.5%NaOCl as

final rinse) shows the maximum efficiency aganist

enterococcus faecalis, followed by group D ( use

of 2.5% NaOCl as initial rinse and MTAD as final

rinse), followed by group A (MTAD alone was used

as irrigant), followed by group E (use of MTAD as

initial rinse and 2% chlorhexidine as final rinse),

followed by group F (use of 2.5% NaOCl as initial

rinse and 2% chlorhexidine as final rinse), followed

by group B( only 2.5% NaOCl was used as irrigant)

and the least effective is the control group G where

only saline was used as irrigant.

DISCUSSIONChemo-mechanical preparation is of

paramount importance for the success of

endodontic treatment.

Sodium hypochlorite has the ability to dissolve

necrotic pulp tissue, organic remnants and also

exhibits potential antibacterial activity3. But it has

certain adverse effects such as corrosion of

endodontic instruments and its inability to

differentiate between the necrotic and vital tissues

when comes in contact with periapical tissues4.

Chlorhexidine is a cationic bisbiguanide. It is

bacteriostatic at lower concentrations and

bactericidal at higher concentrations

(2%Chlorhexidine) and shows the property of

substantivity5.Despite its usefulness as a root canal

irrigant, it cannot be advocated as the main irrigant

in standard endodontic procedures as it lacks the

property to dissolve necrotic tissue remnants and

fails to remove smear layer6, so it can be advocated

only as a final irrigant.

MTAD is commercially available as Powder-

Liquid system. Part A is liquid and is supplied in

syringes(5ml,20ml-single, multiple doses).It

contains 4.25% citric acid and 0.5% polysorbate

80 detergent( Tween 80).

Its low PH 2.157 contributes to its role as a

calcium chelator, thereby causing root surface

demineralization thus helps in the removal of smear

layer.

Tween 80(polyoxyethylene sorbitan

monooleate), is a detergent present in MTAD and

is a nonionic surfactant, helps in reducing the suface

tension of distilled water, NaOCl and EDTA,

Thereby enhancing the flow and penetration of

irrigating solutions like MTAD deeper into the

dentinal tubules.8 It has a pH of 7.0 and is a

biologically acceptable material 9.

Part B is powder supplied in bottles (single,

multiple doses-150mgs,600mgs).

It contains Doxycycline hyclate which is a

broad spectrum antibiotic effective against a wide

range of microorganisms. It is bacteriostatic and

shows the property of substantivity and

anticollagenase activity10.

Mix the powder and liquid according to the

manufacturer’s instructions following which the

final product of BioPure MTAD will be ready to

use.

In Group C and Group D, we could identify

the formation of a brown coloured solution when

MTAD and 2.5% NaOCl comes in contact with

each other when used as irrigating solutions, even

though between these two chemical irrigants, sterile

distilled water was used as an intermediate irrigant.

It was reported2 that when MTAD was used


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as an initial rinse followed by the use of various

concentrations of NaOCl ( 2.6%,5.25%) as final

rinses, in the presence of light or in the absence of

light, brownish discolouration was noticed at the

point of contact of these two solutions on teeth

and it is suggested to be caused by the dentinal

absorption and release of doxycycline present in

MTAD solution.

Bench top reproductions of the phenomenon

revealed that the reaction between MTAD and

NaOCl is a Redox reaction.11

Franklin R.Tay, Noriko Hiraishi2 demonstrated

that the use of 1.3% NaOCl as an initial rinse

followed by the use of MTAD as a final rinse result

in a 30% reduction in the antimicrobial

substantivity of MTAD, but it was seen that erosion

of dentinal tubules was less when compared to that

when MTAD was used as an initial rinse.

In our study when we compared the

antibacterial efficiency of Group A with Group E,

their was no significant difference even though in

Group E we used MTAD, a bacteriostatic agent as

an initial rinse and 2% chlorhexidine, a bactericidal

agent as a final rinse and the reason for their lack

of antibacterial efficiency may be that 2%

chlorhexidine completely gets inactivated in the

presence of any detergent and as MTAD contains

detergent in the form of Tween 80, an nonionic

surfactant. It is reported that in the presence of non-

ionic surfactant, even 2% chlorhexidine gets

inactivated5,12,.

In our study when we compared the

antibacterial efficacy of Group B with Group F,

there was significant difference and Group F has a

better antibacterial combination than Group B, and

it may be due to the combined antibacterial

efficiency of 2.5% NaOCl and 2% chlorhexidine.

Matthias Zehnder reported6 that when sodium

hypochlorite was used as initial irrigant and in case

if it remains in the canal, the subsequently added

chlorhexidine will precipitate in the form of reddish-

brown mass.

Copious amounts of chlorhexidine irrigant

should thus be administered to secure proper action

of the chlorhexidine and to prevent discolouring

of the tooth by these precipitates.

