antibacterial effectiveness of a final rinse with mtad and intracanal medication with 2 ...

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"ANTIBACTERIAL EFFECTIVENESS OF A FINAL RINSE WITH MTAD AND
INTRACANAL MEDICATION WITH 2% CHLORHEXIDINE GEL IN TEETH
WITH APICAL PERIODONTITIS"
By
Gevik Malkhassian, DDS
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Graduate Department of Dentistry
University of Toronto
© Copyright by Gevik Malkhassian 2007
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ABSTRACT
"ANTIBACTERIAL EFFECTIVENESS OF A FINAL RINSE WITH MTAD AND
INTRACANAL MEDICATION WITH 2% CHLORHEXIDINE GEL IN TEETH
WITH APICAL PERIODONTITIS"
Master of Science 2007
Gevik Malkhassian, DDS
Graduate Department of Dentistry, Discipline of Endodontics
University of Toronto
This randomized-controlled and double-blinded clinical study assessed the antibacterial
efficacy of (I) final rinse with BioPure™MTAD™, and (2) intracanal-medication with
2% chlorhexidine gel (CHX) in teeth with apical periodontitis in consenting humans.
Thirty teeth were chemomechanically prepared using NaOCl, then rinsed with MTAD, or
coloured saline. Bacteriological samples were obtained from canals before preparation
(IA), after preparation (lB), and after the final rinse (IC). After 7d medication with CHX
canals were flushed, sampled (2A), further irrigated and re-sampled (2B). Bacteria were
enumerated by colony-forming-unit (CFU) counts after I4d incubation and
epifluorescence-microscopy.
High counts in IA samples were >99% lower in IB samples. Lower counts in IB, IC, 2A
and 2B samples were not significantly different from each other. Microscopic counts
were higher than CFU counts.
A final rinse with MTAD and intracanal-medication with CHX did not reduce bacterial
counts in infected canals beyond levels achieved by chemomechanical preparation using
NaOCl.
11
Acknowledgements
This thesis is the result of a scientific study that has been carried out from 2004 to 2007
in fulfillment of a Masters of Science degree in the Discipline of Endodontics, Faculty of
Dentistry, University of Toronto, Canada.
This work would not have been possible without the invaluable help, advice, and support
of my research supervisor and advisory committee members Dr. Bettina Basrani, Dr.
Shimon Friedman, Dr. Ted Fillery, and Dr. Richard Ellen.
At first I would like to express my gratitude to Dr. Bettina Basrani, my principal
supervisor, for her great support and advice during my research project.
I am also deeply indebted to my mentor, Dr. Shimon Friedman, for his continuous
guidance, encouragement, invaluable insights and his precious time that he has dedicated
to the writing of this thesis. I could have never embarked and started all this without his
uncompromising support.
I would like to extend my gratitude to Dr. Ted Fillery whose supervision and constructive
criticism have been crucial for the development of this project.
It is a pleasure to pay tribute to Dr. Milos Legner who has always kindly granted me his
time and provided me with guidance.
I also convey special acknowledgement to Dr. Richard Ellen for accepting to be a
member of the advisory committee, and for providing me with invaluable advice and
input.
Many thanks goes to Dr. Aldo Manzur for his time and assistance in the laboratory as
well as his exceptional support and friendship.
iii
I wish to acknowledge people who assisted me in my research project: Miss Sheela
Manek whose invaluable help facilitated the long process of the lab work, Ms. Debbie
Flynn whose assistance in the clinic has been invaluable and Ms. Helen Grad who kindly
provided me with the medication needed for the project.
I would like to add my gratitude to all staff in Endo department who have always been
nice and helpful to me. I especially wish to thank Mrs. Heather Hyslop, Lily Kaganovsky,
and Maryann Lisotti.
My special appreciation goes to my colleagues Joe, Cris, Gaelle, Craig, Greg, and Amir
for their help, encouragement and friendship during this study.
I cannot express my full gratitude to my wife Varvareh whose endless support,
unconditional love, and persistent confidence in me has always been my source of
encouragement.
Finally, I would like to thank everybody who was important to the successful realization
of this thesis, and to express my apologies for not being able to mention all the names one
by one.
This project was supported by the grants from The American Association of Endodontists
Foundation, The Canadian Academy of Endodontics Endowment and Dentsply-Tulsa
Dental.
