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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