In our study there was significant difference

in the antibacterial efficiency of Group A with that

of Group B, which shows that MTAD a newly

introduced root canal irrigant used in Group A is

highly potent antibacterial agent against

enterococcus faecalis than 2.5% NaOCl used in

Group B which was routinely used in endodontics.

CONCLUSIONUnder the limitations of this study the

following conclusion can be inferred:

(1) MTAD as an initial rinse followed by the

use of 2.5% NaOCl as final rinse, The antibacterial

efficiency against E.faecalis is much higher and

better than:

(2) MTAD as a final rinse following the use of

2.5% NaOCL as the initial rinse(or)

(3) Using MTAD alone as in Group:A (or)

Using 2.5%NaOCl alone as in Group:B (or)

(3) Using a combination of MTAD as initial

rinse followed by the use of 2%Chlorhexidine as

final rinse (or) using 2.5% NaOCl as initial rinse

followed by use of 2%Chlorhexidine as final rinse.

Further studies are required to prove the efficiency

of MTAD in clinical situations.


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One way ANOVA shows there is significant difference between the root canal irrigants. To find exactly which root canal irrigant

differs from the other, LSD ( Least Significant Difference) Post Hoc test was done.

MTAD - Mixture Tetracycline Citric Acid and Detergent. NaOCl - Sodium hypochlorite. CHX - Chlorhexidine

Fig no: 2 Barr diagram

MTAD - Mixture Tetracycline Citric Acid and Detergent.NaOCl - Sodium hypochlorite. CHX - Chlorhexidine

ILLUSTRATIONSFig no: 1

GROUPS MEAN+/-S.D P-VALUE SIGNIFICANT GROUPS AT NON SIGNIFICANT

5% LEVEL GROUPS

A. MTAD 59.80+/-3.967 A Vs B,C,D,G. A Vs E, F

B. 2.5% NaOCl 81+/- 4.949 B Vs A,C,D,E,F,G.

C. MTAD + 2.5%NaOCl 26+/-2.044 P=.001 (SIGN) C Vs A,B,D,E,F,G.

D. 2.5%NaOCl+ MTAD 38+/-3.676 D Vs A,B,C,E,F,G.

E. MTAD+2% CHX 60+/-4.095 E Vs B,C,D,G.

F. 2.5%NaOCl+2%. CHX 63+/-4.498 F Vs B,C,D,G

G. Saline 115+/-10.252 G Vs A,B,C,D,E,F.

Fig 3: MTAD (Mixture Tetracycline Citric Acid and Detergent)

Fig 4: No of bacterial colony forming units in Group C

REFERENCES1. Vahdaty A. Pittford TR, Efficacy of Chlorhexidine indisinfecting tubules in vitro. Endod Dent Traumatol1993;9:243-248.

2. Franklin R. Tay, Noriko Hiraishi: Reduction in antimicrobialsubstantivity of MTAD after initial Sodium Hypochloriteirrigation; JOE: Oct:2006:Vol 32:No:10;970-975.

3. Spangberg L, Engstrom.b: Biologic effects of dentalmaterials;Toxicity and antimicrobial effects of Endodonticantiseptics in vitro; Oral Surgery:1973;36:856-871.

4. Seltzer S, Farber P.A: Microbiological factors inendodontics: Oral Surgery: 1994;78:634-645.

5. Ching S. Wang, Ronald R. Arnold: Clinical efficiency of


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2%chlorhexidine gel in reducing intracanal bacteria; JOE:Nov2007:Vol 33,No 11:1283-1289.

6. Matthias Zehnder; Root canal irrigants; JOE;May 2006;Vol32;No 5:389-398.

7. Mahmoud Torabinejad, Yongbum: The effect of variousconcentrations of sodium hypochlorite on the ability of MTADto remove the smear layer; JOE: April 2003:Vol 29;No 4:233-240.

8. Fugen Tasman, Zafer C.Cehreli; Surface tension of rootcanal irrigants: JOE: Oct 2000;Vol 26; No 10;586-587.

9. Marvan Abou-Rass, Frank J.Patonai; The effects of

decreasing surface tension on the flow of irrigating solutionsin narrow root canals; Oral Surgery; May:1982;524-526.

10. Shabahang S,Paresmail M, Torabinejad M: Invitroantimicrobial efficacy of MTAD and sodium hypochlorite;JOE:2003;29:450-452.

11. Franklin R. Tay, Annalisa Mazzoni: Potential IatrogenicTetracycline Staining of Endodontically treated teeth viaNaOCl/MTAD irrigation: A Preliminary report: JOE: April2006;Vol 32;No 4:354-358.

12. Ahmed Zamany, Spangberg; An effective method ofinactivating chlorhexidine: OOO: May 2002;Vol 93; No5:617-620.


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Comparison of the Antibacterial Efficiency of MTAD, NaOCl, CHX Against Enterococcus Faecalis