A COMPARISON BETWEEN THE

ANTIBACTERIAL AND CLEANING

EFFECTS OF MTAD AND DIFFERENT

ANTIBIOTIC SOLUTIONS

(AN IN VITRO STUDY)

A thesis submitted to the Faculty of Oral and Dental

Medicine, Cairo University.

In partial fulfillment of the requirements for Master

Degree in Endodontics

By

Dalia Mohamed Makawi

BDS October 6th

University

2007

Faculty of Oral and Dental Medicine

Cairo University

2012

SUPERVISORS

Prof. Dr. Maged M. Negm

Professor of Endodontics

Faculty of Oral and Dental Medicine

Cairo University.

Dr. Yousra M. Nashaat

Lecturer in Endodontics

Faculty of Oral and Dental Medicine

October 6th

University.

Dr. Salwa I. Youssef

Lecturer in Bacteriology

Faculty of Medicine

October 6th

University.

ACKNOWLEDGEMENTS

My deepest gratitude, thanks, appreciation and respect go to Prof. Dr.

Maged M. Negm. Professor of Endodontics, Faculty of Oral and Dental

Medicine, Cairo University, for his unsurpassed kindness, thoughtful

guidance, extraordinary decency, unlimited help, care and support.

Countless thanks to Dr. Yousra M. Nashaat, Lecturer in Endodontics

Faculty of dDentistry 6th

October University, for her unlimited kindness,

care, concern , her valuable cooperation and helpful remarks.

Many thanks to Dr. Salwa I. Youssef, Lecturer in bacteriology, Faculty

of Medicine, 6th October University, for her friendly spirit, great help and

care.

DEDICATION

This work is dedicated to my beloved family; my husband, my son, my

dear parents, my sister and my brother, for their unlimited support. No

words can describe my gratitude to them.

"Love you all, may God bless you"

