ENDODONTOLOGY

Original Research - 6

Comparison of the antibacterial efficiency of MTAD, 2.5% sodium hypochlorite and 2%chlorhexidine against enterococcus faecalis - An ex vivo study.
SRIKUMAR G. P. V. *# K. RAVI VARMA **# K. HARISH KUMAR SHETTY ***# VIDYA ****##

ABSTRACT
Objectives: To evaluate the antibacterial efficiency of MTAD, 2.5%NaOCl and 2%Chlorhexidine when used as root canal irrigants against enterococcus faecalis. Materials and methods: 70 extracted human mandibular single rooted premolar teeth were selected and the teeth were decoronated. An innoculum of 10lit of 24hrs pure culture suspension of enterococcus faecalis was inserted into root canals of all the teeth. After incubation for 48hrs at 37c of temperature all the teeth were randomly divided into 7 groups of 10 teeth in each group. The teeth were instrumented in step back technique and canals were enlarged till size 40. During the process of instrumentation respective root canal irrigants were used as per the groups. In Group A- only MTAD was used, Group B-only 2.5%NaOCl was used, Group C-MTAD followed by the use of 2.5%NaOCl, Group D-2.5%NaOCl followed by the use of MTAD, Group E-MTAD followed by the 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 except between 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 as root canal irrigants showed the maximum antibacterial efficiency against enterococcus faecalis followed by Group 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

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

41

ENDODONTOLOGY
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

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

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 37c. 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
42

MATERIALS AND METHODS

MTAD, 2.5%NaOCL, 2%Chlorhexidine, Sterile physiological saline, sterile distilled water, Enterococcus faecalis were the materials used in this study.

PREPARATION OF SPECIMENS
70 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 121c 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 36.5c. An innoculum of 10 lit, 24hrs pure culture suspension of E.faecalis was suspended into each canal of all the teeth by using a sterile micropipette inside the

ENDODONTOLOGY

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

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 37c 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

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

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
43

RESULTS
One 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.

ENDODONTOLOGY
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.

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

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 p H 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 anticollagenase activity .
10

DISCUSSION
Chemo-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 tissues .
4

and

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 Powder44

Mix the powder and liquid according to the manufacturers 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

ENDODONTOLOGY

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

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 nonionic 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
45

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 reddishbrown 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.

CONCLUSION
Under 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.

ENDODONTOLOGY
ILLUSTRATIONS Fig no: 1
GROUPS MEAN+/-S.D P-VALUE

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

SIGNIFICANT GROUPS AT 5% LEVEL

NON SIGNIFICANT GROUPS A Vs E, F

A. MTAD B. 2.5% NaOCl C. MTAD + 2.5%NaOCl D. 2.5%NaOCl+ MTAD E. MTAD+2% CHX F. 2.5%NaOCl+2%. CHX G. Saline

59.80+/-3.967 81+/- 4.949 26+/-2.044 38+/-3.676 60+/-4.095 63+/-4.498 115+/-10.252 P=.001 (SIGN)

A Vs B,C,D,G. B Vs A,C,D,E,F,G. C Vs A,B,D,E,F,G. D Vs A,B,C,E,F,G. E Vs B,C,D,G. F Vs B,C,D,G G Vs A,B,C,D,E,F.

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 4: No of bacterial colony forming units in Group C Fig no: 2 Barr diagram MTAD - Mixture Tetracycline Citric Acid and Detergent. NaOCl - Sodium hypochlorite. CHX - Chlorhexidine

REFERENCES
1. Vahdaty A. Pittford TR , Efficacy of Chlorhexidine in disinfecting tubules in vitro. Endod Dent Traumatol 1993;9:243-248. 2. Franklin R. Tay, Noriko Hiraishi: Reduction in antimicrobial substantivity of MTAD after initial Sodium Hypochlorite irrigation; JOE: Oct:2006:Vol 32:No:10;970-975. 3. Spangberg L, Engstrom.b : Biologic effects of dental materials;Toxicity and antimicrobial effects of Endodontic antiseptics in vitro; Oral Surgery:1973;36:856-871. 4. Seltzer S, Farber P.A : Microbiological factors in endodontics: Oral Surgery: 1994;78:634-645.

Fig 3: MTAD (Mixture Tetracycline Citric Acid and Detergent) 46

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

ENDODONTOLOGY

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

2%chlorhexidine gel in reducing intracanal bacteria; JOE:Nov 2007:Vol 33,No 11:1283-1289. 6. Matthias Zehnder; Root canal irrigants; JOE;May 2006;Vol 32;No 5:389-398. 7. Mahmoud Torabinejad, Yongbum: The effect of various concentrations of sodium hypochlorite on the ability of MTAD to remove the smear layer; JOE: April 2003:Vol 29;No 4:233240. 8. Fugen Tasman, Zafer C.Cehreli; Surface tension of root canal 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 solutions in narrow root canals; Oral Surgery; May:1982;524-526. 10. Shabahang S,Paresmail M, Torabinejad M : Invitro antimicrobial efficacy of MTAD and sodium hypochlorite; JOE:2003;29:450-452. 11. Franklin R. Tay, Annalisa Mazzoni: Potential Iatrogenic Tetracycline Staining of Endodontically treated teeth via NaOCl/MTAD irrigation: A Preliminary report: JOE: April 2006;Vol 32;No 4:354-358. 12. Ahmed Zamany, Spangberg; An effective method of inactivating chlorhexidine: OOO: May 2002;Vol 93; No 5:617-620.

47