Gevik Malkhassian
Toronto, 2007
IV
1. INTRODUCTION .......................................................................................................... 2
2. REVIEW OF LITERATURE ......................................................................................... 4
2.1. Bacteria in Endodontic Disease ................................................................................... 4
2.1.1. Microbial Flora in Infected Root Canals with Apical Periodontitis ......................... 4
2.1.2. Responses of Pulp and Periapical Tissues to Invasion of Bacteria ........................... 5
2.1.2.1. Invasion of Bacteria ............................................................................................... 5
2.1.2.2. Pulpal Response ..................................................................................................... 5
2.1.2.3. Periapical Response, Apical Periodontitis ............................................................. 6
2.1.3. Etiological Role of Bacteria ...................................................................................... 7
2.1.4. Outcome Investigations ............................................................................................ 8
2.1.5. Reduction of Bacteria and Disinfection of the Root Canal System .......................... 9
2.1.5.1. Mechanical Preparation ......................................................................................... 9
2.1.5.2. Chemomechanical Preparation ............................................................................ 11
2.2. MTAD ........................................................................................................................ 13
2.2.1. Protocol for Use ...................................................................................................... 14
2.2.2. Properties of MTA D ............................................................................................... 14
2.2.2.1. Mechanism of Action ........................................................................................... 14
2.2.2.2. Cytotoxicity ofMTAD ........................................................................................ 15
2.2.2.3. Surface Tension ................................................................................................... 16
2.2.2.4. Smear Layer Removal. ......................................................................................... 16
2.2.2.5. Antibacterial Efficacy .......................................................................................... 17
2.2.3. In vivo Clinical Trial ............................................................................................... 20
2.3. Intracanal Medication ................................................................................................ 20
2.3 .1. Limitations of Calcium Hydroxide ......................................................................... 21
2.3.2. Chlorhexidine (CHX) .............................................................................................. 21
2.3 .2.1. History. ................................................................................................................. 22
2.3.2.2. Molecular Structure ............................................................................................. 22
2.3.2.3. Mode of Action .................................................................................................... 22
2.3.2.4. Substantivity ........................................................................................................ 23
2.3.2.5. Cytotoxicity. ......................................................................................................... 23
2.3.2.6. Chlorhexidine Application in Dentistry ............................................................... 25
2.3.2.7. Chlorhexidine Application in Endodontics .......................................................... 25
2.3.2.8. Chlorhexidine as an Endodontic Irrigant ............................................................. 26
2.3.2.9. Chlorhexidine as an Intracanal Medication ......................................................... 28
2.3.3. Root Canal Microbial Investigations ...................................................................... 33
2.3.4. Previous Work and Background of the Project.. ..................................................... 37
3. AIMS OF THE STUDY ............................................................................................... 40
3 .1. Principle Aim ............................................................................................................. 40
3.2. Secondary Aims ......................................................................................................... 40
3.3. Specific Aims ............................................................................................................. 40
3.4. Null Hypothesis ......................................................................................................... 40
4. MATERIALS AND METHODS .................................................................................. 42
4.1. Design ........................................................................................................................ 42
v
4.1.1. Randomization ........................................................................................................ 42
4.1.2. Blinding. .................................................................................................................. 42
4.2. Sample Size Calculation ............................................................................................ 42
4.3. Study Cohort and Final Sample Size of the Study ..................................................... 44
4.4. Ethics and Scientific Merit Reviews .......................................................................... 44
4.5. Pre-Clinical Procedures ............................................................................................. 44
4.5.1. Laboratory Procedures ............................................................................................ 44
4.5.2. Preparation of 2% Chlorhexidine Gel.. ................................................................... 45
4.6. Clinical Procedures .................................................................................................... 46
4.6.1. Isolation and Field Disinfection .............................................................................. 46
4.6.2. Inactivation of Disinfecting Solutions .................................................................... 47
4.6.3. Sample Acquisition ................................................................................................. 47
4.6.3.1. Sterility Control Samples: .................................................................................... 47
4.6.3.2. Access Cavity and Root Canal Samples: ............................................................. 48
4.6.4. Root Canal Volume Measurement: ......................................................................... 49