LISTS OF CONTENTS

Introduction………………………………………… 1

Review of Literature

BioPure MTAD …………………………….... 2

Antibiotics …………………………………... 17

Antimicrobial effect of different irrigants on E. faecalis 24

Smear layer removal and cleanliness of dentinal walls 31

Aim of the Study……………………………………. 35

Materials and Methods

Materials ……………………………………. 36

Methods …………………………………….. 40

Statistical analysis ……………………………. 50

Results……………………………………………… 51

Discussion…………………………………………… 89

Summary and Conclusion…………………………….. 99

References…………………………………………... 103

Arabic Summary…………………………………….. 119

List of Figures

Fig.1. Unasyn 1500 mg…………………………………… 36

Fig.2. Cefobid 1 gm………………………………………. 37

Fig.3. Zithromax 1200 mg………………………………… 37

Fig.4. Tavanic 500 mg…………………………………………….. 38

Fig.5. Doxymycin 100 mg…………………………………………. 38

Fig.6. BioPure MTAD…………………………………………….. 39

Fig.7. Sterile micropipettes used for bacterial transfer to the root

canals prepared lumens…………………………………………… 41

Fig.8. Serial bacterial dilution……………………………………. 45

Fig.9. Muller Hinton agar plates…………………………………. 45

Fig.10. Incubator used for plates storage………………………… 45

Fig.11. Vortex in which test tubes were vortexed for 20 seconds.. 46

Fig.12. Standardized cropping of images…………………………. 49

Fig.13. Total surface area of all images measured by image J

software…………………………………………………………….. 49

Fig.14. Adjusting the threshold to measure surface area of dentin

debris……………………………………………………………….. 49

Fig.15. Bar chart representing the bacterial count within group 1 before

and after application of treatment…………………………………. 52

Fig.16. Bar chart representing the bacterial count within group 2 before

and after application of treatment…………………………………. 53

Fig.17. Bar chart representing the bacterial count within group 3 before

and after application of treatment…………………………………. 54

Fig.18. Bar chart representing the bacterial count within group 4

before and after application of treatment……………………………… 55

Fig.19. Bar chart representing the bacterial count within group 5

before and after application of treatment…………………………. 56

Fig.20. Bar chart representing the bacterial count within group 6

before and after application of treatment…………………………. 57

Fig.21. Bar chart representing a comparison between the differences in

the bacterial count after treatment of all groups……………………58

Fig.22. Bar chart comparing the range of killed bacteria after the

treatment between group 1 (Unasyn) and group 2 (Cefobid)……...59

Fig.23. Bar chart comparing the range of killed bacteria after the

treatment between group 1 (Unasyn) and group 3 (Zithromax)…. 60

Fig.24. Bar chart comparing the range of killed bacteria after the

treatment between group 1 (Unasyn) and group 4 (Tavanic)……..61

Fig.25. Bar chart comparing the range of killed bacteria after the

treatment between group 1 (Unasyn) and group 5 (Doxymycin)…62

Fig.26. Bar chart comparing the range of killed bacteria after the

treatment between group 1 (Unasyn) and group 6 (MTAD)…….. 63

Fig.27. Bar chart comparing the range of killed bacteria after the

treatment between group 2 (Cefobid) and group 3 (Zithromax)…. 64

Fig.28. Bar chart comparing the range of killed bacteria after the

treatment between group 2 (Cefobid) and group 4 (Tavanic)……. 65

Fig.29. Bar chart comparing the range of killed bacteria after the

treatment between group 2 (Cefobid) and group 5 (Doxymycin)... 66

Fig.30. Bar chart comparing the range of killed bacteria after the

treatment between group 2 (Cefobid) and group 6 (MTAD)………67

Fig.31. Bar chart comparing the range of killed bacteria after the

treatment between group 3 (Zithromax) and group 4 (Tavanic)…..68

Fig.32. Bar chart comparing the range of killed bacteria after the

treatment between group 3 (Zithromax) and group 5 (Doxymycin).69

Fig.33. Bar chart comparing the range of killed bacteria after the

treatment between group 3 (Zithromax) and group 6 (MTAD)…...70

Fig.34. Bar chart comparing the range of killed bacteria after the

treatment between group 5 (Doxymycin) and group 4 (Tavanic)……71

Fig.35. Bar chart comparing the range of killed bacteria after the

treatment between group 6 (MTAD) and group 4 (Tavanic)……...72

Fig.36. Bar chart comparing the range of killed bacteria after the

treatment between group 5 (Doxymycin) and group 6 (MTAD)….73

Fig.37. Bar chart presenting the % reduction in bacterial counts in the six

groups………………………………………………………………..79

Fig.38. SEM evaluation of smear layer in coronal section of group 1

(Unasyn)……………………………………………………………..80

Fig.39. SEM evaluation of smear layer in apical section of group 1

(Unasyn)……………………………………………………………. 80

Fig.40. SEM evaluation of smear layer in apical section of group 1

(Unasyn)……………………………………………………………. 80

Fig.41. SEM evaluation of smear layer in coronal section of group 2

(Cefobid)……………………………………………………………..81

Fig.42. SEM evaluation of smear layer in middle section of group 2

(Cefobid)……………………………………………………………..81

Fig.43. SEM evaluation of smear layer in apical section of group 2

(Cefobid) …………………………………………………………….81

Fig.44. SEM evaluation of smear layer in coronal section of group 3

(Zithromax)…………………………………………………………..82

Fig.45. SEM evaluation of smear layer in middle section of group 3

(Zithromax)……………………………………..……………………82

Fig.46. SEM evaluation of smear layer in apical section of group 3

(Zithromax)……..……………………………………………………82

Fig.47. SEM evaluation of smear layer in coronal section of group 4

(Tavanic)……………………………………………………………..83

Fig.48. SEM evaluation of smear layer in middle section of group 4

(Tavanic)……………………………………………………………. 83

Fig.49. SEM evaluation of smear layer in apical section of group 4

(Tavanic)……………………………………………………………. 83

Fig.50. SEM evaluation of smear layer in middle section of group 5

(Doxymycin)…………………………………….…………………..84

Fig.51. SEM evaluation of smear layer in middle section of group 5

(Doxymycin)…………………………………………………………84

Fig.52. SEM evaluation of smear layer in apical section of group 5

(Doxymycin)…………………………………………………………84

Fig.53. SEM evaluation of smear layer in apical section of group 6

(MTAD)………………………………………………………………85

Fig.54. SEM evaluation of smear layer in apical section of group 6

(MTAD)………………………………………………………………85

Fig.55. SEM evaluation of smear layer in apical section of group 6

(MTAD)……………………………………………………………....85

Fig.56. Box and Whisker Plot showing smear layer score among different

groups………………………………………………………………...86

Fig.57. showing smear layer score among different groups……...…87

List of Tables

Table (1) A comparison of bacterial counts before and after treatment of

group 1…………………………………………………………………51

Table (2): A comparison of bacterial counts before and after treatment of

group 2………………………………………………………………….52

Table (3): A comparison of bacterial counts before and after treatment of

group 3…………………………………………………………………53

Table (4): A comparison of bacterial counts before and after treatment of

group 4…………………………………………………………………54

Table (5): A comparison of bacterial counts before and after treatment of

group 5…………………………………………………………………55

Table (6): A comparison of bacterial counts before and after treatment of

group 6………………………………………………………………….56

Table (7): A comparison of bacterial counts before and after treatment

between all groups……………………………………………………..57

Table (8): A comparison of bacterial counts before and after treatment

within group 1 (Unasyn) and group 2 (Cefobid)……………………..59

Table (9): A comparison of bacterial counts before and after treatment

within group 1 (Unasyn) and group 3 (Zithromax)…………………..60

Table (10): A comparison of bacterial counts before and after treatment

within group 1 (Unasyn) and group 4 (Tavanic)………………………61

Table (11): A comparison of bacterial counts before and after treatment

within group 1 (Unasyn) and group 5 (Doxymycin)…………………..62

Table (12): A comparison of bacterial counts before and after treatment

within group 1 (Unasyn) and group 6 (MTAD)……………………….63

Table (13): A comparison of bacterial counts before and after treatment

within group 2 (Cefobid) and group 3 (Zithromax)…………………..64

Table (14): A comparison of bacterial counts before and after treatment

within group 2 (Cefobid) and group 4 (Tavanic)……………………..65

Table (15): A comparison of bacterial counts before and after treatment

within group 2 (Cefobid) and group 5 (Doxymycin)………………….66

Table (16): A comparison of bacterial counts before and after treatment

within group 2 (Cefobid) and group 6 (MTAD)……………………….67

Table (17): A comparison of bacterial counts before and after treatment

within group 3 (Zithromax) and group 4 (Tavanic)……………………68

Table (18): A comparison of bacterial counts before and after treatment

within group 3 (Zithromax) and group 5 (Doxymycin)……………… 69

Table (19): A comparison of bacterial counts before and after treatment

within group 3 (Zithromax) and group 6 (MTAD)……………………70

Table (20): A comparison of bacterial counts before and after treatment

within group 5 (Doxymycin) and group 4 (Tavanic)………………….71

Table (21): A comparison of bacterial counts before and after treatment

within group 6 (MTAD) and group 4 (Tavanic)………………………72

Table (22): A comparison of bacterial counts before and after treatment

within group 5 (Doxymycin) and group 6 (MTAD)………………….73

Table (23) Summarizing comparisons between all the groups……….74

Table (24) A comparison of the percentage reduction in bacterial count in

all groups………………………………………………………………78

Table (25) A comparison between all groups showing the significance

in percentage reduction………………………………………………..78

Table (26) Mean values ± standard deviation of smear layer scores of

different groups…………………………………………………………86

Table (27) Percentage of surface area of dentine debris in different

groups………………………………………………………………….88

Introduction

1

INTRODUCTION

Microorganisms play a fundamental role in the etiology of pulp and

periradicular lesions. Successful root canal therapy relies on a triad of

instrumentation, disinfection and obturation. Disinfection of the root canal

is a major determinant in the healing of periapical tissues. Although the

chemomechanical preparation and use of antimicrobials are effective in

reducing the bacterial load, some bacteria can still persist. Enterococcus

faecalis is one among the facultative organisms which is persistently

found in root canal failures, and is resistant to various intracanal

medicaments.