4.6.5. First Treatment Appointment. ................................................................................. 49
4.6.6. Second Treatment Appointment ............................................................................. 50
4.7. Post-clinical Procedures ............................................................................................. 51
4.7.1. Bacteriological Procedures ..................................................................................... 51
4.7.1.1. CPU ...................................................................................................................... 51
4.7.1.2. Microscopy .......................................................................................................... 52
4.7.1.2.1. BacLight vs. DHET and DAPI ......................................................................... 52
4.7.1.3. Baclight ................................................................................................................ 52
4. 7 .1.4. DAPI and Dihydroethidium ................................................................................. 53
4.7.2. Adopting the LEICA DMIRE2 Microscopy System .............................................. 54
4.7.3. Modifications to the Microscopic Counting Technique of Paquette ...................... 56
4.8. Effects of Food Colouring ......................................................................................... 57
4.9. Analysis ...................................................................................................................... 58
5. RESULTS ..................................................................................................................... 61
5.1. Access Cavity Samples .............................................................................................. 62
5.2. Sterility Control Samples ........................................................................................... 63
5.3. Root Canal Samples Before and After Preparation ................................................... 63
5.4. Root Canal Samples After Final Irrigation/immersion .............................................. 64
5.5. Root Canal Samples After Intracanal Medication ..................................................... 65
5.6. Root Canal Samples After Final Preparation Before Root Filling ............................ 66
5.7. Comparison of Microscopic Techniques for the Detection of Live and Dead Bacteria
··········································································································································· 67
5.8. Comparison ofUnmerged vs. Merged (Composite) images in Baclight Staining .... 68
5.9. Comparison of Automated (Macro) vs. Visual (manual) Enumeration Methods ...... 68
5.10. Effects of Food Colouring ....................................................................................... 69
6. DISCUSSION ............................................................................................................... 71
6.1. Study Methodology .................................................................................................... 71
6.1.1. Design ..................................................................................................................... 71
6.1.2. Inclusion/exclusion Criteria .................................................................................... 72
6.1.3. Prevention of False Positive Root Canal Samples .................................................. 72
Vl
6.1.4. Prevention of False Negative Root Canal Samples ................................................ 76
6.1.5. Quantification of Bacterial Densities ...................................................................... 79
6.1.6. Clinical Procedures and Materials .......................................................................... 81
6.1. 7. Outcome Measures .................................................................................................. 82
6.2. The Findings .............................................................................................................. 82
6.2.1. Differences in Enumeration Methods ..................................................................... 82
6.2.2. Efficacy of Canal Preparation with 1.3% NaOCl Irrigation ................................... 85
6.2.3. Efficacy of Final Irrigation/immersion with MTAD .............................................. 87
6.2.4. Efficacy oflntracanal Dressing with 2% Chlorhexidine Gel ................................. 89
6.2.5. Efficacy of Additional 1.3% NaOCl Irrigation in the Second Treatment Session. 91
6.3. General Discussion .................................................................................................... 92
7. CONCLUSIONS ......................................................................................................... 101
8. REFERENCE LIST .................................................................................................... 104
9. TABLES ..................................................................................................................... 126
10. FIGURES .................................................................................................................. 142
11. APPENDICES .......................................................................................................... 148
vii
INTRODUCTION
1
2
1. INTRODUCTION
The ultimate biological aim of root canal treatment is either to prevent or cure apical
periodontitis (1 ). Several researches have clearly demonstrated the involvement of
bacteria in the development and persistence of apical periodontitis. Therefore, elimination
of bacteria has been the main focus of root canal treatment. Numerous studies have been
conducted over half a century primarily to gain a better understanding of the microbial
flora and the ecological conditions in infected root canals and secondly to find the ideal
means of eliminating bacteria from the root canal system (2-34).
For many years, sodium hypochlorite has been used as an intracanal irrigating solution
(25, 35-39) and calcium hydroxide as an intra canal inter-appointment medicament (5,
33, 40-44). Combined with mechanical preparation they have provided a good outcome
for endodontic therapy. Recent studies, however, indicate that a small number of bacteria
still survive at the time of root filling in many teeth (11, 26, 33, 34).
Currently, there is an extensive research activity investigating new methods and materials
to be used for the irrigation and disinfection of the root canal system. The effectiveness of
these new materials can be tested using various in vitro and in vivo techniques. The
identification and enumeration of bacteria in vivo is one of the most important methods of
testing the efficacy of the new materials. The sampling and culture method is the most
frequently used technique. However, it has been demonstrated that a large number of
bacteria are not cultivable, and only viable bacteria can be detected by this method (16).
To overcome the limitations of the culture technique some research groups have adopted