Sodium hypochlorite (NaOCl) is a commonly used root canal

irrigant. However, it has an unpleasant odor and taste; it does not

consistently disinfect the root canal system and is toxic when extruded

into the periradicular tissues.

In vitro tests have shown that bacteria are usually killed rapidly

when they come in direct contact with various irrigants and medicaments.

Sometimes an infection is resistant to normal treatment, and the

therapy cannot be successfully completed. Therefore, a search for better

root canal irrigant continues.

Review of literature

2

REVIEW OF LITERATURE

BioPure MTAD:

BioPure MTAD is a mixture of a tetracycline isomer, citric acid, and a

detergent. BioPure MTAD represents an innovative approach for the

simultaneous removal of smear layer and disinfection of the root canal

system. It is commercially available as a two-part set that is mixed on

demand. A biocompatible material and possesses similar solubilizing

effects on pulp and dentin to those of EDTA. It has superior bactericidal

activity compared with NaOCl when tested against Enterococcus faecalis.

In addition to these desirable properties, BioPure MTAD is an effective

solution for the removal of the smear layer.(98)

Shabahang et al (2003) (1)

, compared the antimicrobial effect of

MTAD (a mixture of a tetracycline isomer, an acid, and a detergent) with

that of NaOCl with and without EDTA. Eighty-five extracted human teeth

were contaminated with E. faecalis for 4 weeks. After biomechanical

instrumentation using 1.3% or 5.25% NaOCl as root canal irrigant, the

root canal and the external surface of each tooth were exposed to a 5-min

application of MTAD, 1.3% NaOCl, 5.25% NaOCl or a 1-min application

of EDTA followed by irrigation with 5 ml of 1.3% NaOCl or 5.25%

NaOCl. Teeth or dentin shavings were cultured to determine presence or

absence of the tested bacteria. Fisher’s exact test showed that the

combination of 1.3% NaOCl as a root canal irrigant and MTAD as a final

rinse was significantly more effective against E. faecalis than the other

regimens.

Review of literature

3

Torabinejad et al (2003) (2)

, investigated the effect of various

concentrations of sodium hypochlorite (NaOCl) as an intracanal irrigant

before the use of MTAD as a final rinse to remove the smear layer. Ten

operators, using a combination of passive step-back and rotary 0.04 taper,

nickel-titanium files, prepared 80 single- and multirooted human teeth.

Distilled water, four different concentrations of NaOCl, or MTAD were

used as intracanal irrigants. The canals were then treated for 2 min with 5

ml of one of the following solutions as a final rinse: 5.25% NaOCl, sterile

distilled water, 17% EDTA, or MTAD. The presence or absence of smear

layer and the amount of erosion on the surface of the root canal walls at

the coronal, middle, and apical portions of each canal were examined

under a scanning electron microscope. The results showed that although

MTAD removes most of the smear layer when used as an intracanal

irrigant, some remnants of the organic component of the smear layer

remained scattered on the surface of the root canal walls. The

effectiveness of MTAD to completely remove the smear layer was

enhanced when low concentrations of NaOCl were used as intracanal

irrigant before the use of MTAD as a final rinse.

Torabinejad et al (2003) (3)

, Investigated the effect of MTAD as a

final rinse on the surface of instrumented root canals. Forty-eight

extracted maxillary and mandibular single-rooted human teeth were

prepared by using a combination of passive step-back and rotary 0.04

taper nickel-titanium files. Sterile distilled water or 5.25% sodium

hypochlorite were used as intracanal irrigants. The canals were then

treated with 5 ml of one of the following solutions as a final rinse: sterile

distilled water, 5.25% sodium hypochlorite, 17% EDTA and MTAD. The

presence or absence of smear layer and the amount of erosion on the

Review of literature

4

surface of the root canal walls at the coronal, middle, and apical portion

of each canal were examined under a scanning electron microscope. The

results show that MTAD is an effective solution for the removal of the

smear layer and does not significantly change the structure of the dentinal

tubules when canals were irrigated with sodium hypochlorite and

followed with a final rinse of MTAD.

Torabinejad et al (2003) (4)

, Tested the ability of MTAD to kill

Enterococcus faecalis and compared its efficacy to that of sodium

hypochlorite (NaOCl) and ethylene diamine tetraacetic acid (EDTA). The

zones of inhibition and minimum inhibitory concentrations were

measured for these solutions. Measurement of zones of inhibition and

determination of the minimum inhibitory concentrations showed that

MTAD is as effective as 5.25% NaOCl and significantly more effective

than EDTA. Furthermore, MTAD was significantly more effective in

killing E. faecalis than NaOCl when the solutions were diluted.

Measurement of the minimum inhibitory concentrations demonstrated

that although MTAD is still effective in killing E. faecalis at 200x

dilution, NaOCl ceased to exert its antibacterial activity beyond 32x

dilution. EDTA did not exhibit any antibacterial activity. Based on the

results of this study, it seems that MTAD is an effective solution in

eradicating E. faecalis

Tay et al (2006) (5)

, examined the effect of NaOCl-MTAD

interaction on the antimicrobial substantivity of MTAD in dentin. Dentin

cores previously irrigated with either MTAD, or in conjunction with 1.3%

NaOCl as an initial irrigant were placed on blood agar plates inoculated

with Escherichia faecalis at 105 cfu/ml. Dentin cores irrigated with 1.3%

Review of literature

5

NaOCl only, and autoclaved dentin disks were used as the respective

positive and negative controls. After anaerobic incubation, the mean

diameter of bacterial inhibition zones formed around the MTAD group

was significantly larger than the NaOCl/MTAD group, which in turn, was

not significantly different from the NaOCl positive control. Oxidation of

MTAD by NaOCl resulted in the partial loss of antimicrobial substantivity

in a manner similar to the peroxidation of tetracycline by reactive oxygen

species.

Baumgartner et al (2006) (6)

, compared the antimicrobial efficacy

of irrigation 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. Bilaterally matched human teeth were sterilized

and inoculated with E. faecalis. After chemomechanical root canal

preparation, the root-ends were resected and pulverized in liquid nitrogen

to expose E. faecalis in dentinal tubules or other recesses away from the

main root canal system. The number of colony forming units (CFU) of E.

faecalis per mg was determined from the pulverized root-ends. No

significant differences were seen between the number of colony forming

units of E. faecalis for teeth irrigated with 5.25% NaOCl/15% EDTA

versus those teeth irrigated with 1.3% NaOCl/BioPure MTAD .This study

demonstrated that there was 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.

Review of literature

6

Portenier et al (2006) (7)

, investigated the antibacterial activity of

MTAD and chlorhexidine towards two strains of Enterococcus faecalis

and the inhibitory effects of dentine and bovine serum albumin on the

antibacterial activity. Survival of bacteria exposed to the medicaments in

the presence or absence of inhibitors was monitored in an in vitro model.

Full concentration (100%) MTAD and 0.2% chlorhexidine rapidly killed

both strains. Combining chlorhexidine with cetrimide further reduced the

time required for killing. The presence of dentine or BSA caused a

marked delay in killing by both medicaments. The two E. faecalis strains

tested showed minor differences in their susceptibility to the disinfectants.

Krause et al (2007) (8)

, compared the antimicrobial effect of

MTAD, two of its components, doxycycline and citric acid, and sodium

hypochlorite (NaOCl) in two in vitro models on E. faecalis. In the bovine

tooth model, the lumens of 30 bovine dentin discs were infected with E.

faecalis for 2 weeks before treating with either one of the experimental

irrigants or saline. Bacteria in the shavings were collected with two sizes

of burs and enumerated after overnight culturing. Zones of inhibition were

recorded in the agar diffusion model for each irrigant. In the tooth model,

NaOCl and doxycycline were more effective than control in killing E.

faecalis at the shallow bur depth, but at the deeper bur depth only NaOCl

was superior. In the agar diffusion model, NaOCl produced less inhibition

than MTAD or doxycycline.

Newberry et al (2007) (9)

, investigated the antimicrobial effect of

MTAD as a final irrigant on eight strains of Enterococcus faecalis and

measured the minimum inhibitory concentration (MIC) and the minimum

lethal concentration (MLC) of MTAD. The roots of 240 extracted human

Review of literature

7

teeth were instrumented using 1.3% NaOCl and 17% EDTA. The roots

were divided into eight groups and contaminated with one of eight strains

of E. faecalis. After irrigating with 1.3% NaOCl, the root canal and the

external surfaces were exposed to MTAD for 5 minutes. Roots or dentin

shavings were cultured to determine the growth of E. faecalis. The results

showed that this treatment regimen was effective in completely

eliminating growth in seven of eight strains of E. faecalis. The

MIC/MLC tests showed that MTAD inhibited most strains of E. faecalis

growth when diluted 1:8192 times and killed most strains of E. faecalis

when diluted 1:512 times.

Davis et al (2007) (10)

, investigated the antimicrobial action of

Dermacyn , BioPure MTAD , 2% chlorhexidine , and 5.25% sodium

hypochlorite (NaOCl) against Enterococcus faecalis. Eighteen Petri

dishes of BHI agar were inoculated with E faecalis. Each Petri dish had

five saturated paper disks placed. Four of the disks were saturated with a

different test solution, and the last paper disk served as the control and

was saturated with sterile distilled water. The plates were randomly

distributed into two groups. Group one (n=9) was incubated aerobically

and group 2 (n=9) was incubated anaerobically for 48 hours at 37 degrees

C. The largest diameter of the zones of microbial inhibition was measured

in millimeters and recorded. Statistical analysis was performed with

repeated-measures analysis of variance. BioPure MTAD showed

significantly more zones of microbial inhibition than 5.25% NaOCl, 2%

CHX, and Dermacyn. Sodium hypochlorite and CHX showed

significantly more zones of microbial inhibition than Dermacyn. The

zone of inhibition between NaOCl and CHX was not significant. The

control group showed no microbial inhibition.

Review of literature

8

Johal et al (2007) (11)

, compare the antimicrobial efficacy of 1.3%

NaOCl/BioPure MTAD to 5.25% NaOCl/15% EDTA for root canal

irrigation. Twenty-six bilaterally matched pairs of human teeth were

collected. The teeth were incubated with Enterococcus faecalis for 4

weeks. The teeth were divided into two experimental groups and one

positive control group. The canals were instrumented and irrigated with

either 5.25% NaOCl/15% EDTA or 1.3% NaOCl/BioPure MTAD.

Bacterial samples were collected after instrumentation/irrigation and after

additional canal enlargement. Statistical analysis of the data using the

Wilcoxon Signed Rank test showed significant differences between the

experimental groups. The first bacterial samples revealed growth in 0 of

20 samples with 5.25% NaOCl/15% EDTA irrigation and in 8 of 20

samples with 1.3% NaOCl/BioPure MTAD irrigation. Samples taken

after additional canal enlargement revealed growth in 0 of 20 samples in

5.25% NaOCl/15% EDTA and in 10 of 20 samples in 1.3%

NaOCl/BioPure MTAD group. This investigation showed consistent

disinfection of infected root canals with 5.25% NaOCl/15% EDTA. The

combination of 1.3% NaOCl/BioPure MTAD left nearly 50% of the

canals contaminated with E. faecalis.

Ahangari et al (2008) (12)

, compared the antimicrobial effects of

2.5% Sodium hypochlorite (NaOCl), 2% Chlorhexidine Gluconate

(CHX) and BioPure MTAD (MTAD) on Enterococcus faecalis-

contaminated root canals of human extracted teeth. Seventy human intact

extracted single-rooted teeth with straight root canals were randomly

divided into 5 groups: positive control (n=5), negative control (n=5),

2.5% NaOCl (n=20), 2% CHX (n=20), and MTAD (n=20). Each tooth

was instrumented using the passive step-back technique hand and rotary